the key role of the lectin pathway enzyme masp-3 in the ... · ii abstract the key role of the...

The key role of the Lectin Pathway enzyme

MASP-3 in the innate immune protection against

Neisseria meningitidis

Thesis submitted for the degree of

Doctor of Philosophy

at the University of Leicester

By

Saleh Alshamrani

Department of Infection, Immunity and Inflammation

University of Leicester

2016

I

Statement of originality

This accompanying thesis submitted for the degree of PHD entitled (The

key role of the Lectin Pathway enzyme MASP-3 in the innate immune

protection against Neisseria meningitidis) is based on work conducted by

the author at the University of Leicester mainly during the period between

January 2012 and January 2015

All the work recorded in this thesis is original unless otherwise

acknowledged in the text or by references.

None of the work has been submitted for another degree in this or any other

University

Signed: Date:

II

Abstract

The key role of the Lectin Pathway enzyme MASP-3 in the innate

immune protection against Neisseria meningitidis

Neisseria meningitidis infections pose a worldwide threat to human health being a

major cause of morbidity and mortality. The bacterium can often be found to live as a

commensal organism in the upper respiratory-tract. However, under disease promoting

circumstances it may cause invasive infections such as bacterial meningitis with a

mortality rate of up to 10% in patients with sepsis. The complement system plays a vital

role in immune protection from Neisseria meningitidis infections and ongoing research

in our laboratories has recently observed that the serum of mice deficient in the lectin

pathway of complement effector enzyme MASP-2 had a higher bactericidal activity

towards Neisseria meningitidis as compared to MASP-2 sufficient serum. This work

also revealed a key role of the lectin pathway components MBL and MASP-3 in driving

serum bacteriolytic activity against Neisseria meningitidis and has identified a novel

link between MASP-3 and the alternative pathway of complement activation. The work

described in this thesis highlights the critical role that MASP-3 plays in the innate

immune response to this pathogen using in vitro models of serum bactericidal activity

and in vivo mouse models of Neisseria meningitidis infection. The failure of MASP-3

deficient non immune serum to lyse Neisseria meningitidis serotype A and serotype B

was restored by adding a recombinant enzymatically active MASP-3 fragment to this

serum while the therapeutic systemic injection of recombinant murine MASP-3

zymogen convincingly restored the defective alternative pathway functional activity and

with that repaired the high susceptibility of MASP-1/3 deficient mice to Neisseria

meningitidis infections. In line with the essential role that the alternative pathway plays

in driving the innate immune response against Neisseria meningitidis, the early results

of my study showed the therapeutic utility of enhancing the alternative pathway

functional activity through the addition of recombinant murine properdin to WT mice

sera and significantly increased the lytic activity against Neisseria meningitidis.

III

Acknowledgement

My greatest thanks go to my creator for his blessing and help throughout my life.

All glory and praise is due to Allah

I would like to express my deepest gratitude to my supervisor, Professor Wilhelm

Schwaeble, who has guided me during this project. His encouragement and support

have had a deep impact on my finishing this project successfully. He has always been

very patient with me and never hesitated to answer all my queries.

I would like to give my sincere thanks to my co supervisor Professor Peter Andrew for

his scientific help and advice. Many thanks go out to Dr Mohammad Y. Ali and Dr

Nicholas Lynch for their expert advice during this journey.

Next, it is a pleasure to offer particular thanks to Dr Sarah Glen for her continuous help

and advice during training in the Neisseria lab. Also, I am very thankful to all the staff

and members from lab 231; I was lucky enough to have great friends there and I have

enjoyed working with you.

Finally, I would like to express my deepest gratitude for the constant support,

remarkable patience, encouragement and love I received from my wife, Sawsan, and my

son, Abdulmalik. No words would be enough to thank my parents and siblings for

everything they have given and did for me throughout my life and especially during this

period.

IV

List of contents

Table of contents

CHAPTER 1: INTRODUCTION .................................................................................. 1

1.1 THE IMMUNE SYSTEM ............................................................................................ 1

1.2 THE COMPLEMENT SYSTEM ................................................................................... 3

1.2.1 Classical Pathway ........................................................................................................... 5

1.2.2 Alternative Pathway ...................................................................................................... 6

1.2.2.1 Complement components of alternative pathway ................................. 8

1.2.2.1.1 Factor B ............................................................................................. 8

1.2.2.1.2 Factor D ............................................................................................ 9

1.2.2.1.3 Properdin ........................................................................................... 9

1.2.3 Lectin Pathway .............................................................................................................. 10

1.2.3.1 Lectin pathway components ............................................................... 11

1.2.3.1.1 Mannan Binding Lectin .................................................................. 11

1.2.3.1.2 Ficolin ............................................................................................. 12

1.2.3.1.3 Collectin .......................................................................................... 13

1.2.3.1.4 Mannan Binding Lectin associated serine proteases ...................... 14

1.2.4 Membrane Attack Complex ...................................................................................... 17

1.2.5 Functions of the Complement System ................................................................... 18

1.2.6 Regulators of Complement System ......................................................................... 20

1.2.6.1 Fluid Phase Regulators ....................................................................... 20

1.2.6.2 Membrane-Bound Regulators: ............................................................ 21

V

1.2.7 Complement deficiency .............................................................................................. 24

1.2.7.1 Defects of the Classical Pathway ........................................................ 24

1.2.7.2 Defects of the Alternative Pathway .................................................... 25

1.2.7.3 Defects of the Lectin Pathway ............................................................ 25

1.2.7.4 Defects of terminal complement components .................................... 26

1.2.7.5 Defects of complement regulatory components ................................. 26

1.3 NEISSERIA MENINGITIDIS ....................................................................................... 28

1.3.1 The Virulence Factors of Neisseria meningitidis ................................................ 30

1.3.1.1 Pili and Pilus Subunits ........................................................................ 30

1.3.1.2 Outer Membrane Proteins ................................................................... 31

1.3.1.3 Capsule and Lipo-oligosaccharide (LOS) ........................................... 32

1.3.2 Colonization and invasion by Neisseria meningitidis ....................................... 35

1.3.3 The Immune System and Neisseria meningitidis ................................................ 37

1.3.4 The Complement System and Neisseria meningitidis ....................................... 38

1.4 THESIS AIMS ........................................................................................................ 44

CHAPTER 2: MATERIALS AND METHODS ........................................................ 45

2.1 MATERIALS .......................................................................................................... 45

2.1.1 Chemicals and materials............................................................................................. 45

2.1.2 Antibodies/proteins ...................................................................................................... 47

2.1.3 Media and buffers ........................................................................................................ 49

2.2 METHODS ............................................................................................................ 51

2.2.1 In vitro experiments ..................................................................................................... 51

2.2.1.1 Preparation of mouse and human serum ............................................. 51

VI

2.2.1.2 Preparation of Neisseria meningitidis for Enzyme Linked

Immunosorbent Assay (ELISA) ..................................................................... 51

2.2.1.3 C1q deposition assays ......................................................................... 52

2.2.1.4 MBL-A, MBL-C, Ficolin-A and CL-11 binding assays ..................... 53

2.2.1.5 C3 deposition assays ........................................................................... 54

2.2.1.6 Alternative pathway mediated C3 deposition assays .......................... 55

2.2.1.7 Lectin pathway mediated C4 deposition assays ................................. 56

2.2.1.8 Serum Bactericidal Assay (SBA) ....................................................... 57

2.2.1.9 Preparation of Neisseria meningitidis for FACS analysis .................. 57

2.2.1.10 FACS analysis for detect C3 deposition on Neisseria meningitdis .... 58

2.2.2 In vivo experiments ..................................................................................................... 59

2.2.2.1 Genotyping of fB and MASP-1/3 deficient mice ............................... 59

2.2.2.1.1 Isolation of genomic DNA from mouse ear snips .......................... 59

2.2.2.1.2 Polymerase Chain Reaction (PCR) ................................................. 59

2.2.2.2 Preparation of Neisseria meningitidis passage ................................... 61

2.2.2.3 Virulence testing of passaged stocks of Neisseria meningitidis ......... 62

2.2.2.4 Infection of mice with Neisseria meningitidis .................................... 63

2.2.2.5 Determination of Blood bacterial burden ........................................... 63

2.2.2.6 MASP-3 reconstitution experiment .................................................... 64

2.2.3 Statistical analysis ........................................................................................................ 65

CHAPTER 3: IN VITRO STUDY ............................................................................... 66

3.1 RESULTS: ............................................................................................................. 66

3.1.1 Complement pathway specific Enzyme Linked Immune Sorbent Assays

(ELISAs) ....................................................................................................................................... 66

VII

3.1.1.1 Binding of the classical pathway molecule C1q to Neisseria

meningitidis ......................................................................................................... 67

3.1.1.2 Binding of the lectin pathway recognition component MBL to

Neisseria meningitidis ......................................................................................... 68

3.1.1.3 Binding of the lectin pathway Collectin-11 to Neisseria meningitidis 69

3.1.1.4 Binding of the lectin pathway ficolin-A to Neisseria meningitidis .... 70

3.1.2 C3 deposition assays ................................................................................................... 71

3.1.3 C4 deposition assays ................................................................................................... 78

3.1.4 Serum Bactericidal Assays ........................................................................................ 82

3.1.5 Effect of recombinant properdin on complement mediated killing of

Neisseria meningitidis ............................................................................................................... 97

3.1.5.1 Recombinant properdin has the ability to enhance C3 deposition on

the surface of Neisseria meningitidis .................................................................. 97

3.1.5.2 Recombinant properdin has the ability to enhance the killing of

Neisseria meningitidis ....................................................................................... 100

3.2 DISCUSSION ....................................................................................................... 107

3.2.1 Binding of Neisseria meningitidis to different complement recognition

molecules .................................................................................................................................... 108

3.2.2 Activation of the complement system on the surface of Neisseria

meningitidis requires a close cooperation between the lectin and alternative

pathway ....................................................................................................................................... 111

3.2.3 Recombinant properdin enhances the serum bacteriolytic activity against

Neisseria meningitidis ............................................................................................................. 118

VIII

CHAPTER 4: IN VIVO STUDY ................................................................................ 121

4.1 RESULTS: ........................................................................................................... 121

4.1.1 Genotyping of factor B deficient mice and MASP-1/3 deficient mice ....... 121

4.1.2 The optimal infective dose ...................................................................................... 125

4.1.3 Survival of factor B deficient mice and factor B sufficient mice following

experimental Neisseria meningitidis infection ................................................................. 126

4.1.3.1 The viable bacterial load of Neisseria meningitidis in the blood of

infected mice ................................................................................................... 128

4.1.4 Survival of MASP-1/3 deficient mice and MASP-1/3 sufficient mice

following experimental Neisseria meningitidis infection ............................................. 130


infected mice ..................................................................................................... 132

4.1.5 Effect of full length recombinant MASP-3 on reconstituting the absence of

the alternative pathway functional activity in MASP-1/3 deficient mice ................ 134

4.1.6 Effect of full length recombinant MASP-3 administration on mortality in a

mouse model of Neisseria meningitidis infection ........................................................... 143


infected mice ..................................................................................................... 147

4.2 DISCUSSION ....................................................................................................... 149

4.2.1 Mice deficient in the alternative pathway functional activity show

dramatically higher susceptibility to Neisseria meningitidis infection ..................... 149

4.2.2 The therapeutic application of recombinant full-length MASP-3

dramatically improves the survival of MASP-1/3 deficient mice from Neisseria

meningitidis infection .............................................................................................................. 153

IX

CHAPTER 5: CONCLUSION AND FUTURE WORK ......................................... 158

5.1 CONCLUSION ..................................................................................................... 158

5.1.1 Binding of complement system recognition molecules to Neisseria

meningitidis ................................................................................................................................ 160


meningitidis requires a close cooperation between the lectin and alternative

pathways ...................................................................................................................................... 162

5.1.3 Recombinant properdin enhances the serum bacteriolytic activity against

Neisseria meningitidis ............................................................................................................. 166

5.1.4 Mice deficient in the alternative pathway functional activity show

dramatically higher susceptibility to Neisseria meningitidis infection ..................... 167


dramatically improves the survival of MASP-1/3 deficient mice from Neisseria

meningitidis infection .............................................................................................................. 169

5.2 FUTURE WORK ................................................................................................... 172

5.2.1 Assess the therapeutic benefit of recombinant MASP-3 in fighting other

microbial infection ................................................................................................................... 172

5.2.2 Assess the ability of recombinant properdin in restoring the killing of

properdin-deficient sera .......................................................................................................... 172

CHAPTER 6: BIBLIOGRAPHY .............................................................................. 173

X

List of tables

Table ‎2.1 The severity scores of disease with clinical signs of infected mice ............... 63

Table ‎3.1 Statistically significant differences between serum bactericidal assay of

different mouse sera (C1q-/-

, WT and HIS) against Neisseria meningitidis serogroup A

strain Z2491 ................................................................................................................... 83


different mouse sera (C1q-/-

, WT and HIS) against Neisseria meningitidis serogroup B

strain MC58 . .................................................................................................................. 84

Table ‎3.3 Statistically significant differences between the serum bactericidal assay of

different mouse sera (FB-/-

, MASP-1/3-/-

, WT and HIS) against Neisseria meningitidis

serogroup A strain Z2491 . ............................................................................................. 86

Table ‎3.4 Statistically significant differences between the serum bactericidal assay of

different mouse sera (FB-/-, MASP-1/3-/-, WT and HIS) against Neisseria meningitidis

serogroup B strain MC58 ................................................................................................ 87


different mouse sera (MASP-2-/-

, MBL-/-


serogroup A strain Z2491 ............................................................................................... 89


different mouse sera (MASP-2-/-, MBL-/-


serogroup B strain MC58. ............................................................................................... 90


different mouse sera (MASP-1/3-/-

+ truncated rMASP-3 (10µg/ml), MASP-1/3-/-

, WT

and HIS) against Neisseria meningitidis serogroup A strain Z2491. ............................. 91

XI


different mouse serum (MASP-1/3-/-


, WT

and HIS) against Neisseria meningitidis serogroup B strain MC58 ............................... 93


different human sera (3MC + truncated rMASP-3 (6µg/ml), 3MC, MBL-/-

, Immune,

NHS and HIS) against Neisseria meningitidis serogroup A strain Z2491 ..................... 95


different human sera (3MC + truncated rMASP-3 (6µg/ml), 3MC, MBL-/-

, Immune,

NHS and HIS) against Neisseria meningitidis serogroup B strain MC58 ...................... 96


mouse serum with or without recombinant murine properdin against Neisseria

meningitidis serogroup A strain Z2491 ........................................................................ 101


mouse serum with or without recombinant murine properdin against Neisseria

meningitidis serogroup B strain MC58 ......................................................................... 102


human serum with or without recombinant human properdin against Neisseria

meningitidis serogroup A strain Z2491 ........................................................................ 104



meningitidis serogroup B strain MC58 ......................................................................... 106

Table ‎4.1 The design of the MASP-3 reconstitution experiment .............................. 135

Table ‎4.2 Statistically significant differences for the C3 deposition assay of different

mouse sera (MASP-1/3-/-

+ full-length rMASP-3 (40µg/mouse), MASP-1/3-/-

+ full

length rMASP-3 (20µg/mouse), MASP-1/3-/-, WT and HIS) ...................................... 137

XII

Table ‎4.3 Statistically significant difference assessed between serum bactericidal

assay of different mouse serum (MASP-1/3-/-

+ full length rMASP-3 (treated with two

doses of 20µg/mouse each given 96 and 24 hours), MASP-1/3-/-

+ full length rMASP-3

(20µg/mouse given 96 hours prior to bleeding), MASP-1/3-/-

(untreated), WT and HIS)

against Neisseria meningitidis serogroup A strain Z2491 ............................................ 140

Table ‎4.4 Statistically significant difference assessed between serum bactericidal

assay of different mouse serum (MASP-1/3-/-

+ full length rMASP-3 (treated with two

doses of 20µg/mouse each given 96 and 24 hours), MASP-1/3-/-


(20µg/mouse given 96 hours prior to bleeding), MASP-1/3-/-

(untreated), WT and HIS)

against Neisseria meningitidis serogroup B strain MC58 ............................................ 142

XIII

List of figures

Figure 1.1 Complement system activation pathways. ..................................................... 4

Figure 1.2 Structure of the C1 Complex of the classical pathway. .................................. 5

Figure 1.3 The domain structure and oligomerization of MBL. .................................. 11

Figure 1.4 The domain structure and oligomerization of ficolins ................................ 13

Figure 1.5 MASP and Map domain organization ......................................................... 15

Figure 1.6 MASPs activation. ....................................................................................... 16

Figure 1.7 Membrane attack complex pathway ........................................................... 17

Figure 1.8 Regulation of complement activation. ......................................................... 23

Figure 1.9 Neisseria meningitidis cell membrane ........................................................ 30

Figure 1.10 Different stages in the pathogenesis of N. meningitidis. .......................... 36

Figure 3.1 C1q binding on the surface of different Neisseria meningitidis strains. .... 67

Figure 3.2 MBL-A binding on the surface of different Neisseria meningitidis

strains. ............................................................................................................................. 68

Figure 3.3 MBL-C binding on the surface of different Neisseria meningitidis strains 69

Figure 3.4 CL-11 binding to the surface of different Neisseria meningitidis strains.. . 70

Figure 3.6 C3 deposition assay on the surface of different Neisseria meningitidis

strains under specific condition (high serum dilution in BBS buffer) allows the

activation through classical and lectin pathways ............................................................ 72


strains under alternative pathway permissive conditions (high serum concentration in

EGTA buffer) .................................................................................................................. 73

XIV


strains under specific condition (high serum dilution in BBS buffer) using MASP-2+/+

and MASP-2-/-

serum ...................................................................................................... 74

Figure 3.9 C3 deposition assay on the surface of Neisseria meningitidis strain A

serogroup Z2491 under alternative pathway permissive condition (high serum

concentration in BBS buffer) using sera of MASP-2+/+

and MASP-2-/-

mice................. 75

Figure 3.10 C3 deposition a assay on the surface of Neisseria meningitidis strain B

serogroup MC85 under alternative pathway permissive condition (high serum

concentration in BBS buffer) using sera of MASP-2+/+

and MASP-2-/-

mice................. 76


serogroup Z2491 under alternative pathway permissive conditions .............................. 77

Figure 3.12 C3 deposition assay on the surface of Neisseria meningitidis strain B

serogroup MC58 under alternative pathway permissive conditions ............................... 78


strains under lectin pathway specific condition (high serum dilution in MBL binding

buffer) ............................................................................................................................. 79


serogroup Z2491 under specific condition (high serum dilution in BBS buffer) ........... 80


serogroup MC58 under specific condition (high serum dilution in BBS buffer) ........... 81

Figure 3.16 Bactericidal activity of different mouse sera (C1q-/-

, WT and HIS)

against Neisseria meningitidis serogroup A strain Z2491. ............................................. 83

Figure 3.17 Bactericidal activity of different mouse sera (C1q-/- , WT and HIS)

towards Neisseria meningitidis serogroup B strain MC58.. ........................................... 84

XV

Figure 3.18 Bactericidal activity of different mouse sera (FB-/-

, MASP-1/3-/-

, WT and

HIS) against Neisseria meningitidis serogroup A strain Z2491. .................................... 85


, MASP-1/3-/-

, WT and

HIS) against Neisseria meningitidis serogroup B strain MC58.. .................................... 86

Figure 3.20 Bactericidal activity of different mouse sera (MASP-2-/-

, MBL-/-

, WT and

HIS) against Neisseria meningitidis serogroup A strain Z249.. ..................................... 88


, MBL-/-

, WT and

HIS) against Neisseria meningitidis serogroup B strain MC58 ...................................... 89

Figure 3.22 Bactericidal activity of different mouse sera (MASP-1/3-/-

+ truncated

rMASP-3 (10µg/ml), MASP-1/3-/-


serogroup A strain Z2491. .............................................................................................. 91


+ truncated

rMASP-3 (10µg/ml), MASP-1/3-/-



Figure 3.24 Bactericidal activity of different human sera (3MC + truncated rMASP-3

(6µg/ml), 3MC, MBL-/-, Immune, NHS and HIS) against Neisseria meningitidis

serogroup A strain Z2491 ............................................................................................... 94


(6µg/ml), 3MC, MBL-/-

, Immune, NHS and HIS) against Neisseria meningitidis



serogroup Z2491 under alternative pathway permissive conditions (high serum

concentration in EGTA buffer) ....................................................................................... 98

XVI


serogroup MC58 under alternative pathway permissive conditions (high serum

concentration in EGTA buffer). ...................................................................................... 99

Figure 3.28 FACS analysis of C3 deposition on the surface of Neisseria meningitidis

serogroup B strain MC58 using human and mouse serum with or without recombinant

properdin. ...................................................................................................................... 100

Figure 3.29 Bactericidal activity of mouse serum with or without recombinant murine

properdin against Neisseria meningitidis serogroup A strain Z2491.. ......................... 101


properdin against Neisseria meningitidis serogroup B strain MC58. ........................... 102

Figure 3.31 Bactericidal activity of human serum with or without recombinant human

properdin against Neisseria meningitidis serogroup A strain Z2491. .......................... 103


properdin against Neisseria meningitidis serogroup B strain MC58 ............................ 105

Figure 3.33 Lectin pathway effector arms. ................................................................ 117

Figure 4.1 Genotyping results for the FB targeted mouse line showed the amplified

products ......................................................................................................................... 123

Figure 4.2 Genotyping results for MASP1/3 mice showed the amplified products . 124

Figure 4.3 Survival of C57BL/6 wild-type mice following an i.p. injection with

different doses of N.meningitidis serogroup B strain MC58. ....................................... 125

Figure 4.4 Survival of wild type (on C57/BL6 background) and Factor B deficient

mice (on C57/BL6 background) following an i.p. injection with a low dose (1×105) of

Neisseria meningitidis serogroup B strain MC58. ........................................................ 127

XVII

Figure 4.5 Average illness score of wild type and Factor B deficient mice following an

i.p. injection with a low dose (1×105) of Neisseria meningitidis serogroup B strain

MC58. ........................................................................................................................... 128

Figure 4.6 Bacterial load in the blood of Factor B deficient mice and wild-type mice

given an i.p. injection of a low dose (1x105

CFU/mouse) of Neisseria meningitidis

serogroup B strain MC58 .............................................................................................. 129

Figure 4.7 Survival of WT and MASP-1/3 deficient mice (both on C57/BL6

background) following an i.p. injection with a low dose (1×105) of Neisseria

meningitidis serogroup B strain MC58.. ....................................................................... 131

Figure 4.8 Average illness score of wild type and MASP-1/3 deficient mice following

an i.p. injection with a low dose (1×105) of Neisseria meningitidis serogroup

B strain MC58 ............................................................................................................... 132

Figure 4.9 Bacterial load in blood of MASP-1/3 deficient mice and wild-type mice

given an i.p. injection of a low dose (1x105 CFU/mouse) of Neisseria meningitidis

serogroup B strain MC58. ............................................................................................. 133

Figure 4.10 C3 deposition of MASP-1/3 deficient mice treated with full length

recombinant murine MASP-3. ...................................................................................... 136

Figure 4.11 Serum Bactericidal Activity of the different mouse sera (MASP-1/3-/-

+

full length rMASP-3 (treated with two doses of 20µg/mouse each given 96 and 24

hours), MASP-1/3-/-

+ full length rMASP-3 (20µg/mouse given 96 hours prior to

bleeding), MASP-1/3-/-

(untreated) and WT mouse blood and heat-inactivated WT

mouse blood, i.e. HIS) against Neisseria meningitidis serogroup A strain Z2491. ...... 139

XVIII


+

full length rMASP-3 (treated with two doses of 20µg/mouse each given 96 and 24

hours), MASP-1/3-/-

+ full length rMASP-3 (20µg/mouse given 96 hours prior to

bleeding), MASP-1/3-/-

(untreated) and WT mouse blood and heat-inactivated WT

mouse blood, i.e. HIS) against Neisseria meningitidis serogroup B strain MC58 ....... 141

Figure 4.13 Survival of wild type mice, MASP-3 reconstituted MASP-1/3 deficient

mice (receiving 2 doses of full length recombinant MASP-3 (20µg/mouse at time points

96 hours and time point 24 hours prior to infection) and non-treated MASP-1/3 deficient

mice (all on C57/BL6 background) were infected by i.p. injections with a low dose

(1×105) of Neisseria meningitidis serogroup B strain MC58. ...................................... 145

Figure 4.14 Average illness score for wild type, treated MASP-1/3 deficient mice and

non-treated MASP-1/3 deficient mice following an i.p. injection with a low dose

(1×105) of Neisseria meningitidis serogroup B strain MC58 ....................................... 146

Figure 4.15 Bacterial load in blood of wild-type mice, treated MASP-1/3 deficient

mice and non-treated MASP-1/3 deficient mice given an i.p. injection of a low dose

(1x105 CFU/mouse) of Neisseria meningitidis serogroup B strain MC58 .................. 148

I

Abbreviations

Α Alpha

Β Beta

AP Alkaline phosphate

AP Alternative pathway

BBS Barbital buffer saline

BHI Brain heart infusion

Bp Base pair

BSA Bovine serum albumin

CCP Complement control protein

CED Carbohydrate recognition domain

CF Catalytic fragment

CFS Cerebrospinal fluid

CNL Capsule null locus

CP Classical Pathway

CPS Capsule synthesis

CRD Carbohydrate recognition domain

CrgA Contact-regulated gene A

II

DIC Disseminated intravascular coagulation

DNA Deoxyribonucleic acid

dNTPs Deoxynucleotides

EDTA Ethylenediaminetetra acetic acid

EGF Epidermal growth factor

EGTA Ethylene glycol tetraacetic acid

ELISA Enzyme Linked Immune Sorbent Assay

ELISA Enzyme Linked Immunosorbent Assay

FACS Fluorescent activated cell sorter

FB Factor B

FBG Fibrinogen-like carbohydrate recognition domain

FCS Foetal calf serum

FCS Foetal calf serum

Fd Factor D

G Grams

gDNA Genomic deoxyribonucleic acid

GNA Genome derived neisserial antigen

HAE Hereditary angioedema

HIS Heated inactivated serum

III

Ig Immunoglobulin

Kb Kilobase

kDa Kilodalton

KDO 2-keto-3-deoxyoctulosonic acid

KO Knockout

LNnt Lacto-N-neotetraose

LOS Lipo-oligosaccharide

MASP MBL- associated serine proteases

MBL Mannan Binding Lectin

MIDS Metal ion dependant adhesion site

Min Minutes

NHS Normal human serum

NK Natural killer cell

NspA Niesserial surface protein A

OD Optical density

OMPs Outer membrane proteins

Opa Outer membrane protein A

Opc Outer membrane protein C

PAMPS Pathogen associated molecular patterns

IV

PBS Phosphate buffered saline

PCR Polymerase chain reaction

PEA Phosphoethanolamine groups

PorA Meningococcal porin A

PorB Meningococcal porin B

rMASP-3 Recombinant MASP-3

RT Room temperature

SBA Serum bactericidal assay

SCRs Short consensus repeats

Sec Seconds

Tfp Type IV pili

TSRs Thrombospondin structural homology repeats

vWA Van Willebrand factor A

WT Wild-type

µ Micro

Chapter 1: Introduction

1


1.1 The Immune System

Throughout our life we are exposed to various microbial organisms as well molecular

species such as toxins and allergic substances that may impact on the normal

physiological function and integrity of our body. This exposure leads to the activation

of different defence mechanisms that protect our body by eliminating or neutralizing the

effects of these molecules and invading organisms. Together, these defence mechanisms

compose the immune system, which consists of a large number of proteins, cells, tissues

and organs that have evolved to protect the human body (Chaplin, 2010). The immune

system comprises two main systems, namely the innate immune system and the

adaptive immune system. The innate immune system, which is phylogenetically older

than the latter, provides the first line of defence against invading organism and toxic

substances (Medzhitov, 2007). It is characterized by the absence of memory function

but has the ability to mount a rapid response against invading microbes, resulting in

isolation and destruction of invading organisms (Borghesi & Milcarek, 2007). This

system works at many levels of anatomical and physiological barriers, for example, the

skin and mucosal membranes, by the production of protective chemicals such as

stomach acidity and proteins that recognise structural proteins and other molecules on

the surface of microbes known as pathogen associated molecular patterns (PAMPS).

Following recognition, the organism is then able to eliminate the microbe by a process

known as phagocytosis utilising various cells such as macrophages (Goldsby et al.,

2003; Hoffman et al., 1999; Medzhitov, 2007). The adaptive immune system, referred


2

to as the antigen-specific immune response, is more complex compared to the innate

immune system. It starts with the binding of antigens to immune cells, followed by a

series of complex steps which lead to the destruction of the invading microbe. Unlike

innate immunity, adaptive immunity has memory cells, which are established after the

primary exposure to the microbe and provide a more rapid response on secondary and

further exposures (Reid 1983). The adaptive immune system is divided into two parts,

cell-mediated immunity and humoral immunity. Humoral immunity develops when B-

lymphocytes produce antibodies after exposure to antigens while cell-mediated

immunity involves activation of immune cells (such as natural killer cells (NK) and

antigen specific cytotoxic T-lymphocytes) and the release of cytokines (Holmskov, et

al., 2003; Medzhitov, 2007).

The complement system is also an important component of the immune system. Its

largely thought of as being a part of the innate immune system but it also helps to link

innate immunity with adaptive immunity by its interaction with antibodies (Ricklin &

Lambris, 2007).


3

1.2 The Complement System

The complement system is a vital part of the immune system and plays a crucial role in

fighting against invading microbes (Trouw & Daha, 2011). The complement system

was firstly described as heat labile components found in the serum or plasma which had

the ability to destroy bacteria by complementing the ability of antibodies. Paul Ehrlich

later coined this term (complement) for these components (Holmskov, et al., 2003;

Medzhitov, 2007; Fujita et al., 2004).

The complement system consists of more than 35 proteins present in both plasma and

on the surface of cells, which interact with each other to form a network to give the

body protection against microbes. Most components are present in their inactive

proenzyme form (i.e. zymogen), which will be converted and cleaved to their active

enzymatic form during a series of sequential steps of complement activation (Sarma &

Ward, 2011; Walport, 2001). The complement system can be activated via three

different pathways, named the classical pathway, the alternative pathway and the lectin

pathway (Figure 1.1). While the activation of the classical pathway depends on the

presence of antibodies and commences once the multi-molecular C1 complex binds to

an antigen-antibody complex, the other two pathways, the alternative and the lectin

pathway, are antibody-independent, representing the linkage between innate immunity

and adaptive immunity. The activation of the lectin pathway depends on the association

of the carbohydrate recognition molecules with the key enzyme of the lectin pathway, a

serine protease called Mannan binding lectin serine protease-2 (MASP-2). The

alternative pathway forms an amplification loop of activation that supports the functions

of the classical and lectin pathways by amplifying the activation of the most abundant

third complement component called C3. C3 activation, a key step during complement


4

activation, contributes to the formation of the bactericidal membrane attack complex.

Membrane attack complexes (MAC) are initiated once the fifth complement component

C5 is cleaved via a C5 convertase to C5a and C5b. C5b is the initial component of

forming the terminal C5b-9 complement complex that leads to bacterial lysis (Stover et

al., 1999; Köhl, 2001; Schneider et al., 2006). The complement system has many

functions, such as opsonisation, lysis of microbes and cells and antigen antibody

complex clearance and it is controlled by proteins found in the serum (fluid phase

regulators) or on the surface of cells (surface bound regulators) (Unsworth et al., 2011).

Figure 1.1 Complement system activation pathways (Figure courtesy of Professor W

Schwaeble, University of Leicester, UK).


5

1.2.1 Classical Pathway

The activation of the classical pathway begins when the multimolecular C1 complex

binds to immune complexes in the presence of calcium (Gál et al., 2009). The

multimolecular C1 complex consists of the recognition subcomponent C1q (composed

of hexamers of the heterotrimeric C1q chains C1q-A, C1q-B and C1q-C) and a

heterotetramer of the classical pathway specific serine proteases C1r and C1s,

C1s:C1r:C1r:C1s complex (Wallis et al., 2010). A single C1q molecule consists of six

similar subunits that bind to collagenous stalks, ending in globular head domains

(Figure 1.2). Three homologous genes (A, B, and C), which are located in one gene

locus, encode the three homologous polypeptide chains that form each subunit of the

C1q (Arlaud et al., 2002).

Figure 1.2 Structure of the C1 Complex of the classical pathway showing the homodimers of

C1r and C1s with the C1q molecule (Pflieger et al., 2010).


6

The activation of the classical pathway starts once C1q binds indirectly to the Fc region

of the immunoglobulins IgG and IgM or directly to the surface of bacteria. This binding

leads to an auto-activation of C1r which cleaves and activates C1s. C1s in turn cleaves

C4 into two portions: a small portion C4a, which dissociates in the fluid phase as an

anaphylatoxin, and a large portion, C4b, which is bound to C2 on the surface of the

microbe (Wallis et al. 2007). C2 then cleaves into a small particle C2b released into the

fluid phase and a large particle C2a by the action of C1s. The large particle C2a will

stay attached to C4b to form the classical pathway C3 convertase (C4b2a) which works

on the most abundant component in the plasma, C3, and converts it to C3a which is

released as an anaphylatoxin and C3b that opsonises the pathogen and helps in

phagocytosis. C3b will bind to C3 convertase, leading to the formation of C5 convertase

that cleaves C5 to C5a that is released as an anaphylatoxin and C5b that binds to the

surface of the pathogen and initiates the formation of the membrane attack complex

leading to lysis of the pathogen (Arlaud et al., 2002; Schwaeble et al., 2011; Vorup-

Jensen et al., 2000).

1.2.2 Alternative Pathway

Alternative pathway activation takes place when the C3, which is abundantly available

in blood, is spontaneously hydrolysed to form C3(H2O) that binds to Factor B (FB) in

the presence of Mg+2, leading to the creation of a zymogen complex C3(H2O)B. This

binding of C3(H2O) to FactorB (FB) allows complexed FB to be cleaved by factor D

(fD) into its two activation products: Ba and Bb. While the fragment Ba is released from


7

the complex, the other fragment Bb will stay attached to the complex, forming the

alternative pathway C3 convertase C3(H2O)Bb. This C3 convertase cleaves C3 into C3a

and C3b, which binds to the pathogen surface. Factor B will bind to the newly generated

C3b to form a new C3 convertase C3bBb after cleavage of factor B by Factor D

(Thurman & Holers, 2006). Properdin, which is a positive regulatory component of the

alternative pathway, promotes alternative pathway activation by stabilizing the C3

convertase C3bBb and prevent the decay of this convertase complex as an antagonist to

the regulatory plasma component factor H. The action of properdin can increase the

half-life of the AP C3 and C5 convertase complexes by 5-10 fold. Properdin directly

blocks the downregulatory activity of factor H, which binds to C3b bound in the C3bBb

or C3bB complexes and decays these complexes and also acts as a cofactor in the factor

I –mediated conversion of C3b to iC3b, the inactive form of C3b, which subsequently

inactivates the alternative pathway C3 convertase (Schwaeble and Reid, 1999).

Recently, it was postulated that properdin can bind like a recognition molecule to

pathogen surfaces to act as receptor for C3b and initiate the formation of C3bBb

complexes and target AP activation to the surface of pathogens (Hourcade, 2006).

Another way to activate the alternative pathway amplification loop would be by the

provision of C3b from either the classical pathway or the lectin pathway which after the

binding to factor B initiated the formation of the alternative pathway C3 convertase

complex resulting in a pronounced amplification of complement activation (Schwaeble

and Reid, 1999). Host cells protect themselves from overshooting complement

activation by expressing a large number of complement regulatory components, such as

complement receptors CR-1, CR-3 or membrane bound regulators, such as MCP or


8

DAF, or fluid-phase regulators like factor H that interacts with host membrane

structures and increases its affinity towards C3b by the binding of its carboxy-terminal

domains to autologous cell surfaces (Wallis et al., 2007).

As described before for the classical pathway, the alternative pathway C3 convertase

(C3bBb) switches its substrate specificity from cleaving C3 to cleaving C5 if several

molecules of C3b bind in close proximity to C3 convertase, forming the AP C5

convertase (C3bBb(C3b)n). This C5 convertase cleaves C5 into C5a and C5b which

initiates the terminal activation cascade leading to the formation of the membrane attack

complex (Farries et al., 1988).

1.2.2.1 Complement components of alternative pathway

1.2.2.1.1 Factor B

Alternative pathway factor B (FB) is a proenzyme composed of two fragments Ba and

Bb. Bb fragment comprises van Willebrand factor A (vWA) domain that binds C3b to

FB via a metal ion dependant adhesion site (MIDS) in the presence of magnesium

Mg+2 and C-terminal serine protease (SP) domain. Ba fragment comprises three N-

terminal complement control protein (CCP) domains linked to fragment Ba via a 45

residue long linker (Pryzdial and Isenman, 1987).


9

1.2.2.1.2 Factor D

Factor D (FD) is a small serine protease consisting of a single serine protease with a

plasma concentration of around 2µg/ml. Factor D is a critical component of the

alternative pathway and is produced in different tissues, mainly in adipose tissue

(Barnum et al., 1984; Stanton et al., 2011; Volanakis and Narayana, 1996). The main

form of factor D is mature factor D. However, the rest of factor D, about 1%, is

profactor D that is converted to mature factor D after its biosynthesis (Lesavre and

Muller-Eberhard, 1978; Yamauchi et al., 1994).

1.2.2.1.3 Properdin

Properdin is a glycoprotein known as a positive regulator of complement activation and

found in its soluble form in the blood (Pillemer et al., 1991). Its normal plasma

concentration is 5-15µg/ml (Schwaeble and Reid, 1999). It is composed of a monomer

formed of six homologous structural units called thrombospondin structural homology

repeats (TSRs) and an N terminal domain. This monomer is linked together head to tail

to form different forms of properdin, which is dimer, trimer and tetramer (Perdikoulis

et al., 2001; Schwaeble and Reid, 1999; Smith et al., 1984). The functional activity of

the different forms is variable as functionality increases by increasing the size of the

polymers. Therefore, the tetramer has ten times the activity of the dimer (Pangbum,

1989). Properdin has the ability to bind to soluble C3b and cell bound C3b. However,

the affinity of properdin binding to cell bound C3b is greater than that for soluble C3b.

Moreover, properdin binds to C3b and the C3bBb complex with a greater affinity to cell

bound C3 convertase rather than to cell bound C3b (Farries et al., 1989). Properdin has

an important role in alternative pathway activation, as lack of properdin in serum leads


10

to decreased ability of serum to activate the alternative pathway. However, adding

properdin can restore the activation of the alternative pathway (Schwaeble and Reid,

1999). Furthermore, it has been found that properdin can bind to the alternative pathway

activator and initiate the activation of the alternative pathway. Moreover, it has been

claimed that properdin can bind directly to the bacterial surface (Neisseria gonorrhoeae

and Escherichia coli) and enhance C3 deposition on the bacterial surface after its

addition to properdin deficient serum (Spitzer et al., 2007).

1.2.3 Lectin Pathway

Lectin pathway activation takes place when one or more of the lectin pathway

components (Mannan-binding lectin (MBL), ficolin and CL-11) bind to the pathogen-

associated molecular patterns (PAMPs), such as polysaccharides/carbohydrates and

acetylated sugars, on the pathogens (Schwaeble et al., 2011). As a result of this binding,

mannan-binding lectin-associated serine proteases 1, 2, 3 (MASPs) and Map19, which

is a non-enzymatic truncated product of MASP-2, become active.

Activation of MASP-2 leads to the cleavage of C4 into two portions: the large one, C4b,

which remains attached to the cell surface and the small portion, C4a, which is released

as an anaphylatoxin. C2 is also cleaved by active MASP-2 into two portions: the large

portion C2a and the small portion C2b. The large portion of the cleaved C2 (C2a) binds

to the large portion of the cleaved C4 (C4b) on the pathogen to form the lectin pathway


11

C3 convertase (C4b2a) on the surface of the pathogen (Fujita, 2002). As seen in the

classical and alternative pathways, the lectin pathway C3 convertase cleaves C3, which

forms C5 convertase (C4b2a(C3b)n) that then initiates the formation of the membrane

attack complex, as described earlier for the classical and alternative pathways

(Schwaeble et al., 2002; Thiel et al., 2000).

1.2.3.1 Lectin pathway components

The activation of the lectin pathway is more complex than the classical and alternative

pathways because of the interaction between the lectin pathway components (Sorenson

et al., 2005). These components are multimeric carbohydrate recognition

subcomponents (Mannan Binding Lectin (MBL) and ficolin) and Mannan Binding

Lectin associated serine proteases (MASP-1, MASP-2, MASP-3 and MAP19) (Stover et

al., 1999; Takahshi et al., 1999).

1.2.3.1.1 Mannan Binding Lectin

Mannan Binding Lectin (MBL) is a member of collectin proteins produced mainly from

the liver. It circulates in serum as a large oligomeric complex (trimers, tetramers and

hexamers) (Brouwer et al., 2008; MacMullen et al., 2006). It is composed of multimers

of three identical polypeptide chains (homotrimers). Each polypeptide chain is

comprised of a short N-terminal cysteine-rich domain (collagen-like domain) that links

MASPs with the MBL neck region and a globular head part that contains the

carbohydrate recognition domain (CRD) (Figure 1.3). Mannose and N-acetyl-

glucosamine (GIcNAc) show affinity for MBL while other carbohydrates show no


12

affinity for MBL. In addition to this, IgM can bind to MBL and activate the lectin

pathway directly (McMullen et al., 2006). Recent reports by Ogden et al. (2001) and

Jack et al. (2005) claimed that MBL works as a complement independent opsonin.

Moreover, Jack et al. (2001) and Jack et al. (2005) show that MBL works as a

dependent opsonin by enhancing the uptake and killing of Neisseria meningitidis

bacteria by human phagocytes. While humans have one MBL gene, rodents have two

genes, MBL-A and MBL-C (Sastry et al., 1995).

Figure 1.3 The domain structure and oligomerization of MBL (Garred et al., 2009).

1.2.3.1.2 Ficolin

Ficolins are carbohydrate recognition molecules composed of three identical

polypeptide chains. Each polypeptide chain consists of a short N-terminal cysteine-rich

domain with many cysteine residues, a collagen-like domain, a neck region and a

fibrinogen-like carbohydrate recognition domain (FBG) (Figure 1.4). Most ficolins are

present in serum and activate the lectin pathway by binding to N-acetyl glucosamine


13

sugars and lipoteichoic acids of gram positive bacteria (Garred et al., 2009; Matsushita

et al., 2001; Endo et al., 2005; Lynch et al., 2004).

Ficolins in humans have three different forms which are L-ficolin, M-ficolin, and H-

ficolin. Unlike L-ficolin and H-ficolin, which are found in serum and activate the lectin

pathway, M-ficolin is found on the surface of leukocytes (neutrophils and monocytes)

and activates the lectin pathway by forming a complex with MASP-1 and MASP-2 (Liu

et al., 2005; Matsushita et al., 2000; Matsushita and Fujita, 2001). Mice have two

different forms of ficolin: ficolin-A which is found in serum and looks like human

ficolin-L and ficolin-B which is found in bone marrow cells. Although ficolin-B has a

similar structure to ficolin-A, it has no ability to activate the lectin pathway because it

does not bind to MASP-2 (Endo et al., 2005; Runza et al., 2008).

Figure 1.4 The domain structure and oligomerization of ficolins (Garred et al., 2009).

1.2.3.1.3 Collectin

Collectin 11 (CL11) is a member of the collectin family produced in many organs,

especially the kidney, adrenal gland and liver, with a plasma concentration of about 2.1


14

µg/ml. It is composed of an N-terminal domain, collagen-like domain, neck domain and

a carbohydrate recognition domain (CRD). Collectin 11 can interact with MASP-1 and

MASP-3 in the plasma. It can also bind to D-mannose and L-fucose terminal

saccharides on the surface of microbes and acts as a recognition molecule of the lectin

pathway (Hansen et al., 2010).

1.2.3.1.4 Mannan Binding Lectin associated serine proteases

Mannan Binding Lectin associated serine proteases are members of the serine protease

family and are homologous to the serine proteases C1r and C1s of the classical pathway.

In mammals, three forms of MASPs are found: MASP-1, MASP-2 and MASP-3. In

addition to the MASPs, MAP19 is a truncated product generated from the MASP-2

gene and Map44 is a truncated product generated from the MASP-1 gene, which are

non-enzymatic products and act as inhibitors of lectin pathway activation (Schwaebleet

al., 2002; Stover et al., 1999; Wallis, 2007).

MASP-1, MASP-3 and Map19 are generated from the MASP-1 gene on chromosome 3

in humans. MASP-2 and Map19 are generated from the MASP-2 gene on chromosome

1 in humans and 4 in mice (Dahl et al., 2001; Sorenson et al., 2005; Stover et al., 1999).

MASP-2 is produced only by the liver, MASP-1 is also mainly produced by the liver

and MASP-3 is produced by the liver, spleen, lung and other tissues. All MASPs share

the same domain organization which is N-terminal CUB 1, an epidermal growth factor

(EGF)-like domain, CUB 2 domain, two complement control protein domains (CCP1

and CCP2), also known as short consensus repeats (SCRs), and a chymotrypsin-like

serine protease domain (Figure 1.5). Although MASP-1 and MASP-3 have the same N-


15

terminal domain they have a different serine protease domain (Lynch et al., 2005;

Sorenson et al., 2005; Thiel, 2007).

Figure 1.5 MASP and Map domain organization as described by Yongqing et al. (2012).

Binding between MASPs, MBL and ficolin occur by the binding of CUB1 and the EGF-

like domain from MASPs and the collagen-like domain of MBL and ficolin (Wallis et

al., 2004). The enzymatically active form of MASPs occurs when there is cleavage

between CCP-2 and the serine protease domain which leads to the formation of a heavy

chain (N-terminal domains) and light chain (serine protease domain) liked together by a

disulfide bridge (Matsushita and Fujita, 1995).

Lectin pathway activation occurs only by MASP-2 which cleaves C4 and C2 that bound

to C4b to form the lectin pathway C3 convertase and neither MASP-1 nor MASP-3 can

restore the activation of the lectin pathway (Matsushita et al., 2000; Rossi et al., 2001;


16

Vorup-Jensen et al., 2000). Therefore, murine MASP-2 deficiency leads to the loss of

lectin pathway activation (Schwaeble et al., 2011). Takahshi et al. (2010) reported that

MASP-1 plays a role in activation of the alternative pathway by converting factor D to

an enzymatically active form (Figure 1.6). A recent study of Iwaki et al. (2011)

reported an important role of MASP-3 in the activation of the alternative pathway,

showing that a complex of recombinant MASP-3 and recombinant MBL has the ability

to activate the alternative pathway by cleaving C3b bound factor B on the surface of

bacteria.

Figure 1.6 MASPs activation results in the formation of a heavy and a light chain held together

through a disulfide bond (Fujita, 2002).


17

1.2.4 Membrane Attack Complex

Any of the complement pathway C3 convertases (classical pathway and lectin pathway

C3 convertase (C4b2a) or alternative pathway C3 convertase (C3bBb)) facilitate the

formation of the membrane attack complex by cleaving C3 into a small fragment C3a

and a large fragment C3b. Accumulation of C3b leads to the formation of C5

convertase, C4b2a(C3b)n for the classical and lectin pathways, or C3bBb(C3b)n for the

alternative pathway which cleaves C5 into C5b and C5a. The larger cleavage product

C5b binds to the surface of the pathogen, leading to the formation of a C5b-8 complex.

This results in C9 polymerisation that forms pores in the lipid bilayer of the pathogen

cell membrane which, in turn, disturbs the balance of salts and metabolites inside the

pathogen (Figure 1.7) (Podack et al., 1982).

Figure 1.7 Membrane attack complex pathway (Fujita, 2002).


18

1.2.5 Functions of the Complement System

The complement system plays several roles in the normal functioning of the immune

system, especially in the process of protection against pathogens, and links the non-

specific immune system with the specific immune system. Also, waste products (e.g.

apoptotic cells and debris) are removed by the complement system (Markiewski &

Lambris, 2007). In addition, the opsonisation of bacteria, which plays an important role

in the recognition of the presence of bacterial infection, is controlled by the complement

system through C3b, C4b, iC3b and iC4b which can attach to the surface of pathogens.

Phagocytosis can be enhanced by attracting leucocytes via C3b which uses complement

receptors 1 and 3 (CR1 and CR3) to bind pathogens. Moreover, phagocytic cells are

able to bind to MBL and L-ficolin to establish the process of phagocytosis (Matsushita,

2010).

Inflammatory cells (i.e. leucocytes such as mast cells, phagocytes and neutrophils) are

attracted and activated at the site of infection by C3a and C5a through establishing a

strong pro-inflammatory reaction. The C3a and C5a complement components are called

anaphylatoxins and they are produced during the activation of the complement system

due to the cleaved products of C3 and C5. C3a and C5a are able to attract leucocytes to

inflammatory sites as a consequence of raising the vascular permeability of endothelial

cells. Subsequently, pathogens can be recognised and excluded (Markiewski &

Lambris, 2007).


19

Moreover, C5a was shown to enhance the activation and production of some

chemokines (e.g. tumour necrosis factor (TNF)) and interleukins,e.g. IL-1 and IL-8

(Markiewski & Lambris, 2007). In addition, using a mouse model, it was shown that

C5a and C3a have many functions in releasing macrophage inflammatory protein-2 and

monocyte chemo-attractant protein-1 by exciting endothelial cells (Laudes et al., 2002).

Furthermore, the complement system has an important role in direct bacterial lysis

mediated by the MAC complex, particularly those that have thin cell walls such as

gram-negative bacteria (Wu et al., 2009).

In addition to MAC complex-mediated killing, the complement system has another

crucial role in clearing immune complexes, necrotic cells and apoptotic cells.

Phagocytic cells like macrophages have complement receptors CR3 and CR4 on their

surfaces, which mainly control this clearance. Phagocytosis happens after CR3 and CR4

identify the apoptotic cells that bind to C1q and create immune complexes (Taylor et

al., 2000). In addition, because RBCs have CR1 receptors that link to the C3b

opsonized C1 complex, immune complexes can be found in the bloodstream associated

with‎ RBC’s. Ultimately, the spleen and liver remove RBC’s‎ with attached immune

complexes via macrophages and the reticulo-endothelial system (Frank, 2010).

Finally, the specific immune response by B cells is activated by the complement system.

B cells have CR1 and CR2 that attach to iC3b, C3dg and C3d and leads to the activation

of B cells (Molina et al., 1994). As a result of this molecular linkage, antibody

production can be enhanced because of the lowering of the threshold required for

activation of B cells (Chen et al., 2000).


20

1.2.6 Regulators of Complement System

Initiating and controlling the activation of the complement system needs to be done

cautiously as un-regulated, activation of the complement system can lead to potentially

dangerous outcomes such as host tissue damage and inflammatory diseases (Trouw et

al., 2008). In practice, soluble regulators (fluid phase) and membrane-bound regulators

are both used to control the activation of the complement system (Figure 1.8)

(Kirschfink & Mollnes, 2003).

1.2.6.1 Fluid Phase Regulators

Regulation of the activation of classical pathway (CP) and lectin pathway (LP) occurs

via a regulatory protein called C1 inhibitor (C1-INH). C1 inhibitor can be attached to

C1 complexes to produce (C1-INH-C1r2-C1s2-C1-INH) in the classical pathway and

can be attached to LP complexes to produce (MASP-1-C1-INH and MASP-2-C1-INH)

in the lectin pathway (Ehrnthaller et al., 2011). However, C1-INH does not bind to

MASP-3 (Yongqing et al., 2012).

In addition to C1 inhibitor (C1-INH) the C4-binding protein, C4bp, is another crucial

regulator of the complement system. It works by reducing C4b-bound C2 to block C3

convertase which is required for the production of CP and LP. Moreover, by enhancing

the production of I-factor, which plays a role as a cofactor in factor I-mediated

conversion of C4b to C4dg and C3b to iC3b and C3dg, is another function of the C4-

binding protein C4bp (Jurianz et al., 1999).


21

Furthermore, control of the AP C3 convertase and C3 convertase pathways by Factor H

(which is one of the most copious fluid phase mediatory components (150 mg/L)) plays

a vital role in preventing unwanted complement system activation (Ehrnthaller et al.,

2011).

Moreover, clusterin and S-protein mediate the terminal pathway. They block pore

formation within the cell membrane through attaching to the C5b-7 complex, which

reduces the lytic activity of the MAC complex. Finally, the inflammatory role of the

anaphylatoxins within the complement system, C3a and C5a, can be blocked by

carboxypeptidase N (Ehrnthaller et al., 2011).

1.2.6.2 Membrane-Bound Regulators:

Basically, the main complement mediators that are bound to the membrane are CD35

(complement receptor 1 (CR1), CD46 (membrane cofactor protein, (MCP), CD55

(decay accelerating factor (DAF) and CD59 (protectin) (Ehrnthaller et al., 2011).

Complement receptor 1 (CR1), which can be found on RBCs (erythrocytes) and WBCs

(leukocytes) retards the activity of C3 and C5 convertase. Also, CR1 can work as a

cofactor for factor I in C3b and C4b cleavage.


22

In addition, membrane factor protein (MCP) can attach to C3b and support factor I in its

deactivation. Moreover, decay accelerating factor (DAF) can attach to C2a in the CP

and LP and Bb in the AP to retard the formation of C3 and C5 convertase (Frank, 2010).

Finally, protectin (CD59) can stop production of the MAC complex by interacting with

C8 and C9 and prevent them from attaching to the C5b-7 complex (Ehrnthaller et al.,

2011).


23

Figure 1.8 Regulation of complement activation by membrane bound and fluid phase

regulators as described by Mollnes et al. (2002).


24

1.2.7 Complement deficiency

The complement system plays a crucial role in fighting microbial infection and clearing

the body of immune complexes and apoptotic cells (Langer et al., 2010). This role is

preformed via a network of proteins which either initiate complement pathway

activation or regulate its activation. Therefore, deficiency in any component of the

complement system or in complement regulators may lead to changes in body

homeostasis and health, resulting in increased susceptibility to pathogens or the

induction of autoimmune disease (Mayilyan, 2012).

1.2.7.1 Defects of the Classical Pathway

Immune complex/autoimmune disease is often associated with the classical complement

pathway deficiency. Deficiency of the classical pathway C1q component leads to

increases in the risk of Systemic Lupus Erythematosus (SLE). This indicates that C1q

plays an important role in the clearance of immune complexes and apoptotic cells

(Leffler et al., 2014). Deficiency of C1s, C1r, and C4 is also associated with SLE

disease but is lower than with C1q deficiency. Classical pathway deficiency is

associated with recurrent bacterial infection (Brown et al., 2002). C2 deficiency is

associated with increased susceptibility to S.pneumoniae in children (Jonsson et al.,

2005). Even though, the deficiency of C3 is uncommon, it leads to autoimmune disease

and increases the risk of getting recurrent severe infection caused by encapsulated

bacteria like Haemophilus influenza and pneumococci (Ross & Densen, 1984; Singh

and Rai, 2009). In addition to this, defects in innate immunity and adaptive immunity


25

have been reported in a C3 deficient patient due to the functional impairment of the

function of B cells, T cells, and dendritic cells (Botto et al., 2009).

1.2.7.2 Defects of the Alternative Pathway

Alternative pathway malfunction is most often associated with deficiency of factor D,

factor B and properdin. Alternative pathway factor D deficiency is associated with

increased severity of meningococcal infection. Factor B deficiency also leads to

recurrent meningococcal and pneumococcal infection (Slade et al., 2013; Sprong et al.,

2006). Properdin deficiency, which is the most common deficiency of alternative

pathway, increases the risk of meningococcal disease and pneumonia (Schejbel et al.,

2009; Fijien et al., 1999). In addition, properdin deficient individuals are up to 250

times more likely to get meningococcal infection compared to normal individuals.

Furthermore, the rate of morbidity and mortality in meningococcal infection increases

due to properdin deficiency, illustrating the crucial role of properdin against this

infection (Fijien et al., 1999).

1.2.7.3 Defects of the Lectin Pathway

Lectin pathway MBL deficiency is most commonly caused by polymorphism in the

MBL gene. Even though 10% of the population has been found to be MBL deficient,

this population remains healthy and shows no increasing susceptibility to bacterial

infection and morbidity compared to normal people. However, under certain conditions,

their susceptibility to bacterial infection increases, especially when it is associated with


26

immunocompromised conditions like HIV infection (Dahl et al., 2004; Peterslund et al.,

2001; Sorensen et al., 2005; Super et al., 1989). In addition to this, MBL deficiency is

associated with impairment of opsonisation and it has been found that MBL deficiency

increases the risk of rheumatoid arthritis, atherosclerosis and arterial thrombosis

(Jacobsen et al., 2001; Ohlenschlaeger et al., 2004). L-ficolin deficiency is associated

with recurrent respiratory infection and allergies in children (Atkinson et al., 2004).

Autoimmune disease and recurrent severe infections have been found to be associated

with MASP-2 deficiency, which is a rare condition (Stengaard-Pedersen et al., 2003).

1.2.7.4 Defects of terminal complement components

Terminal complement components (C5, C6, C7, C8 and C9) play an important role in

the lysis of bacteria. Deficiency of one or more of terminal complement components

impairs this function and increases the susceptibility to recurrent meningitis (Figuera

and Densen, 1991). C7 and C9 deficiency are associated with an increased risk of

meningococcal infection. However, the risk of meningococcal infection increases 1000

fold in C9 deficient individuals and 1400 fold in C7 deficient individuals compared to

the general population (Nagata et al., 1989).

1.2.7.5 Defects of complement regulatory components

C1 inhibitor is important not only in complement regulation but also plays a role in

regulating blood clotting pathways. Therefore, deficiency of C1 inhibitor is associated

with hereditary angioedema (HAE), increased vascular permeability to plasma and


27

excessive lymphoproliferation in autoimmune disease and lymphoma. In

lymphoproliferation, the concentration of C1 inhibitor is normally high while the

concentration of C1q is often low (Cancian, 2014; Cugno et al., 2009; Markovic et al.,

2000). Deficiency of the alternative pathway regulators, Factor H and Factor I, is

associated with immune associated diseases such as SLE and atypical haemolytic

uraemic syndrome (Reis et al., 2006; Thurman and Holers, 2006). It is also associated

with increased susceptibility to infection because of continuous alternative pathway

activation that depletes levels of factor B and C3 (Reis et al., 2006).


28

1.3 Neisseria meningitidis

There are twelve species of Neisseria genus that have been isolated from humans. These

species of Neisseria can be divided into two groups according to their colony

characteristics and morphology. The species in the first group grow as non-pigmented

and translucent colonies, such as Neisseria meningitidis, Neisseria gonorrhoeae, and

Neisseria lactamica. The species in the second group grow as opaque and yellow

pigment colonies, such as Neisseria muocsa, Neisseria subflava and Neisseria sicca.

Among these, only two species are considered to be pathogenic: Neisseria meningitidis

and Neisseria gonorrhoeae (Barrett and Sneath, 1994; Knapp, 1988).

Neisseria meningitidis is the main cause of bacterial meningitis throughout the world,

significantly contributing to increased mortality (Emonts et al., 2003). While its

diagnosis, vaccination and treatment have improved considerably in recent years,

infection is still spreading worldwide, with a mortality rate of up to 10% (Connolly and

Noah, 1999).

Neisseria meningitidis is a gram negative, bean-shaped diplococcus, which attacks the

upper respiratory tract as commensals in 10-40% of population, may cause systemic

diseases. Meningococcal disease is spread between the hosts primarily through an

aerosol transmission. Although some individuals infected with Neisseria meningitidis

suffer severe disease, other individuals have it as commensals. However, it is not clear

yet why some individuals develop the severe disease (Yazdankhah et al., 2004). Due to


29

the gradual loss of passive immunity children under five years old can be at risk of

meningococcal disease (Edwards and Baker, 1981).

Neisseria meningitidis is enclosed by three layers: outer membrane lipids, outer

membrane proteins (OMPs) and lipo-oligoccharides (LOS), which are covered by a

polysaccharide capsule on the pathogenic Neisseria meningitidis (Figure 1.9) (van

Deuren et al., 2000). Neisseria meningitidis can be identified according to the

polysaccharide capsule into 13 serogroups (A, B, C, E-29, H, I, K, L, M, W-135, X, Y

and Z) (Choudhury et al., 2008). However, five strains (A, B, C, Y and W-135) are

responsible for most diseases (Spinosa et al., 2007). While the serogroups B and C are

most common in industrialized countries, the serogroups A and C are most common in

the third world (Takahashi et al., 2008). Each year, serogroup A causes an outbreak of

meningococcal disease in Sub-Saharan Africa, with an incidence rate of up to

1,000/100,000 (Moore, 1992; Riedo et al., 1995). Northern China was the source of an

outbreak of meningococcal disease caused by serogroup A, which spread throughout the

world (Wang et al., 1992). Serogroup Y caused the most cases of meningococcal

disease in the United States (Racoosin et al., 1998). Northwestern Europe is affected by

meningococcal disease caused by serogroup B, with an attack rate of up to 1 to

50/100,000. In the UK, more than 2000 people are affected each year, with 10%

mortality rate. People who recover from meningococcal disease may suffer permanent

damage, like deafness and mental retardation (Baraff et al., 1993).


30

Figure 1.9 Neisseria meningitidis cell membrane (Rosenstein et al., 2001).

1.3.1 The Virulence Factors of Neisseria meningitidis

1.3.1.1 Pili and Pilus Subunits

Type IV pili (Tfp) and pilus subunits are very important factors that play a significant

role in the interaction of Neisseria meningitidis with the host cells, resulting in a

productive infection (Ieva et al., 2005). The type IV pili subunit is made up of

thousands of main pilus subunits, PilE proteins, and a small number of pilus-associated

proteins, for example PilV and PilC, which contain two proteins PilC1 and PilC2 that

are very important in the formation of pili. During the initiation of interaction between

the epithelial mucosa and Neisseria meningitides, the expression of PilC1 will be

upregulated by control of the sequence 150-bp that is located upstream of PilC1 known

as CREN. Contact-regulated protein A (CrgA) protein, as a regulatory protein, works

when the Neisseria meningitidis starts adhesion to the host epithelial cells (Tettelin et

al., 2000). PilT is an important component of pilus that induces intimate attachment


31

once Neisseria has attached to the host cell by promoting pilus retraction, which is

required for twitching motility (Pujol et al., 1999).

1.3.1.2 Outer Membrane Proteins

Outer membrane proteins (OMPs) are other important virulence factors of Neisseria

meningitidis that facilitate the Neisseria’s adhesion to and invasion of the host cells.

These proteins are divided into five classes (class 1, 2, 3, 4 and 5) depending on the size

of proteins on sodium dodecyl sulphate polyacrylamide gels (SDS- polyacrylamide gel).

However, recently, these classes of protein have been reordered according to the

producer genes into two groups, (1) ProA, which includes proteins classes 1, 4, and 5

and (2) ProB, which includes protein classes 2 and 3 (Tsai et al., 1981; Hitchcock,

1989).

Meningococcal porins (ProA and ProB) are the most frequent types of proteins found in

the meningococcal membrane. The molecular size of these proteins varies across

different strains of meningococcal. These proteins are found in trimer form and act to

allow the small hydrophilic nutrients to enter the cell (Tommassen et al., 1990; Frasch

et al., 1985). In addition, they are used to classify meningococcal disease serologically

because of their abundance in the outer membrane. While ProA, known as class 1 porin,

has the ability to enhance the generation of B cells and the interaction between T cells

and B cells, the two forms of ProB, ProB2 known as class 2 porin and ProB3 known as

class 3 porin, have the ability to activate T cells and dendritic cells. Additionally, ProB

acts to differentiate T cells into Th2 cells which facilitate the production of antibodies

(Mackinnon et al., 1999). Meningococcal opacity proteins (Ops) comprise two proteins


32

Opa and Opc, with Opa being more abundant compared to Opc (De Vries et al., 1998).

CEACAM and heparin sulphate proteoglycan receptors that are found on the host cells

are bound to the membrane opacity proteins A (Opa), initiating the adhesion (Albiger et

al., 2003).

1.3.1.3 Capsule and Lipo-oligosaccharide (LOS)

Capsule and lipo-oligosaccharide (LOS), non-protein factors, play a crucial role in

Neisseria meningitidis infection, Neisseria protection from complement-mediated

killing and Neisseria protection from the phagocytosis by the immune cells (Takahashi

et al., 2008). Because the lipid A portion of the lipo-oligosaccharide, which mediates

the endotoxic shock, leads to the activation of macrophages and produces various

cytokines and chemokines, lipo-oligosaccharide is considered the most important

virulence factor (Albiger et al., 2003).

Neisseria meningitidis serogroups differ in their polysaccharides capsules, for example,

serogroups B, C, Y, and W-135 have polysaccharides capsules comprising mainly sialic

acid attached to glucose or galactose while the serogroup A polysaccharide capsule

comprises N-acetyl mannosamine-1-phosphate (Swartley et al., 1997). Serogroups B

and C have a similar structure of their polysaccharides capsules and they have the

ability to change their lipo-oligosaccharides to variable degrees (Mandrell et al., 1991).

Neisseria meningitidis capsule synthesis gen (cps) has five different regions. Region A

has the gene responsible for making the polysaccharides capsule. Different serogroups


33

have different genes, for example, serogroups B, C, Y, W-135 have the siaD gene which

is required to make a capsule containing sialic acid, whereas serogroup A has the myn

gene which is required to make the serogroup A capsule (Edwards et al., 1994). The

gene on region B facilitates lipid modifications (Frosch & Müller, 1993). Region C has

the Ctr gene that is responsible for polysaccharide transport and is also found conserved

in most isolated Neisseria meningitidis. It is also used as a target to clinically detect

Neisseria (Guiver and Borrow, 2001). Region D contains a gene that is responsible for

making lipopolysaccharides. Region E contains the tex gene the function of which is not

yet clear (Claus et al., 2002).

However, it has been claimed that some Neisseria meningitidis strains can be present in

the human nasopharyngeal transmission system without a gene responsible for

synthesising the capsule and replace it with the capsule null locus (cnl) sequence that

has been found at least in four different groups of Neisseria meningitidis (Claus et al.,

2002). Uncapsulated meningococcal strains show increased ability to adhere to mucosal

epithelium. Moreover, uncapsulated meningococcal strains are more susceptible to be

killed by human serum compared to capsulated meningococcal strains because of the

presence of the capsule provides a protection against human serum (Dolan-Livengood et

al., 2003; Vogel et al., 1996).

Lipooligosaccharide (LOS) is one of the Neisseria meningitidis virulence factors which

provides bacterial protection from serum and plays a role in the colonization and

invasion of host cells. Endotoxic shock is associated with the Lipid A portion of LOS. It


34

leads to the activation of macrophages which produce many of cytokines and

chemokines involved in inflammation (Albiger et al., 2003; Rosenstein et al., 2001).

Additionally, the levels of endotoxin are associated with the severity of meningococcal

disease (Brandtzaeg et al., 1992). LOS is characterized by a lack of O-antigen and

having short chains of polysaccharide comprising two to five sugars linked to the inner

core of Neisseria meningitidis through two 2-keto-3-deoxyoctulosonic acid (KDO)

molecules (Gamian et al., 1992). Some studies have shown that changing the Neisseria

meningitidis LOS by phosphoethanolamine groups (PEA) and O-acetyl groups increases

Neisseria meningitidis adhesion to epithelial or endothelial cells (Takahashi et al.,

2008). LOS has been used to serologically divide meningococcal meningitis into 12

immunotypes (L1-L12),‎depending‎on‎ the‎group‎of‎α‎and‎β‎chains‎added‎ to‎ the‎ inner‎

core region (Mandrell and Zollinger, 1977). However, some Neisseria menigitidis have

more than one immunotype-specific epitope and are classified as the L3, 7, 9 or L2, 4

immunotypes (Tsai et al., 1983). The L3, 7, 9 meningococcal immunotype is the most

common cause of meningococcal disease (Griffiss et al., 2000).

Recent studies have claimed that in spite of the expression of PilC and Pili, Neisseria

meningitidis that lacks LOS is not able to adhere to epithelial cells and significantly

decreases the competence of DNA transformation (Albiger et al., 2003). The ability of

LOS in complement activation has been studied, showing that Neisseria menigitidis are

able to activate the complement system effectively even when they are LOS mutants

(Sprong et al., 2004).


35

1.3.2 Colonization and invasion by Neisseria meningitidis

Neisseria meningitidis is a human pathogen transmitted by aerosol and other infected

secretions. The human nasopharyngeal compartment is the normal habitat for Neisseria

menigitidis. It can colonize the mucosal surface of the upper respiratory track without

causing any symptoms in a phenomenon called carriage. However, 10% of the

population are asymptomatic carriers (Broome, 1986). The susceptibility of the host and

concomitant viral infection particularly when found in an epidemic area and the

meningococcal virulence factors all play a role in the development of meningococcal

disease (Anderson et al., 1998). The rates of neisserial carriage increase by up to 40% in

closed‎and‎semi‎closed‎populations‎like‎universities‎(Ala’Aldeen‎et al., 2000; Caugant

et al., 1992).

Meningococcal colonization starts on the surface of mucosal epithelium and

intraepithelially. Subsequently, it penetrates the blood stream as result of endocytosis

caused by phagocytosis. This penetration causes various clinical symptoms, ranging

from sepsis and mild meningococcal infection to meningococcal septicemia and

meningitis (Figure 1.10) (Van Deuren et al., 2000). If the level of bacteria in the blood

is low the bacteria will be cleared out spontaneously. However, if the bacteria is not

cleared out and the level of bacteria is high (could be up to 106

colony forming units per

ml), this leads to vascular and tissue damage (Brandtzaeg et al., 1995). Endotoxins are

released during the bacteremic stage of the disease in the form of vesicular outer

membrane structures (composed of LOS, OMPs, lipids and polysaccharides capsule),

which promotes the production of inflammatory factors in the blood. This inflammatory


36

response is responsible for the severe symptoms of meningococcal disease (Hong et al.,

2008).

Although it is rare for Neisseria meningitidis to cause other diseases, it can lead to

septic arthritis, sinusitis, conjunctivitis and otitis (Tzeng and Stephens, 2000).

Meningococcal LPS can stimulate the coagulation system by upregulation of tissue

factors, which leads to disseminated intravascular coagulation (DIC) (Hardaway, 1982).

Additionally, meningococcus has the ability to cross to cerebrospinal fluid (CFS)

through blood brain barrier. Meningococcal replication and an acute inflammatory

response in CFS can lead to severe forms of diseases, like septicemia or meningitis

(Van Deuren et al., 2000). The symptoms of acute meningitidis include headache, fever,

stiff neck, vomiting, photophobia and changes in mental status (Rosenstein et al., 2001).

DIC and Shock are characteristic of human meningococcal sepsis. In the case of

Neisseria meningitidis, septic shock, an extreme immune response, will result in

hypotension, organ failure and finally death (Kahler Stephens, 1998).

Figure 1.10 Different stages in the pathogenesis of N. meningitidis (Virji, 2009).


37

1.3.3 The Immune System and Neisseria meningitidis

Neisseria meningitidis infection stimulates the innate immune system to clear the

Neisseria meningitidis. Short febrile flu can be observed because of low levels of

bacteremia, which usually clear out spontaneously. The induction of specific adaptive

immunity, in the form of antibodies, is more effective in the development of resistance

to Neisseria meningitidis. However, because it is a delayed response, the innate immune

response plays a crucial role in fighting against Neisseria meningitidis infection (van

Deuren et al., 2000).

Some factors, such as the complement system, cationic antimicrobial peptides (CAMPs) and

other components of the inflammatory response, work to limit the growth of Neisseria

meningitidis in the host. A potent inflammatory response which emerges following a Neisseria

meningitidis invasion is responsible for the symptoms of meningococcal disease (Hong et al.,

2008).

The host produces different antimicrobial substances after invasion from pathogens.

One of them is cationic antimicrobial peptide (CAMP). Cationic antimicrobial peptide

(CAMP) is one of the important components of innate immunity produced by

phagocytic cells and expressed by epithelial cells after pathogen invasion (Hancock,

2001). Neisseria meningitidis shows a high resistance to cationic antimicrobial peptide

through different mechanisms, such as a modification of lipid A. The Neisseria

meningitidis capsule also shows resistance to the cationic antimicrobial peptide

(Takahashi et al., 2008).

Another way of controlling Neisseria meningitidis infection has been shown by human

dendritic cells through their ability to phagocytose the pathogens (Kolb-Mäurer et al.,

2001).


38

1.3.4 The Complement System and Neisseria meningitidis

The complement system is a vital part of the innate immune system and plays a crucial

role in fighting against invading microbes (Trouw & Daha, 2011). It consists of more

than 35 proteins present in both plasma and on the surface of cells which interact with

each other to form a network of proteins that protect against microbes. The complement

system is activated by three different pathways which are the classical pathway, the

alternative pathway and the lectin pathway. This activation leads to the cleavage of C3

which is a central component of complement activation, to C3b, which binds to the

microbe and opsonises it for phagocytosis by immune cells. Moreover, C3 activation

leads to the formation of the bactericidal membrane attack complex, which can lead to

the lysis of the microbe. The membrane attack complex is initiated once the C5

molecule is cleaved by C5 convertase to C5a and C5b which are the initial components

of forming the terminal C5b-9 complement complex that leads to bacterial lysis (Kohl,

2001; Schneider et al., 2006; Stover et al., 1999; Walport, 2001). The complement

system plays a crucial role in fighting meningococcal infection so deficiency in any

complement component will increase the susceptibility to meningococcal infection

(Rosa et al., 2004; Rossi et al., 2001).

The meningococcal polysaccharide capsule is a very important structure that protects

the Neisseria meningitidis from complement mediated killing. Therefore, the absence of

the meningococcal capsule increases the susceptibility of the meningococcal to lysis by

the complement system (Schneider et al., 2006; Geoffroy et al., 2003). A study

conducted by Vogel et al. (1996) has used a modified meningococcal serogroup B strain

in which they inactivated the gene responsible for capsule expression found in the wild-


39

type strain and then compared the survival in human serum. The study showed that the

wild-type was less susceptible to killing than is more than the non-capsulated strain due

to the absence of capsule. However, they found no difference in the C3 deposition on

the surface of both strains which indicates that the absence of capsule does not affect the

cleavage of C3 on both strain surfaces (Vogel et al., 1996).

Meningococcal lipopolysaccharide is another important structure and is composed of

two chains linked to the lipid A portion of the outer membrane and provides protection

against complement mediated killing (Brandtzaeg et al., 1995). Meningococcal

serogroup B and C Strains have increased sialylation of LOS by addition of sialic acid

to Lacto-N-neotetraose (LNnt) to increase the serum resistance and avoid

meningococcal lysis by the complement system (Estabrook et al., 1997). Additionally,

serum resistance of Neisseria meningitidis can be enhanced through binding of

meningococcal Opc to vitronectin which is a very important regulator of terminal

complement activation (Viriji et al., 2008).

The classical pathway is a complement pathway that initiates binding to natural IgM

antibodies during meningococcal infection and is a potent activator of complement.

Following meningococcal colonization both specific and cross reactive antibodies may

be generated (Finne et al., 1987; Hoff and Hoiby, 1978; Liu et al., 2008). A previous

study has shown that the C4 binding (C4b) protein of the fluid-phase complement

proteins of classical and lectin pathways can bind to Neisseria meningitides and this

requires meningococcal porin A. However, the role of the C4bpin both meningococcal


40

binding and evasion of the immune system is not yet clear as it is seen in only non-

physiological sodium conditions (Jarva et al., 2005).

The alternative pathway factor H is a regulatory protein that works as a cofactor for

factor I mediating the cleavage of C3b to iC3b which is an enzymatically inactive decay

fragment (Pangburn et al., 2000). It has been suggested that Neisseria meningitidis can

bind to factor H via specific factor H binding protein (fHbp), also called genome

derived neisserial antigen (GNA), limiting complement activation on the surface of

Neisseria meningitidis (Pangburn et al., 2000; Schneider et al., 2006). Recent studies

have shown that factor H binds to Neisseria meningitidis via the neisserial surface

protein A (NspA) which has the ability to provide serum resistance to Neisseria

meningitidis even without presence of factor H binding protein (Lewis et al., 2010). In

addition, it has been reported that the sialylation of meningococcal serogroup B and C

increases the affinity of factor H to factor B or Bb bound C3b, leading to a decay of the

formed C3 convertase complex which results in the inhibition of complement activation

via the alternative pathway (Van Deuren et al., 2000).

The alternative pathway factor D is a serine protease that plays an important role in the

initiation and amplification of C3 convertase of the alternative pathway (Choy and

Spiegelman, 1996; Matsumoto et al., 1997). Sprong et al. (2006) reported that a defect

in the factor D allele could lead to factor D deficiency, which was associated with

increased invasive meningococcal disease (Sprong et al., 2006).


41

The alternative pathway includes properdin which is a serum glycoprotein that acts as a

positive regulator of alternative pathway activation (Nolan et al., 1992). Deficiency of

components in the alternative pathway have been associated with increased

susceptibility to severe meningococcal infection and thus an increased rate of mortality

compared to normal individuals (Braconier et al., 1983; Densen et al., 1987; Morgan

and Walport, 1991; Fijien et al., 1995; Spath et al., 1999). Agarwal et al (2010)

conducted a study in which they showed that native properdin could not bind to

meningococcus directly. However, unfractionated properdin also has the ability to bind

directly to meningococcus after incubation in properdin depleted serum and enhances

the level of C3 deposition on the surface of the meningococci (Agarwal et al., 2010).

Additionally, the importance of properdin has been reported in an in vivo model of

polymicrobial septic peritonitis which showed that the survival of properdin deficient

mice was decreased compared to their wild-type littermates (Stover et al., 2008).

Garred et al. (1993) studied the relationship between the MBL/lectin pathway and

meningococcal disease and found that there was no relationship between meningococcal

disease (caused by serogroup B and C) and low concentrations of lectin pathway MBL

(Garred et al., 1993). On the other hand, the Department of Paediatrics, Imperial

College‎ School‎ of‎ Medicine‎ at‎ St‎ Mary’s‎ UK,‎ conducted‎ two‎ independent‎ studies,‎

showing that the susceptibility to meningococcal disease increased in children with

genetic variants of MBL (Hibberd et al., 1999). In addition to that, MBL deficiency in

early childhood increases the risk of meningococcal disease (Eisen et al., 2003; Faber et

al., 2007; Tully et al., 2006).


42

Although MBL binding varies in different serogroups of meningococcal, it has been

reported that LOS expression on meningococci is responsible for the major binding of

MBL and the meningococcal serogroups B and C show high activation of MBL even at

low concentrations (Jack et al., 2001; Van Emmerik et al., 1994; Kuipers et al., 2003).

It has also been shown that the binding affinity of MBL to Neisseria meningitidis

increased with low sialylation and decreased with high sialylation (Jack et al., 2001). In

a recent study it was reported that MBL binds to the meningococcal outer membrane

proteins Opa and ProB (Estabrook et al., 2004). This binding is not calcium dependent,

is not inhibited with mannose and is sensitive to 0.5M NaCl. Bjerre et al. (2002) carried

out a study using a whole blood model to study complement activation by

meningococcal strains and showed that meningococcal strains activate the complement

system through both the alternative and lectin pathways.

In summary, Neisseria meningitidis is the main causes of bacterial meningitis

throughout the world, and causes a significant rate of mortality (Emonts et al., 2003).

While there has been considerable progress in its diagnosis, vaccination and treatment

in recent years, the rates of Neisseria meningitidis infection are still increasing

worldwide, with a mortality rate of up to 10% (Connolly and Noah, 1999).

The complement system is a vital part of the immune system (activated by three

different pathways, which are the classic pathway, the alternative pathway and the lectin

pathway) and plays a crucial role in fighting microbial infection (Trouw and Daha 2011;

Schwaeble et al., 2002). Previous studies have highlighted the important role of the


43

complement system in fighting Neisseria meningitidis infection so this study has

focused on delineating the roles of defined complement component deficiencies in

increasing the susceptibility to meningococcal infection in mice and therefore to further

define the complement pathways that are involved in providing protection against

infection and disease progression (Jarva et al., 2005; Rosa et al., 2004; Rossi et al.,

2001)


44

1.4 Thesis Aims

Previous work in our laboratories has recently observed that serum of mice deficient of

the lectin pathway of complement effector enzyme MASP-2 has a higher bactericidal

activity towards Neisseria meningitidis as compared to MASP-2 sufficient serum. It

also showed that MASP-2 deficient mice were significantly protected against

meningococcal infection compared to MASP-2 sufficient mice. These findings

suggested that lectin pathway specific enzyme MASP-1 and/or MASP-3 has ability to

drive the alternative pathway mediated complement activation to fight N.meningitidis

infections. Therefore, this work aimed to achieve the following goals:

To assess the role of different complement pathways in innate immune response

against Neisseria meningitidis using in vitro assays

To assess the role of the alternative complement pathway in meningococcal

infection in experimental models of infection in a transgenic mouse strain with a

total deficiency of alternative pathway functional activity

To investigate the therapeutic benefits of recombinant MASP-3 (a lectin

pathway specific serine protease) in a murine model of meningococcal infection

To determine the potential benefits of recombinant properdin towards Neisseria

meningitidis using in vitro assays

Chapter 2: Materials and methods

45


2.1 Materials

2.1.1 Chemicals and materials

37% Formaldehyde solution Sigma-Aldrich

Agar-Agar Lab M

Barbital Sigma-Aldrich

Bovine serum albumin (BSA) Sigma-Aldrich

Brain heart infusion (BHI) medium Oxoid

Calcium chloride Sigma-Aldrich

Defibrinated Horse blood Oxoid

Ethylene glycol tetraacetic acid (EGTA) Sigma-Aldrich

Foetal calf serum Harlan

Heparin Sigma-Aldrich

Iron Dextran Sigma-Aldrich


46

Magnesium chloride Sigma-Aldrich

Mannan Sigma-Aldrich

N-acetyl BSA Promega

Phosphate Buffered Saline (PBS) Oxoid

Sigma Fast p-Nitrophenyl Phosphate tablet Sigma-Aldrich

Sodium chloride Fisher Scientific

Tris-HCl Sigma-Aldrich

Tween 20 Sigma-Aldrich

Zymosan Sigma-Aldrich


47

2.1.2 Antibodies/proteins

FITC-conjugated rabbit anti-human

C3c

Dako

Goat anti-human C1q polyclonal

antibody

Atlantic Antibodies Scarborough (USA)

Chicken anti-human C4c-alkaline

phosphatise

Immunsystem AB

Donkey anti-goat IgG (whole

molecule) alkaline phosphatase

antibody

Sigma-Aldrich

Goat anti-rabbit IgG (whole molecule)

Alkaline phosphatase antibody

Sigma-Aldrich

Rat anti-mouse MBL-A monoclonal

antibody

Hycult

Rat anti-mouse MBL-C monoclonal

antibody

Hycult

Rabbit anti-human C3c polyclonal

antibody

Dako

Rabbit anti-mouse Ficolin A Prof. T. Fujita, Department of Immunology,

Fukushima Medical University School of

Medicine, Fukushima, Japan

Rabbit anti-mouse IgG (whole

molecule) Alkaline phosphatase

antibody

Sigma-Aldrich


48

Recombinant murine MASP-3 Dr Sadam Yassin

Department of infection, Immunity &

inflammation, University of Leicester (UK)

Recombinant murine properdin Dr Youssif Mohammed Ali

Department of infection, Immunity &

inflammation, University of Leicester (UK)

Rat anti-mouse CL-11 antibody

Dr. Soren Hansen

Department of Cancer and Inflammation

Research,

University of Southern Denmark (Denmark)


49

2.1.3 Media and buffers

Levinthal‟s‎supplement‎

400ml of Brain heart infusion (BHI) was mixed

with 200 ml defibrinated horse blood and heated

at 45°C for 40 minutes. Then mixtures was

cooled for 15 minutes at room temperatures and

centrifuged‎at‎5350‎xg‎for‎25‎minutes‎at‎4◦C.‎

Then the supernatant of mixture was aliquoted

into 40 mland kept at -20◦C.‎

Coating buffer 15 mM Na2CO3

35 mM NaHCO3

pH 9.6

Tris buffer saline (TBS) 10 mMTris-HCL

140 mMNaCl

pH 7.4

BSA-TBS blocking buffer TBS with 1% (w/v) BSA

pH 7.4

Washing buffer TBS with 0.05% tween-20

5mM CaCl2

pH 7.4

Barbital buffer saline (BBS) 4 mM barbital

145 mM NaCl

1 mM MgCl2

2 mM CaCl2

pH7.4


50

MBL binding buffer 20 mM Tris-HCl

10 mM CaCl2

1 M NaCl

pH 7.4

Ethylene glycol tetraacetic acid

(EGTA) buffer

4 mM barbital

145 mM NaCl

1 mM MgCl2

10 mM EGTA

pH7.4


51

2.2 Methods

2.2.1 In vitro experiments

2.2.1.1 Preparation of mouse and human serum

Sera were used in the immune biochemical test was extracted from mouse (by cardiac

Puncture) and healthy human (by vein puncture). For some assays sera was taken from

mice and humans who have a deficient on their complement components. Blood

samples were taken and kept on ice for 5 hours to avoid complement activation and

coagulation factors. Blood was then centrifuged in a cooled microcentrifuge at 14,000

rpm for 7 minutes. Serum was aliquated as 100µl into labelled Eppendorf tubes and

stored at -80°C.

2.2.1.2 Preparation of Neisseria meningitidis for Enzyme Linked Immunosorbent

Assay (ELISA)

Tow strains of Neisseria meningitidis were used (these tow strains are the same strains

that used in the previous study in our laboratories) which are serotype B (BMC-58) and

serotype A (Z2491). Neisseria meningitidis were grown overnight on brain heat

infusion‎medium‎(BHI)‎supplemented‎with‎5%‎Levanthal’s (to enrich the medium with

iron which is an important element for the Neisseria meningitidis growth) at 37⁰C and

5% CO2. Then the medium containing bacteria were centrifuged at 4000 ×g for 10

minutes. The pellet was then washed three times with phosphate buffered saline (PBS),

and 10ml of 0.5 % formalin was added to resuspend the pellet and then incubated for 60


52

minutes at room temperature. The pellet were centrifuged and washed three times with

PBS after the incubation. A loopful of the pellet was grown overnight on BHI agar

supplemented‎with‎5%‎Levanthal’s‎at‎37⁰C and 5% CO2 to ensure the formalin fixation

by the absence of growth. Finally the pellet was resuspended in coating buffer (15 mM

Na2CO3, 35 mM NaHCO3, 0.02% sodium azide, pH 9.6) for later use after adjusting the

optical density (OD) of the resuspension to be at 0.6 (OD550= 0.6).

2.2.1.3 C1q deposition assays

Microtitre‎ ELISA‎ plates‎ were‎ coated‎ with‎ 100μl‎ of‎ different‎ fixed‎ Neisseria

meningitidis strains (OD550=0.6). As positive controls ELISA plate wells were coated

with‎1μg/ml‎bovine‎serum‎albuminutes‎(BSA).‎Also‎as negative controls ELISA plate

wells received 100μl‎of‎the‎coating‎buffer. Then the plates were incubated overnight in

a 4°C fridge. Next day, ELISA plates were incubated at RT for 2hours. Plates were

washed three times by washing buffer (TBS with 0.05% tween-20 and 5mM CaCl2)

before‎ adding‎ 100‎ μl‎ of rabbit anti-BSA‎ (2‎ μg/ml)‎ to‎ positive‎ control‎ wells‎ and‎

incubated for 90 minutes at room temperature. Two fold serial dilutions of mouse serum

were prepared in barbital buffered saline (BBS; 4 mM barbital, 145 mM NaCl, 2 mM

CaCl2, 1 mM MgCl2, pH 7.4) starting from 1/80 (high dilution). Plates were washed

three times by washing buffer (TBS with 0.05% tween-20 and 5mM CaCl2) before

adding‎ the‎ 100μl‎ of‎ serum‎ dilutions‎ in‎ duplicates‎ into‎ corresponding‎ wells‎ and‎

incubating at 37°C for 1hour. Washing buffer was used to repeat the washing followed

by‎ the‎ addition‎ of‎ 100μl‎ of‎ goat‎ anti-human‎C1q‎ (2‎μg/ml).‎ Plates‎were‎ incubated‎ at‎

37°C‎for‎90‎minutes‎before‎repeating‎the‎washing‎again.‎100μl‎of‎secondary‎antibodies‎

(anti-goat IgG-alkaline phosphatase conjugate diluted 1/10000) was added to the wells


53

and incubated again at RT for 90 minutes. Plates were washed again, followed by the

addition of 100μl‎ substrate solution (Fast pNPP tablet (p-Nitrophenyl phosphate Tablet)

sets, Sigma). Hydrolysis of substrates was monitored using BioRad microtitre ELISA

plate reader by measuring the absorption at 405nm.

2.2.1.4 MBL-A, MBL-C, Ficolin-A and CL-11 binding assays



by‎1μg/ml‎mannan‎in‎MBL-A and MBL-C‎assays,‎10μg/ml‎Zymosan‎in‎CL-11 assays,

and‎10μg/ml‎N-acetylated BSA in ficolin-A assays. Also as negative controls ELISA

plate‎ Wells‎ received‎ 100μl‎ of‎ TBS‎ buffer‎ (10‎ mM‎ Tris, 140 mM NaCl, pH 7.4)

containing 1% of bovine serum albuminutes (BSA). Then the plates were incubated

overnight‎in‎a‎4°C‎fridge.‎Next‎day,‎ELISA‎plates‎were‎blocked‎by‎250μl‎of‎TBS‎buffer‎

containing 1% BSA and incubated at room temperature (RT) for 2hours. Two fold serial

dilutions of mouse serum were prepared in MBL binding buffer (20 mM Tris-HCl, 10

mM CaCl2, 1 M NaCl, pH 7.4) starting from 1/20 (low dilution). Plates were washed

three times with washing buffer (TBS with 0.05% tween-20 and 5mM CaCl2) before

added‎100μl‎of‎serum‎dilutions‎in‎duplicates‎into‎corresponding‎wells‎and‎incubated‎at‎

37°C for 1hour. Washing buffer was used to repeat the washing, followed by the

addition‎of‎100μl‎of‎primary‎antibodies‎rat‎anti-mouse MBL-A (Hycult; 1mg/ml stock

solution), rat anti-mouse MBL-C (Hycult; 1mg/ml stock solution), rabbit anti-mouse

Ficolin A (Prof. T. Fujita, Department of Immunology, Fukushima Medical University

School of Medicine, Fukushima, Japan: 0.7 mg/ml stock solution) antibodies diluted

1/1000, rat anti-mouse CL-11 (Dr. Soren Hansen, Department of Cancer and Inflammation


54

Research, University of Southern Denmark: 2.04mg/ml stock solution) antibodies diluted

1/500. Plates were incubated at RT for 90 minutes before repeating the washing again.

100μl‎ of secondary antibody (goat anti mouse IgG-alkaline phosphatase conjugate or

goat anti rabbit IgG-alkaline phosphatase conjugate diluted 1/10000) dilutions were

added to wells and incubated again at RT for 90 minutes. Plates were washed again,

followed by the‎ addition‎ of‎ 100μl‎ substrates‎ solution‎ (Fast‎ pNPP‎ tablet‎ sets,‎ Sigma).‎

Hydrolysis of substrates was monitored using BioRad microtitre ELISA plate reader by

measuring the absorption at 405nm.

2.2.1.5 C3 deposition assays

Microtitre ELISA plates were coated with 100μl‎ of‎ different‎ fixed‎ Neisseria


with‎10μg/ml‎Zymosan‎(Sigma).‎Also‎as‎negative‎controls‎ELISA‎plate‎Wells‎received‎

100μl‎of‎TBS‎buffer‎(10‎mM‎Tris,‎140‎mM‎NaCl,‎pH‎7.4) containing 1% BSA. Then

the plates were incubated overnight in a 4°C fridge. Next day, ELISA plates were

blocked‎by‎250μl‎of TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4) containing of 1%

BSA and incubated at RT for 2hours. Two fold serial dilutions of mouse serum were

prepared in barbital buffered saline (BBS; 4 mM barbital, 145 mM NaCl, 2 mM CaCl2,

1 mM MgCl2, pH 7.4) starting from 1/80 (high dilution). Plates were washed three

times by washing buffer (TBS with 0.05% tween-20 and 5mM CaCl2) before addition

of the‎100μl‎of‎serum‎dilutions‎in‎duplicates‎into‎corresponding‎wells‎and‎incubating‎at‎

37°C for 1hour. Washing buffer was used to repeat the washing followed by the

addition‎ of‎ 100μl‎ of‎ rabbit‎ anti-human C3c antibodies (Dako) diluted 1/5000. Plates

were incubated‎ at‎ 37°C‎ for‎ 90‎minutes‎ before‎ repeating‎ the‎washing‎ again.‎ 100μl‎ of‎


55

secondary antibodies (goat anti rabbit IgG-alkaline phosphatase conjugate (Sigma)

diluted 1/10000) was added to the wells and incubated again at RT for 90 minutes.

Plates were washed‎again,‎ followed‎by‎ the‎addition‎of‎100μl‎ substrates‎ solution‎ (Fast‎

pNPP tablet sets, Sigma). Hydrolysis of substrates was monitored using BioRad

microtitre ELISA plate reader by measuring the absorption at 405nm.

2.2.1.6 Alternative pathway mediated C3 deposition assays



with‎10μg/ml‎Zymosan‎(Sigma).‎Also‎as‎negative‎controls‎ELISA‎plate Wells received

100μl‎of‎TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4) containing 1% BSA. Then

the plates were incubated overnight in a 4°C fridge. Next day, ELISA plates were

blocked‎by‎250μl‎of‎TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4) containing 1%

BSA and incubated at RT for 2hours. Two fold serial dilutions of mouse serum were

prepared in Ethylene glycol tetra acetic acid (EGTA) buffered (4 mM barbital, 145 mM

NaCl, 10 mM EGTA, 1 mM MgCl2, pH 7.4) starting from ½ (low dilution). Plates were

washed three times by washing buffer (TBS with 0.05% tween-20) before adding the

100μl‎of‎serum‎dilutions‎in‎duplicates‎into‎corresponding‎wells‎and‎incubating‎at‎37°C‎

for 1hour. Washing buffer was used to repeat the washing followed by the addition of

100μl‎of‎rabbit‎anti-human C3c antibodies (Dako) diluted 1/5000. Plates were incubated

at‎ 37°C‎ for‎ 90‎ minutes‎ before‎ repeating‎ the‎ washing‎ again.‎ 100μl‎ of‎ secondary‎

antibodies (goat anti rabbit IgG-alkaline phosphatase conjugate (Sigma) diluted

1/10000) was added to the wells and incubated again at RT for 90 minutes. Plates were

washed‎again,‎followed‎by‎the‎addition‎of‎100μl‎substrates‎solution‎(Fast‎pNPP‎tablet‎


56

sets, Sigma). Hydrolysis of substrates was monitored using BioRad microtitre ELISA

plate reader by measuring the absorption at 405nm.

2.2.1.7 Lectin pathway mediated C4 deposition assays



with‎10μg/ml‎Zymosan‎(Sigma).‎Also‎as‎negative‎controls ELISA plate Wells received

100μl‎of‎TBS‎buffer‎(10‎mM‎Tris,‎140‎mM‎NaCl,‎pH‎7.4) containing 1% BSA. Then

the plate incubated overnight in a 4°C fridge. Next day, ELISA plates were blocked by

250μl‎ of‎TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4) containing 1% BSA and

incubated at RT for 2hours. Two fold serial dilutions of mouse serum were prepared in

MBL binding buffer (20 mM Tris-HCl, 10 mM CaCl2, 1 M NaCl, pH 7.4) starting from

1/80 (high dilution). Plates were washed three times by washing buffer (TBS with

0.05% tween-20‎ and‎ 5mM‎ CaCl2)‎ before‎ adding‎ the‎ 100μl‎ of‎ serum‎ dilutions‎ in‎

duplicates into corresponding wells and incubating at 37°C for 1hour. Washing buffer

was‎used‎to‎repeat‎the‎washing‎followed‎by‎the‎addition‎of‎100μl‎of‎1μg/ml‎human‎C4‎

diluted in BBS. Plates were incubated at 37°C for 90 minutes before repeating the

washing‎ again.‎ 100μl‎ of‎ chicken‎ anti-human C4c antibody alkaline phosphatase

conjugate diluted 1/10000 was added to the wells and incubated again at RT for 90

minutes. Plates were‎ washed‎ again,‎ followed‎ by‎ the‎ addition‎ of‎ 100μl‎ substrates‎

solution (Fast pNPP tablet sets, Sigma). Hydrolysis of substrates was monitored using

BioRad microtitre ELISA plate reader by measuring the absorption at 405nm.


57

2.2.1.8 Serum Bactericidal Assay (SBA)

Serum bactericidal assay (after modification on Estabrook et al (1997) was used to

assess the sensitivity of Neisseria meningitidis toward different serum samples.

Neisseria meningitidis was‎grown‎in‎10ml‎BHI‎supplemented‎with‎5%‎Levanthal’s‎and‎

incubated 3 hours at 37⁰C in 5% CO2. After this time, a sample of the growth was taken

and the optical density at 600nm was measured (which corresponds to 109 CFU/ml) to

check the count. Then aliquots of 500µl were made of the growth and kept at -80⁰C.

Viable counts were done to confirm the bacterial count. An aliquot was resuspended in

phosphate buffered saline (PBS) to get the desired count of bacteria. A suspension of a

known concentration of Neisseria meningitidis (105) was mixed with a desired

concentration of serum samples on Barbital buffer saline and incubated at 37oC, with

shaking at 120rpm. Samples of reaction were taken at 0, 30, 60, 90, and 120 min, and

serial dilutions in phosphate buffered saline (PBS) have prepared and plated onto Brain

heart infusion (BHI) agar with‎5%‎Levanthal’s‎supplement and incubated overnight at

37oC in 5% CO2.

2.2.1.9 Preparation of Neisseria meningitidis for FACS analysis

Neisseria meningitidis Strain B serogroup MC58 grew overnight on brain heat infusion

medium (BHI) supplemented with 5% Levanthal’s‎ at‎ 37⁰C and 5% CO2. Then the

medium containing bacteria were centrifuged at 4000 ×g for 10 minutes. The pellet was

then washed three times with phosphate buffered saline (PBS), and 10ml of 0.5 %

formalin was added to resuspend the pellet, which was then incubated it for 60 minutes

at room temperature. Subsequently, the pellet was centrifuged and washed three times


58

with TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4). A loopful of the pellet was

grown‎overnight‎on‎BHI‎agar‎supplemented‎with‎5%‎Levanthal’s‎at‎37⁰C and 5% CO2

to ensure the formalin fixation by the absence of growth. Finally, the pellet was

resuspended in EGTA buffer (4 mM barbital, 145 mM NaCl, 10 mM EGTA, 1 mM

MgCl2, pH 7.4) for later use.

2.2.1.10 FACS analysis for detect C3 deposition on Neisseria meningitdis

Fixed Neisseria meningitidis was washed three times with TBS buffer (10 mM Tris, 140

mM NaCl, pH 7.4) before resuspended the pellets in EGTA buffer (4 mM barbital, 145

mM NaCl, 10 mM EGTA, 1 mM MgCl2, pH 7.4). 100µl of the suspend bacteria were

opsonised with 200µl of 5% normal human serum or 15% wildtype mouse serum at

37°C with or without 5µg/ml of recombinant properdin. Nonopsonised bacteria were

used as negative control. After 1 hour, bacterial samples were centrifuged and washed

three times with TBS buffer. 200µl of FITC-conjugated rabbit anti-human C3c diluted

1/5000 was added to bacteria and incubated at RT for I hour. Bacterial sample was then

centrifuged and washed three times again with TBS buffer before resuspending the

bacteria in 1ml PBS buffer. The fluorescence intensity was measured with a

FACSCalibur cell analyser (BD Biosciences).


59

2.2.2 In vivo experiments

2.2.2.1 Genotyping of fB and MASP-1/3 deficient mice

2.2.2.1.1 Isolation of genomic DNA from mouse ear snips

Around 4mm of mice ear snips were incubated overnight at 55°C and gently mixed in

master mix containing 250µl of Nuclei Lysis Solution, 60µl of 0.5M EDTA solution,

and‎10µl‎of‎Proteinase‎K‎(Qiagen).‎On‎the‎next‎day,‎1.5μl‎of‎RNase‎solution‎(4mg/ml)‎

was added to the mixture and mixed by inverting the tube three times, and incubated at

37°C‎for‎30‎mins.‎The‎sample‎was‎cooled‎to‎room‎temperature‎for‎five‎mins,‎100μl‎of‎

protein precipitation was added to the tube and it was vortexed at high speed for 20 sec.

Ice was used to cool the sample for five mins, then it was centrifuged for four mins at

13,000rpm. Supernatant was taken to a clean labelled‎ tube,‎ then‎300μl‎of‎ isopropanol

was added to the supernatant and then centrifuged for five mins at 13000rpm.

Supernatant was‎discarded‎and‎300μl‎of‎70% ethanol was added. The tube was gently

inverted three times then centrifuged for one min at 13000rpm. The tube was dried from

ethanol‎for‎30‎min‎by‎inverting‎the‎tube‎on‎clean‎absorbent‎paper,‎then‎100μl‎of‎DNA‎

re-hydration solution was added and it was stored at 4°C overnight.

2.2.2.1.2 Polymerase Chain Reaction (PCR)

PCR is a powerful method to amplify a specific region of DNA that lies between known

sequences of DNA. A standard PCR reaction is composed of three main steps

(denaturation, annealing and DNA synthesis), starting with a denaturation step during

which the DNA template is denatured by heating to a high temperature, followed by a

cooling step which allows the primer to link to its target, and finally an amplification

end step by optimal temperature that allows synthesis of the DNA. These steps are

repeated 35 times using an automated thermal cycler.


60

PCR was done to determine the genomic DNA (gDNA) and therefore identify the fB+/+

,

fB+/-

, fB-/-

, MASP-1/3 +/+

, MASP-1/3 +/-

, and MASP-1/3 -/-

mice.

The primers used for genotyping of fB are

Primer name Primer sequence

FB_F2‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎5’- GAAGGACCTAGAAACAGCGCTCA-3’

FB_WTO_R1‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎5’- CTGATCTACCTTCTCAATCAAGTTGGTGA-3’

Neo3_F5 5’-CTGTTGTGCCCAGTCATAGCCGA-3’

The primers used for genotyping of MASP-1/3 are

Primer name Primer sequence

NeoU 5’- CAT CGC CTT CTA TCG CCT TCT TGA-3

M1U‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎5’- CTC CCT GCC TCA GAC TGT TTG ATA-3’

Mil‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎‎5’- GCT GAT GCT GAT GTT AGG ATG GTA TTC-3’

Each PCR reaction tube contains:

Genomic DNA‎(200ng/μl)‎ 1μl‎

Reaction buffer (10x) 1.5μl‎

MgCl2 (2.5mM) 1.5μl‎

dNTP mix (10mM) 0.3μl‎

M2screen_F1 1.5μl‎

M2wto_R1 1.5μl‎

Neo5_R1 1.5μl‎

Taq-DNA polymerase 0.12μl‎

Nanopure distilled water 6.08μl


61

2.2.2.2 Preparation of Neisseria meningitidis passage

Neisseria meningitidis replication depends on the presence of iron as it an important

factor to perform some metabolic functions by acting as a cofactor for some enzymes

including enzymes required for DNA replication, oxygen metabolism and electron

transport. A previous study made it clear that the mice that were injected with iron (iron

dextran or human transferrin) before challenging them with Neisseria meningitidis had

ability to develop a lethal infection. However, the mice that not injected with iron could

not develop a lethal infection (Griffiths, 1999; Holbein, 1980; Holbein et al., 1979).

A mouse was injected with iron intraperitoneally (400mg/kg body weight) 12 hours

before the infective dose. Neisseria meningitidis was grown in 10ml BHI supplemented

with‎5%‎Levanthal’s‎and‎incubated‎for‎3‎hours‎at‎37⁰C in 5% CO2. After this time, a

sample of the growth was taken and the optical density at 600nm was measured (which

corresponds to 109 CFU/ml) to check the count. Subsequently, aliquots of 500µl of the

growth were made and kept at -80⁰C. Viable counts were done to confirm the bacterial

count. An aliquot of it was resuspended in phosphate buffered saline (PBS) and 100µl

of resuspended bacteria (109

CFU/ML) was injected into the mouse intraperitoneally.

On the following day, the uses mouse culled by cervical dislocation once it developed

the sign of terminal illness and a blood sample was collected by cardiac puncture. The

blood sample was streaked on brain heart infusion (BHI) agar supplemented with 5%

Levanthal’s‎and‎incubated‎overnight‎at‎37⁰C in 5% CO2.

A loopful of colonies from the overnight culture was picked up and resuspended in

10ml‎ serum‎BHI‎ (80%‎ BHI‎ supplemented‎with‎ 5%‎ Levanthal’s‎ and‎ 20%‎ foetal‎ calf‎


62

serum (FCS)) and incubated at 37⁰C in 5% CO2 for 3 hours. After this time, a sample of

the growth was taken and the optical density at 600nm was measured (which

corresponds to 109 CFU/ml) to check the count. Afterwards, aliquots of 500µl of the

growth were made and kept at -80⁰C. Viable counts were done to confirm the bacterial

count of the stocks. For each infection experiment, an aliquot of the stock was thawed at

room temperature, spun down, and resuspended in PBS to get the desired concentration

of bacteria. The bacterial concentration was confirmed by doing serial dilution in PBS

and‎plating‎it‎onto‎brain‎heart‎ infusion‎(BHI)‎agar‎supplemented‎with‎5%‎Levanthal’s‎

and incubated overnight at 37⁰C in 5% CO2.

2.2.2.3 Virulence testing of passaged stocks of Neisseria meningitidis

Wild-type mice were used to assess the virulence of the passaged stocks of Neisseria

meningitidis. Mice were injected intraperitoneally with iron dextran (400mg/kg body

weight) 12 hours before infection dose. An aliquot of passaged stock of Neisseria

meningitidis was thawed at room temperature, centrifuged and resuspended in PBS to

obtain the required viable count. Mice were divided to three groups and injected with

required bacterial doses of 1 x105, 1 x10

6, and 1 x10

7 CFU/mouse diluted in sterile 100

µl PBS. Following infection, mice were regularly monitored every six hours for the

illness symptoms. The signs of illness were scored (table 2.1) based on the scheme

(with some modifications) of Fransen etal (2010). Once mice developed signs of

terminal illness, they were culled by cervical dislocation and a blood sample was

collected by cardiac puncture under aseptic conditions for bacterial load determination.


63

Table 2.1 The severity scores of disease with clinical signs of infected mice

Signs Score

Normal 0

Slightly ruffled fur 1

Ruffled fur, slow and sticky eyes 2

Ruffled fur, lethargic and eyes shut 3

Very sick and no movement after

stimulation 4

Dead 5

2.2.2.4 Infection of mice with Neisseria meningitidis

Wild-type mice and mice deficient in their alternative pathway components (fB-/-

or

MASP-1/3-/-

) have been used in the infection studies. Mice ranged in age from 12 to 14

weeks. Mice were injected intraperitoneally with iron dextran (400mg/kg body weight)

12 hours before infection dose. An aliquot of passaged stock of Neisseria meningitidis

was thawed at room temperature, centrifuged and resuspended in PBS to obtain the

required viable count 1 x105 CFU/mouse diluted in sterile 100 µl PBS. Following

infection, mice were monitored every six hours for the illness symptoms. Once mice

developed the signs of terminal illness (very sick and no movement after simulation), it

was culled immediately by cervical dislocation and a blood sample collected by cardiac

puncture following by determined of Blood bacterial burden.

2.2.2.5 Determination of Blood bacterial burden

Following the introducing of the pathogen to the mice intraperitoneally, the course of

infection was monitored by time course of bacteraemia. Every six hours of infection,


64

mice were placed in thermocage at 37°C for 20 minutes to worm the mice and dilate the

veins in mice tails. Mice blood was collected from the tails of mice and serial diluted in

sterile PBS‎and‎plating‎on‎BHI‎agar,‎supplemented‎with‎5%‎Levanthal’s‎and‎incubated‎

overnight at 37⁰C in 5% CO2 to count the bacteria.

2.2.2.6 MASP-3 reconstitution experiment

Wild-type mice and MASP-1/3-/-

mice were divided to three groups: Group A wild type

mice were injected intravenously with PBS, group B MASP-1/3-/-

mice were injected

intravenously with‎20μg/mouse‎of‎recombinant‎mouse‎MASP-3 at 0 hour, and group C

MASP-1/3-/-

mice‎were‎injected‎intravenously‎with‎20μg/mouse‎of‎recombinant‎mouse‎

MASP-3 at 0 hour and 72 hours. Mice were bled at 0, 24, 48, 72 and 96 hours post

reconstitution. Alternative pathway C3 deposition assay was performed. Microtitre

ELISA‎ plates‎ were‎ coated‎ with‎ with‎ 10μg/ml‎ Zymosan‎ (Sigma)‎ and‎ incubated‎

overnight‎in‎a‎4°C‎fridge.‎Next‎day,‎ELISA‎plates‎were‎blocked‎by‎250μl‎of‎1%‎bovine‎

serum albuminutes (BSA) in TBS buffer (10 mM Tris, 140 mM NaCl, pH 7.4) and

incubated at RT for 2hours. Two fold serial dilutions of mice sera were prepared in

Ethylene glycol tetraacetic acid (EGTA) buffered saline (4 mM barbital, 145 mM NaCl,

10 mM EGTA, 1 mM MgCl2, pH 7.4) starting at 13% concentration. Plates were

washed three times by washing buffer (TBS with 0.05% tween-20) before adding the

100μl‎of‎serum‎dilutions‎in‎duplicates‎into‎corresponding‎wells‎and‎incubating‎at‎37°C‎

for 1hour. Wells received only washing buffer were used as negative controls. Washing

buffer‎was‎used‎to‎repeat‎the‎washing,‎followed‎by‎the‎addition‎of‎100μl‎of‎rabbit‎anti-


65

human C3c antibodies diluted 1/5000. Plates were incubated at 37°C for 90 minutes

before‎ repeating‎ the‎ washing‎ again.‎ 100μl‎ of‎ secondary‎ antibodies (α-rabbit IgG-

alkaline phosphatase conjugate diluted 1/10000) was added to the wells and incubated

again at RT for 90 minutes. Plates were washed again, followed by the addition of

100μl‎substrates‎solution‎(Fast‎pNPP‎tablet‎sets,‎Sigma).‎Hydrolysis‎of‎substrates was

measured using BioRad microtitre ELISA plate reader to read an absorbance at 405nm.

2.2.3 Statistical analysis

GraphPad prism version 6 was used to preform experimental statistical analysis

by using unpaired t-test for all experiments except the in vivo survival

experiments where Log-rank (Mantel-Cox) test was used.

Chapter 3: In vitro study

66



fighting against invading microbes including Neisseria meningitidis. However the role

of different complement pathways in fighting Neisseria meningitidis infection needs to

be clarified which could be done by studying the binding of the complement pathways

recognition molecules to the Neisseria meningitidis and by studying the bacteriolytic

activity of different mice sera against Neisseria meningitidis.

3.1 Results:

3.1.1 Complement pathway specific Enzyme Linked Immune Sorbent

Assays (ELISAs)

The initiation of either of the three complement activation pathways on the surface of N.

meningitidis depends on the binding of complement recognition molecules to

meningococcus. The binding of various complement recognition molecules to different

serotypes of Neisseria meningitidis strains (i.e. serotype A strain Z2491 and serotype B

strain MC58) were examined. This results show that meningococcus serotype A strain

Z2491 and serotype B strain MC58 have the ability to bind to C1q, the recognition

molecule of classical pathway, and recognition molecules of the lectin pathway

including mouse MBL-A, MBL-C and CL-11. In contrast, ficolin-A a recognition

molecule of lectin pathway in the mouse showed no binding to any of the

meningococcal serotypes, i.e. serotype A strain Z2491 and serotype B strain MC58.


67

3.1.1.1 Binding of the classical pathway molecule C1q to Neisseria meningitidis

A C1q binding assay was used to investigate the role of the classical pathway in fighting

Neisseria meningitidis infection in the absence of specific antibodies against Neisseria

meningitidis by studying the binding of C1q to the surface of different Neisseria

meningitidis strains. This work showed that C1q bound to the surface of Neisseria

meningitidis strains, indicating that the classical pathway may possibly contribute to the

formation of C3 convertases deposited on the surface of Neisseria meningitidis (See

Figure 3.1).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

1 .8

% s e r u m c o n c e n t r a t io n

OD

At

40

5n

m

B S A im m u n e co m p le x

S e ro A -Z 2 4 9 1

S e ro B -M C 5 8

N e g a tiv e c o n tro l

Figure 3.1 C1q binding on the surface of different Neisseria meningitidis strains.

Mouse serum was diluted in BBS. BSA anti-BSA coating was used as positive control.

The negative control wells received BSA blocking buffer as a coating. Binding of C1q

was detected by using Goat anti-human C1q polyclonal antibody (the data is presented

as a mean of all three independent experiments with duplicate for each +/- SEM).


68

3.1.1.2 Binding of the lectin pathway recognition component MBL to Neisseria

meningitidis

The contribution of the lectin pathway recognition subcomponent Mannan Binding Lectin

(MBL) in fighting Neisseria meningitidis infection was examined by assessing the binding of

the murine lectin pathway recognition molecules MBL-A and MBL-C to the surface of

Neisseria meningitidis. The binding of MBL-A and MBL-C to the surface of Neisseria

meningitidis outer membrane proteins (outer membrane protein A and porin B) had been

reported previously (Estabrook et al., 2004). It has also been reported that the structure and

sialylation of lipooligoccharides (LOS) acts as a binding site for MBL (Jack et al., 1998; Jack et

al., 2001). MBL-A and MBL-C binding assays were carried to assess the relative binding

affinity of the recognition subcomponents to the bacterial surface of different N. meningitidis

serotypes. These assays showed that MBL-A and MBL-C had the ability to bind to the surface

of all tested Neisseria meningitidis strains (see Figures 3.2 and 3.3).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4


OD

At

40

5n

m

M a n n a n , M B L -A

S e ro B -M C 5 8 , M B L -A

S e ro A -Z 2 4 9 1 , M B L -A

N e g a tiv e C o n tro l M B L - A

Figure 3.2 MBL-A binding on the surface of different Neisseria meningitidis strains. Mouse

serum was diluted in MBL binding buffer. Mannan coating was used as a positive control. The

negative control wells received BSA blocking buffer as a coating. Binding of MBL-A was

detected by using rat anti-mouse MBL-A monoclonal antibody (the data is presented as a mean

of three independent experiments with duplicate for each +/- SEM).


69

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

% S e r u m C o n c e n t r a t io n

OD

At

40

5n

m

S e ro B -M C 5 8, M B L -C

S e ro A -Z 2 4 9 1 , M B L -C

M an na n, M B L-C

N e ga tiv e C on tro l, M B L - C

Figure 3.3 MBL-C binding on the surface of different Neisseria meningitidis strains. Mouse

serum was diluted in MBL binding buffer. Mannan coating was used as a positive control. The

negative control wells received BSA blocking buffer as a coating. Binding of MBL-C was

detected by using rat anti-mouse MBL-C monoclonal antibody (the data is presented as a mean

of all three independent experiments with duplicate for each +/- SEM).

3.1.1.3 Binding of the lectin pathway Collectin-11 to Neisseria meningitidis

Collectin-11 (CL-11) is a member of the lectin pathway and has recently been identified

as a recognition molecule of the lectin pathway, which can bind to the surface of the

pathogen (1-fucose and d-mannose sugars) and activate the lectin pathway. Binding of

CL-11 to Neisseria meningitidis has been done to assess the binding of CL-11 to the

Neisseria meningitidis. Using the direct binding assay shown in Figure 3.4 it was shown

that CL-11 had the ability to bind to the surface of the two Neisseria meningitidis strains

tested (see Figure 3.4).


70

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .1

0 .2

0 .3

0 .4

0 .5

0 .6

0 .7

0 .8

0 .9

Z y m o s a n

S e ro A -Z 2 4 9 1

S e ro B -M C 5 8


OD

At

40

5n

m


Figure 3.4 CL-11 binding to the surface of different Neisseria meningitidis strains. Mouse

serum was diluted in MBL binding buffer. Zymosan coating was used as a positive control. The

negative control wells received BSA blocking buffer as a coating. Binding of CL-11 was

detected by using Rat anti-mouse CL-11 antibody (the data is presented as a mean of all three

independent experiments with duplicate for each +/- SEM).

3.1.1.4 Binding of the lectin pathway ficolin-A to Neisseria meningitidis

Ficolin-A is one of the recognition molecules of the lectin pathway belonging to the ficolin

family that can activate the lectin pathway by binding to N-acetylglucosamine on the surface of

pathogens. In mice, there are two types of ficolins, ficolin- A and ficolin-B. While ficolin-B is

synthesised mainly in myeloid cells in the bone marrow, ficolin-A is predominantly synthesised

in the liver. Ficolin A is considered to be the only murine ficolin that can bind to the mannan

binding lectin associated serine proteases (MASPs) to activate the lectin pathway (Endo et al.,

2005; Runza et al., 2008). The binding of ficolin-A to Neisseria meningitidis was assessed to

evaluate the binding of ficolin-A the Neisseria meningitidis. The failure to detect any binding of

ficolin-A to the surface of Neisseria meningitidis strains indicates that deficiency of ficolin-A


71

might not predispose for a higher predisposition to Neisseria meningitidis infections (see Figure

3.5).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6


OD

At

40

5n

m

N -A c e ty l a lb u m in

S e ro B -M C 5 8

S e ro A -Z 2 4 9 1


Figure 3.5 Ficolin-A does not bind to the surface of different Neisseria meningitidis strains.

Mouse serum was diluted in MBL binding buffer. N-Acetyl albumin coating was used as a

positive control. The negative control wells received BSA blocking buffer as a coating. Binding

of the ficolin-A was detected by using rabbit anti-mouse ficolin-A antibody (the data is

presented as a mean of all three independent experiments with duplicate for each +/- SEM).

3.1.2 C3 deposition assays

C3 deposition assays were performed to assess the ability of Neisseria meningitidis to

activate complement pathways by measuring the amount of C3 that was deposited on

the surface of Neisseria meningitidis strains. In these assays, an antibody against C3c,

which is generated from the cleavage of C3 and binds to ELISA plates, was used to


72

detect the cleavage of C3. Specific conditions were chosen to allow activation of the

classical and lectin pathways, but not the alternative pathway (as highly diluted serum

makes the alternative pathway dysfunctional). High levels of C3 deposition was

detected on the surface of the two Neisseria meningitidis strains tested. Serogroup B

strain MC-58 showed more C3 depositions compared to serogroup A strain Z2491 (see

Figure 3.6).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

1 .8


OD

At

40

5n

m

Z y m o s a n

S e ro A -Z 2 4 9 1

S e ro B -M C 5 8

N e g a t iv e c o n tro l

Figure 3.6 C3 deposition assay on the surface of different Neisseria meningitidis strains under

specific condition (high serum dilution in BBS buffer) allows the activation through classical

and lectin pathways. Mouse serum was diluted on BBS binding buffer. Zymosan coating has

used as a positive control while the negative control wells received BSA blocking buffer as a

coating. The binding of the C3 cleavage product (C3c) was detected by using rabbit anti-human

C3c polyclonal antibody (the data is presented as a mean of all three independent experiments

with duplicate for each +/- SEM).

To assess the role of the alternative pathway in fighting Neisseria meningitidis, an

alternative pathway specific C3 deposition assay was designed using high serum


73

concentrations (sera were diluted in and alternative pathway permissive EGTA buffer

that contains Mg++

(in absence of Ca++

complement can only be activated via the

alternative pathway). A high level of C3 deposition was observed on Neisseria

meningitidis serogroup B strain MC58, while a moderate level of C3 deposition was

observed on Neisseria meningitidis serogroup A strain Z2491. These results suggests

that the alternative pathway plays a role in fighting Neisseria meningitidis infection (see

Figure 3.7).

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4


OD

At

40

5n

m

Z y m o s a n

S e ro B -M C 5 8

S e ro A -Z 2 4 9 1

N e g a t iv e c o n tro l


alternative pathway permissive conditions (high serum concentration in EGTA buffer). Mouse

serum was diluted in EGTA buffer. Zymosan coating was used as a positive control while the

negative control wells received BSA blocking buffer as a coating. The binding of the C3

cleavage product (C3c) was detected by using rabbit anti-human C3c polyclonal antibody (the

data is presented as a mean of all three independent experiments with duplicate for each +/-

SEM).


74

The role of the lectin pathway in fighting Neisseria meningitidis has been assessed using a C3

deposition assay. In this assay, MASP-2 sufficient and MASP-2 deficient serum, which lack

MASP-2 dependent lectin pathway functional activity, were used at a low serum concentrations.

This assay demonstrated that MASP-2 sufficient serum showed high C3 deposition on the

surface of Neisseria meningitidis strains while MASP-2 deficient serum showed no C3

deposition, which suggests that the lectin pathway has an important role in the activation of the

complement system on the surface of Neisseria meningitidis (see Figure 3.8).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .5

1 .0

1 .5


OD

At

40

5n

m

Z y m o s a n , M A S P -2+ /+

se ru m Z y m o s a n , M A S P -2- /-

se ru m

A -Z 2 4 9 1 , M A S P -2+ /+

se ru m A -Z 2 4 9 1 , M A S P -2- /-

se ru m

B -M C 58 , M A S P -2+ /+

se ru m B -M C 58 , M A S P -2- /-

se ru m

N e g a t iv e C o n t ,M A S P - 2+ /+

s e ru m N e g a t iv e C o n t ,M A S P - 2-/-

s e ru m


specific condition (high serum dilution in BBS buffer) using MASP-2+/+

and MASP-2-/-

serum.

The different mouse sera were diluted in BBS binding buffer. Zymosan coating was used as a

positive control while the negative control wells received BSA blocking buffer as a coating. The

binding of the C3 cleavage product (C3c) was detected using rabbit anti-human C3c polyclonal

antibody (the data is presented as a mean of all three independent experiments with duplicate for

each +/- SEM).


75

Furthermore, C3 deposition assays were performed using MASP-2 sufficient and MASP-2

deficient mouse sera in high concentrations that allows all three pathways to work. In these

assays, high levels of C3 deposition were observed on the surface of Neisseria meningitidis

strains. However, in MASP-2 deficient serum there was more C3 deposition on the surface of

the Neisseria meningitidis strains tested compared to the MASP-2 sufficient serum (see Figures

3.9 and 3.10). This result supports previously published results, showing that the remaining

enzymes of the lectin pathway MASP-1 and/or MASP-3 can mediate C3 deposition through the

alternative pathway (Iwaki et al., 2011).

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6


OD

At

40

5n

m

Z y m o s a n , M A S P -2+ /+

s e ru m Z y m o s a n , M A S P -2- /-

se ru m

N e g a t iv e C o n t M A S P - 2-/-

s e ru mN e g a t iv e C o n t M A S P - 2+ /+

s e ru m

A -Z 2 4 9 1 , M A S P -2+ /+

s e ru m A -Z 2 4 9 1 , M A S P -2- /-

s e ru m

Figure 3.9 C3 deposition assay on the surface of Neisseria meningitidis strain A serogroup

Z2491 under alternative pathway permissive condition (high serum concentration in BBS

buffer) using sera of MASP-2+/+

and MASP-2-/-

mice. Different mouse sera were diluted in BBS

binding buffer. Zymosan coating was used as a positive control while the negative control wells

received BSA blocking buffer as a coating. The binding of the C3 cleavage product (C3c) was

detected using rabbit anti-human C3c polyclonal antibody (the data is presented as a mean of all

three independent experiments with duplicate for each +/- SEM).


76

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6


OD

At

40

5n

m

Z y m o s a n , M A S P -2+ /+

s e ru m Z y m o s a n , M A S P -2- /-

se ru m

B -M C 58 , M A S P -2+ /+

se ru m

N e g a t iv e C o n t M A S P - 2-/-

s e ru mN e g a t iv e C o n t M A S P - 2+ /+

s e ru m

B -M C 58 , M A S P -2- /-

se ru m

Figure 3.10 C3 deposition a assay on the surface of Neisseria meningitidis strain B serogroup

MC85 under alternative pathway permissive condition (high serum concentration in BBS

buffer) using sera of MASP-2+/+

and MASP-2-/-

mice. Different mouse sera were diluted in BBS

binding buffer. Zymosan coating was used as a positive control while the negative control wells


detected using rabbit anti-human C3c polyclonal antibody (the data is presented as a mean of all

three independent experiments with duplicate for each +/- SEM).

To further investigate the role of the different complement pathways in fighting

Neisseria meningitidis, C3 deposition assays were done on different Neisseria

meningitidis strains using wild-type serum, C1q deficient serum (lacking the classical

pathway), factor B deficient serum (lacking the alternative pathway) and MASP-2

deficient serum (lacking the lectin pathway) in high concentrations that allow all of the


77

three complement pathways to work. The highest level of C3 deposition was shown

with the MASP-2 deficient serum which could be mediated by the alternative and the

classical pathways. C1q deficient serum showed significant amounts of C3 deposition

which could have been mediated by both the lectin and the alternative pathways. Factor

B deficient serum showed a limited amount of C3 deposition which could have been

mediated by both the classical and the lectin pathways. These results suggested that the

alternative pathway plays an role in fighting Neisseria meningitidis (see Figures 3.11

and 3.12).

0 2 0 4 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

M A S P -2- /-

s e ru m

C 1q- /-

s e ru m

W t s e ru m

FB- /-

s e ru m



OD

At

40

5n

m


Z2491 under alternative pathway permissive conditions (high serum concentration in BBS

buffer) the presence of both Ca++

and Mg++

allows the activation through all the complement

pathways. Different mouse sera were diluted in BBS binding buffer. Zymosan coating was used

as a positive control while the negative control wells received BSA blocking buffer as a coating.

The binding of the C3 cleavage product (C3c) was detected by using rabbit anti-human C3c

polyclonal antibody (the data is presented as a mean of all three independent experiments with

duplicate for each +/- SEM).


78

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

M A S P -2- /-

s e ru m

C 1q- /-

s e ru m

W t s e ru m

FB- /-

s e ru m



OD

At

40

5n

m

Figure 3.12 C3 deposition assay on the surface of Neisseria meningitidis strain B serogroup

MC58 under alternative pathway permissive conditions (high serum concentration in BBS

buffer) the presence of both Ca++

and Mg++

allows the activation through all the complement

pathways. Different mouse sera were diluted in BBS binding buffer. Zymosan coating was used

as a positive control while the negative control wells received BSA blocking buffer as a coating.

The binding of the C3 cleavage product (C3c) was detected by using rabbit anti-human C3c

polyclonal antibody (the data is presented as a mean of all three independent experiments with

duplicate for each +/- SEM).

3.1.3 C4 deposition assays

The lectin pathway recognition molecules MBLs and CL-11 are able to bind to surface

of the Neisseria meningitidis. Therefore C4 deposition assays were performed to assess

the ability of Neisseria meningitidis to activate the lectin pathway by measuring the

amount of C4 deposited on the surface of Neisseria meningitidis strains. Wild-type

mouse serum was highly diluted (starting from 1/80) in MBL binding buffer which


79

dissociates the C1q complex through its high salt concentration and prevents

endogenous C4 from activation allowing only the lectin pathway to work. These assays

showed that high levels of C4 deposition were observed on Neisseria meningitidis

strains. Serogroup A-Z2491 showed more C4 deposition on its surface compared to

serogroup B-MC58 (see Figure 3.13).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6


OD

At

40

5n

m

Z y m o s a n

S e ro A -Z 2 4 9 1

S e ro B -M C 5 8


Figure 3.13 C4 deposition assay on the surface of different Neisseria meningitidis strains

under lectin pathway specific condition (high serum dilution in MBL binding buffer) allows the

activation of complement to occur through the lectin pathways. Mouse serum was diluted in

MBL binding buffer. Zymosan coating has used as a positive control while the negative control

wells received BSA blocking buffer as a coating. Human C4 proteins were added to wells after

washing the ELISA plates from the serum and incubated at 37°C for 90 minutes. C4 was then

detected by using chicken anti-human C4 antibody (the data is presented as a mean of all three



80

In order to investigate the role of the classical pathway in forming the C4 deposition on

the surface of Neisseria meningitidis, C4 deposition assay was done on both strains of

Neisseria meningitidis using wild-type serum and C1q deficient serum (lacking the

classical pathway). These assays showed that lack of a functional classical pathway had

a minimal (but not significant) effect on the formation of C4 deposition on both strains

of Neisseria meningitidis (see Figures 3.14 and 3.15).

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4


OD

At

40

5n

m

Z y m o sa n , W T s e ru m Z y m o sa n , C 1 q- /-

s e ru m

N e g a tiv e c o n t, W T se ru m N e g a tive co n t, C 1 q- /-

s e ru m

A -Z 2 4 9 1 , C 1 q- /-

s e ru mA -Z 2 4 9 1 , W T s e ru m


Z2491 under specific condition (high serum dilution in BBS buffer) using both WT and C1q-/-

serum. Different mice sera were diluted in BBS binding. Zymosan coating was used as a

positive control while the negative control wells received BSA blocking buffer as a coating.

Human C4 proteins were added to the wells after washing the ELISA plates and incubated at

37°C for 90 minutes. C4 was then detected by using chicken anti-human C4 antibody (the data

is presented as a mean of all three independent experiments with duplicate for each +/- SEM).


81

0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4


OD

At

40

5n

m

Z y m o sa n , W T s e ru m Z y m o sa n , C 1 q- /-

s e ru m

N e g a tiv e c o n t, W T se ru m N e g a tive co n t, C 1 q- /-

s e ru m

B -M C 5 8, C 1q- /-

s e ru mB -M C 5 8 , W T se rum


MC58 under specific condition (high serum dilution in BBS buffer) using both WT and C1q-/-

serum. Different mice sera were diluted in BBS binding. Zymosan coating was used as a

positive control while the negative control wells received BSA blocking buffer as a coating.

Human C4 proteins were added to the wells after washing the ELISA plates and incubated at

37°C for 90 minutes. C4 was then detected by using chicken anti-human C4 antibody (the data

is presented as a mean of all three independent experiments with duplicate for each +/- SEM).


82

3.1.4 Serum Bactericidal Assays

Serum bactericidal assays were done to assess the serum bacteriolytic activity of mice

sera against Neisseria meningitidis strains. In these assays, different mice sera, which

were deficient in one of the complement pathways (classical, alternative or lectin

pathways) were compared with the wild-type mice sera in terms of their ability to kill

Neisseria meningitidis strains.

C1q deficient serum (C1q-/-

), which is considered to be a classical pathway deficient

serum, was used to assess the role of the classical pathway in fighting the

meningococcus strains by comparing it to the wild-type (WT) control serum. This assay

showed no significant difference between the C1q deficient serum (classical pathway

deficient serum) and wild-type serum which suggests that the classical pathway might

plays a minimal role in fighting Neisseria meningitidis infection (see Figures 3.16 and

3.17). Additionally, it suggests that meningococcus could be killed through other

complement pathways (the alternative and lectin pathways).


83

0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

4 .8

M i n u t e s

log

cfu

/ml

HIS

W T

C 1q- /-

Figure 3.16 Bactericidal activity of different mouse sera (C1q-/-

, WT and HIS) against

Neisseria meningitidis serogroup A strain Z2491. Bacteria and sera (30% concentration) were

incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 And 90 minutes

and plated out and the viable count was calculated. No significant difference emerged between

the C1q-/-

serum and the WT serum. However, the difference between the C1q-/-

, WT and the

heated inactivated sera (HIS) which was used as a negative control, was significant (the data is


Table 3.1 Statistically significant differences between serum bactericidal assay of different

mouse sera (C1q-/-

, WT and HIS) against Neisseria meningitidis serogroup A strain Z2491 using

the‎Student’s‎t-test at time point 90 minutes.

Student’s‎t-test at time point 90 minutues

Significant? P > 0.05 P value Summary

WT vs C1q-/- NO Ns (0.4385)

WT vs HIS YES * (0.0154)

C1q-/-

vs HIS YES * (0.0133)


84

0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

M i n u t e s

log

cfu

/ml

HIS

W T

C 1q- /-

Figure 3.17 Bactericidal activity of different mouse sera (C1q-/- , WT and HIS) towards

Neisseria meningitidis serogroup B strain MC58. Bacteria and sera (30% concentration) were

incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 and 90 minutes and

plated out and the viable count was calculated. No significant difference emerged between the

C1q-/-

serum and the WT serum. However, the difference between the C1q-/-

, WT and the heated

inactivated sera (HIS) which was used as negative control, was significant (the data is presented

as a mean of all three independent experiments with duplicate for each +/- SEM).


mouse sera (C1q-/-

, WT and HIS) against Neisseria meningitidis serogroup B strain MC58 using

the‎Student’s‎t-test at time point 90 minutes.

Student’s‎t-test at time point 90 minutes


WT vs C1q-/- NO Ns (0.4385)


C1q-/-

vs HIS YES * (0.0289)


85

In order to assess the role of the alternative pathway in fighting Neisseria meningitidis,

Factor B deficient (FB-/-

) serum and MASP-1/3 deficient (MASP-1/3-/-

) serum, a

previous study by Takahashi et al (2010) had showed that MASP-1/3 deficient mice are

lacking the full function of alternative pathway, were used to assess the ability of these

sera to kill Neisseria meningitidis. The assays showed that unlike the wild-type serum,

neither sera (FB-/-

and MASP-1/3-/-

) has the ability to kill meningococcus,

demonstrating the important role of the alternative pathway in fighting Neisseria

meningitidis (see Figures 3.18 and 3.19).

0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

4 .8

M i n u t e s

log

cfu

/ml HIS

W T

FB- /-

M A S P -1 /3- /-


, MASP-1/3-/-

, WT and HIS)

against Neisseria meningitidis serogroup A strain Z2491. Bacteria and sera (30% concentration)

were incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 And 90

minutes and plated out and the viable count was calculated. The ability of FB-/-

and MASP-1/3-/-

deficient serum was disrupted compared to the WT serum. In this assay heated inactivated

serum (HIS) was used as a negative control (the data is presented as a mean of all three



86

Table 3.3 Statistically significant differences between the serum bactericidal assay of different

mouse sera (FB-/-

, MASP-1/3-/-

, WT and HIS) against Neisseria meningitidis serogroup A strain

Z2491‎using‎the‎Student’s‎t-test at time point 90 minutes.



WT vs FB-/- YES ** (0.0079)

WT vs MASP-1/3-/- YES ** (0.0055)

WT vs HIS YES ** (0.0066)

FB-/-

vs MASP-1/3-/-

NO Ns (0.9918)

FB-/-

vs HIS NO Ns (0.3578)

MASP-1/3-/-


0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

M i n u t e s

log

cfu

/ml

HIS

W T

FB- /-

M A S P -1 /3- /-


, MASP-1/3-/-

, WT and HIS)

against Neisseria meningitidis serogroup B strain MC58. Bacteria and sera (30% concentration)


minutes and plated out and the viable count was calculated. The ability of FB-/-

and MASP-1/3-/-

deficient serum was disrupted compared to the WT serum. In this assay heated inactivated




87

Table 3.4 Statistically significant differences between the serum bactericidal assay of different

mouse sera (FB-/-, MASP-1/3-/-, WT and HIS) against Neisseria meningitidis serogroup B

strain‎MC58‎using‎the‎Student’s‎t-test at time point 90 minutes.



WT vs FB-/- YES * (0.0218)

WT vs MASP-1/3-/- YES * (0.0230)


FB-/-

vs MASP-1/3-/-

NO Ns (0.8531)

FB-/-


MASP-1/3-/-


MASP-2 deficient serum (MASP-2-/-

) and MBL deficient serum, lectin pathway

deficient sera, were used to assess the role of the lectin pathway in fighting the

meningococcus strains by comparing them to the wild-type (WT) control serum. The

assays showed that the ability of the MBL deficient serum to kill Neisseria meningitidis

was impaired compared to the wild-type serum. This result is consistent with previous

reports which showed an association between MBL deficiency and Neisseria

meningitidis infection (Faber et al., 2007; Tully et al., 2006). In addition, the assays

showed that the ability of MASP-2 deficient serum to kill Neisseria meningitidis was

significantly higher compared to the wild-type serum which suggests that the functional

activity of the alternative pathway may compensate for the absence of the functional

activity of the lectin pathway (see Figures 3.120 and 3.21). This result is in the line with

previous results, which showed that MASP-2-/-

deficient serum showed high levels of

C3 deposition on the surface of meningococcus strains when used at a high


88

concentration possibly due to the presence of the alternative pathway that compensates

for the absence of lectin pathway (see Figures 3.9 and 3.10). This high level of C3

deposition was dramatically reduced when using a low serum concentration which

allows only the classical and lectin pathways to be active (see Figure 3.8).

0 2 0 4 0 6 0 8 0 1 0 0

3 .6

3 .8

4 .0

4 .2

4 .4

4 .6

4 .8

5 .0

M i n u t e s

log

cfu

/ml

HIS

W T

M A S P -2- /-

M BL- /-


, MBL-/-

, WT and HIS)

against Neisseria meningitidis serogroup A strain Z249. Bacteria and sera (30% concentration)


minutes and plated out and the viable count was calculated. The ability of MASP-2-/-

serum to

kill meningococcus was higher compared to wild-type serum. However the ability of MBL-/-

deficient serum was impaired. In this assay heated inactivated sera (HIS) was used as a negative

control (the data is presented as a mean of all three independent experiments with duplicate for

each +/- SEM).


89


mouse sera (MASP-2-/-

, MBL-/-

, WT and HIS) against Neisseria meningitidis serogroup A strain

Z2491‎using‎the‎Student’s‎t-test at time point 90 minutes.



WT vs MASP-2-/- YES * (0.0168)


WT vs MBL-/- YES * (0.0158)

MASP-2-/-

vs HIS YES ** (0.0053)

0 2 0 4 0 6 0 8 0 1 0 0

4 .0

4 .2

4 .4

4 .6

4 .8

M i n u t e s

log

cfu

/ml

HIS

W T

M A S P -2- /-

M BL- /-


, MBL-/-

, WT and HIS)

against Neisseria meningitidis serogroup B strain MC58. Bacteria and sera (30% concentration)


minutes and plated out and the viable count was calculated. The ability of MASP-2-/-

serum to

kill meningococcus was higher compared to wild-type serum. However the ability of MBL-/-

deficient serum was impaired. In this assay heated inactivated sera (HIS) was used as a negative

control (the data is presented as a mean of all three independent experiments with duplicate for

each +/- SEM).


90


mouse sera (MASP-2-/-, MBL-/-

, WT and HIS) against Neisseria meningitidis serogroup B

strain‎MC58‎using‎the‎Student’s‎t-test at time point 90 minutes.



WT vs MASP-2-/- YES * (0.0371)


WT vs MBL-/- YES * (0.0157)

MASP-2-/-

vs HIS YES ** (0.0091)

To study the role of the alternative pathway in bacteriocidal activity further, MASP-1/3

deficient serum which lacks functional activity of alternative pathway was used

(Takahshi et al., 2010), this showed no killing of Neisseria meningitidis (see Figures

3.18 and 3.19), following the addition of truncated recombinant of MASP-3 which

could restores the functional activity of the alternative pathway to kill Neisseria

meningitidis. The results from these assays showed that the ability to kill

meningococcus; and thus the activity of alternative pathway, was restored by the

addition of truncated recombinant MASP-3 (see Figures 3.22 and 3.23). This result is in

line with previous work by Iwaki et al., (2011) who suggested that MASP-3 of the

lectin pathway had the ability to induce the activity of the alternative pathway by

cleaving C4b bound factor B on the surface of pathogen, forming C3 convertase of

alternative pathway.


91

0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

M i n u t e s

log

cfu

/ml

HIS

W T

M A S P -1 /3- /-

+

rM A S P -3 (1 0 µ g /m l)

M A S P -1 /3- /-


+ truncated rMASP-3

(10µg/ml), MASP-1/3-/-

, WT and HIS) against Neisseria meningitidis serogroup A strain Z2491.

Bacteria and sera with or without truncated recombinant MASP-3 10ug/ml (30% serum

concentration) were incubated at 37°C with shaking. Samples were taken at time points 0, 30,

60 And 90 minutes and plated out and the viable count was calculated. The ability of MASP-

1/3-/-

deficient serum to kill Neisseria meningitidis; thus, the functional activity of the

alternative pathway was restored by adding 6ug/ml of truncated recombinant MASP-3 (rMASP-

3). In this assay heated inactivated serum (HIS) was used as a negative control (the data is

presented as a mean of all three independent experiments with duplicate for each +/- SEM)


mouse sera (MASP-1/3-/-



Neisseria meningitidis serogroup‎ A‎ strain‎ Z2491‎ using‎ the‎ Student’s‎ t-test at time point 90

minutes.



WT vs MASP-1/3-/- YES ** (0.0055)

WT vs MASP-1/3-/-

+rMASP-3 NO Ns (0.0513)


MASP-1/3-/-

vs MASP-1/3-/-

+ rMASP-3 YES * (0.0143)

MASP-1/3-/- vs HIS NO Ns (0.2025)

MASP-1/3-/-

+ rMASP-3 vs

HIS YES * (0.0277)


92

0 2 0 4 0 6 0 8 0 1 0 0

4 .4

4 .6

4 .8

M i n u t e s

log

c

fu

/m

l

HIS

W T

M A S P -1 /3- /-

+

rM A S P -3 (1 0 µ g /m l)

M A S P -1 /3- /-


+ truncated rMASP-3

(10µg/ml), MASP-1/3-/-

, WT and HIS) against Neisseria meningitidis serogroup B strain MC58.

Bacteria and sera with or without truncated recombinant MASP-3 10ug/ml (30% serum

concentration) were incubated at 37°C with shaking. Samples were taken at time points 0, 30,

60 And 90 minutes and plated out and the viable count was calculated. The ability of MASP-

1/3-/-

deficient serum to kill Neisseria meningitidis; thus, the functional activity of the

alternative pathway was been restored by adding 6ug/ml of truncated recombinant MASP-3

(rMASP-3). In this assay heated inactivated serum (HIS) was used as a negative control (the

data is presented as a mean of all three independent experiments with duplicate for each +/-

SEM).


93


mouse serum (MASP-1/3-/-



Neisseria meningitidis serogroup‎ B‎ strain‎ MC58‎ using‎ the‎ Student’s‎ t-test at time point 90

minutes.



WT vs MASP-1/3-/- YES * (0.0112)

WT vs MASP-1/3-/-

+rMASP-3 NO Ns (0.0559)


MASP-1/3-/-

vs MASP-1/3-/-

+ rMASP-3 YES * (0.0349)

MASP-1/3-/- vs HIS NO Ns (0.7007)

MASP-1/3-/-

+rMASP-3 vs

HIS YES * (0.0261)

Furthermore, the serum bactericidal assay was done using HIS serum, MBL-/-

serum,

NHS, immune serum and 3MC serum with or without truncated recombinant MASP-3

6µg/ml (this serum was obtained from a patient, with MASP-1 but not MASP-3, with a

disease called Carnevale, Mingarelli, Malpuech, Michels (3MC) syndrome) to study

their bacteriolytic activity against Neisseria meningitidis. This assay showed that the

ability of MBL deficient serum to kill Neisseria meningitidis was impaired compared to

normal human serum. Additionally this assay showed that the functional activity of the

alternative pathway was restored by the addition of truncated recombinant MASP-3,

which enhanced the killing of Neisseria meningitidis (see Figure 3.24 and 3.25). This

result is consistent with previous results, which showed the importance of the


94

alternative pathway in fighting Neisseria meningitidis through cleavage Factor D by the

action of the lectin pathway MASP-3 that leads to formation of the C3 alternative

convertase on the surface of Neisseria meningitidis.

0 3 0 6 0 9 0 1 2 0 1 5 0

1

2

3

4

5

6

M i n u t e s

log

cfu

/ml

HIS

Im m une

3 M C se rum (P a tie n t 2 )

3 M C se ru m (P a tie n t 2 ) +

rM A S P -3 (6 g/m l)

M BL- /-

NHS


(6µg/ml), 3MC, MBL-/-, Immune, NHS and HIS) against Neisseria meningitidis serogroup A

strain Z2491. Bacteria and sera with or without truncated recombinant MASP-3 6ug/ml (20%

serum concentration) were incubated at 37°C with shaking. Samples were taken at time points

0, 15, 30, 60 and 90 minutes and plated out and the viable count was calculated. The ability of

3MC serum to kill Neisseria meningitidis; thus, the functional activity of alternative pathway,

was restored by adding 6ug/ml of truncated recombinant MASP-3 (rMASP-3). Additionally it

showed that the ability of MBL-/-

deficient serum was impaired. In this assay heated inactivated




95


human sera (3MC + truncated rMASP-3 (6µg/ml), 3MC, MBL-/-

, Immune, NHS and HIS)

against Neisseria meningitidis serogroup A strain Z2491 using the Student’s‎t-test at time point

120 minutes.



3MC vs HIS YES * (0.0108)

NHS vs 3MC +rMASP-3 YES * (0.0443)

3MC vs 3MC + rMASP-3 YES * (0.0102)

NHS vs Immune YES * (0.0450)

0 3 0 6 0 9 0 1 2 0 1 5 0

0

1

2

3

4

5

6

M i n u t e s

log

cfu

/ml

HIS

3 M C (p a tie n t 2 )

NHS

3 M C (p a tie n t 2 ) +

rM A S P -3 (6 µ g /m l)

M BL- /-

Im m une


(6µg/ml), 3MC, MBL-/-

, Immune, NHS and HIS) against Neisseria meningitidis serogroup B

strain MC58. Bacteria and sera with or without truncated recombinant MASP-3 6ug/ml (20%

serum concentration) were incubated at 37°C with shaking. Samples were taken at time points

0, 15, 30, 60 And 90 minutes and plated out and the viable count was calculated. The ability of

3MC serum to kill Neisseria meningitidis; thus, the functional activity of alternative pathway,

was enhanced by adding 6ug/ml of truncated recombinant MASP-3 (rMASP-3). Additionally it

showed that the ability of MBL-/-

deficient serum was impaired. In this assay heated inactivated




96


human sera (3MC + truncated rMASP-3 (6µg/ml), 3MC, MBL-/-

, Immune, NHS and HIS)

against Neisseria meningitidis serogroup B strain MC58 using the Student’s‎t-test at time point

120 minutes.



Immune vs HIS YES ** (0.0078)

NHS vs Immune NO ns(0.0920)

NHS vs 3MC YES * (0.0351)

3MC vs 3MC + rMASP-3 YES ** (0.0084)

3MC vs Immune YES * (0.0121)


97

3.1.5 Effect of recombinant properdin on complement mediated

killing of Neisseria meningitidis

Properdin is a positive regulator of the alternative pathway activation and can bind to

and stabilize C3 convertase by increasing its half-life. In addition, it has an important

role in alternative pathway activation, as properdin deficiency in serum decreases the

ability of the serum to activate the alternative pathway. However adding properdin can

restore normal activation of alternative pathway (Schwaeble and Reid, 1999). In

addition, it has been claimed that properdin can bind directly to the bacterial surface and

enhance C3 deposition on the bacterial surface (Spitzer et al., 2007).

3.1.5.1 Recombinant properdin has the ability to enhance C3 deposition on the

surface of Neisseria meningitidis

In order to assess whether recombinant properdin has the ability to enhance the

formation of C3 deposition on the surface of Neisseria meningitidis, ELISA and FACS

analysis were used to measure C3 deposition on the surface of Neisseria meningitidis

under specific conditions that block all complement pathway activation except the

alternative pathway. Both ways of measurement showed recombinant properdin could

enhance the alternative C3 deposition on the surface of Neisseria meningitidis (see

Figures 3.26, 3.27 and 3.28).


98

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

1 .8


OD

At

40

5n

m

Z y m o s a n , w t Z y m o s a n , w t + rP ro p e rd in (5 µ g /m l)

S e ro A -Z 2 4 9 1 , w t S e ro A -Z 2 4 9 1 , w t + rP ro p e rd in (5 µ g /m l)

N e g a tiv e C o n t, w t N e g a t iv e C o n t, w t + rP ro p e rd in (5 u g /m l)


Z2491 under alternative pathway permissive conditions (high serum concentration in EGTA

buffer). Mouse serum was diluted in EGTA buffer with or without recombinant properdin

(5µg/ml). Zymosan coating was used as a positive control while the negative control wells


detected by using rabbit anti-human C3c polyclonal antibody (the data is presented as a mean of

all three independent experiments with duplicate for each +/- SEM).


99

0 1 0 2 0 3 0 4 0 5 0 6 0

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

1 .2

1 .4

1 .6

1 .8

2 .0


OD

At

40

5n

m

Z y m o s a n , w t Z y m o s a n , w t + rP ro p e rd in (5 µ g /m l)

S e ro B -M C 5 8 , w t S e ro B -M C 5 8 , w t + rP ro p e rd in (5 µ g /m l)

N e g a tiv e C o n t, w t N e g a t iv e C o n t, w t + rP ro p e rd in (5 u g /m l)


MC58 under alternative pathway permissive conditions (high serum concentration in EGTA

buffer). Mouse serum was diluted in EGTA buffer with or without recombinant properdin

(5µg/ml). Zymosan coating was used as a positive control while the negative control wells


detected by using rabbit anti-human C3c polyclonal antibody (the data is presented as a mean of

all three independent experiments with duplicate for each +/- SEM).


100

Figure 3.28 FACS analysis of C3 deposition on the surface of Neisseria meningitidis

serogroup B strain MC58 using human and mouse serum with or without recombinant

properdin. Human serum (5% NHS) and mouse serum (15% WT) were diluted in EGTA buffer

(that blocks all complement pathways except the alternative pathway) to 1×105

Neisseria

meningitidis B-MC58 with or without recombinant properdin. C3 deposition was detected using

FITC-conjugated rabbit anti human C3c (Dako). Purple represents un-opsonised bacteria; green

represents opsonised bacteria with serum; red represents opsonised bacteria with serum plus

recombinant properdin (5µg/ml). (A) Neisseria meningitidis B-MC58 with NHS with or without

recombinant human properdin. (B) Neisseria meningitidis B-MC58 with WT serum with or

without recombinant murine properdin.

3.1.5.2 Recombinant properdin has the ability to enhance the killing of Neisseria

meningitidis

Building on the previous results, which supported the ability of recombinant murine

properdin to enhance the formation of C3 deposition, serum bactericidal assays were done

to assess whether properdin has the ability to enhance the bacteriolytic activity of serum.

These assays showed that recombinant murine properdin has the ability to enhance the

bacteriolytic activity of the serum against Neisseria meningitidis (see Figures 3.29 and

3.30).


101

0 2 0 4 0 6 0 8 0 1 0 0

3 .6

3 .9

4 .2

4 .5

4 .8

M i n u t e s

log

cfu

/ml

H IS + rP ro p e rd in (5 µ g /m l)

W T

W T + rP ro p e rd in (5 µ g /m l)

HIS

Figure 3.29 Bactericidal activity of mouse serum with or without recombinant murine properdin against

Neisseria meningitidis serogroup A strain Z2491. Bacteria and serum with or without recombinant murine

properdin 5µg/ml (30% serum concentration) were incubated at 37°C with shaking. Samples were taken

at time points 0, 30, 60 and 90 minutes and plated out and the viable count was then calculated. It was

shown that the ability of recombinant murine properdin to kill Neisseria meningitidis was enhanced by

adding 5ug/ml of recombinant properdin, which indicates that properdin enhanced the functional activity

of the alternative pathway. In this assay heated inactivated serum (HIS) was used as a negative control

(the data is presented as a mean of all three independent experiments with duplicate for each +/- SEM).

Table 3.11 Statistically significant differences between serum bactericidal assay of mouse serum with or

without recombinant murine properdin against Neisseria meningitidis serogroup A strain Z2491 using the

Student’s‎t-test at time point 90 minutes.



WT vs WT + rProperdin YES * (0.0221)


WT vs HIS + rProperdin YES *(0.0222)

WT + rProperdin vs HIS YES ** (0.0059)

WT + rProperdin vs HIS +

rProperdin YES **(0.0051)

HIS + rProperdin vs HIS NO Ns(0.5536)


102

0 2 0 4 0 6 0 8 0 1 0 0

2 .0

2 .4

2 .8

3 .2

3 .6

4 .0

4 .4

4 .8

M i n u t e s

log

cfu

/ml

HIS

W T

W T + rP ro p e rd in (5 µ g /m l)



properdin against Neisseria meningitidis serogroup B strain MC58. Bacteria and serum with or

without recombinant murine properdin 5µg/ml (30% serum concentration) were incubated at

37°C with shaking. Samples were taken at time points 0, 30, 60 and 90 minutes and plated out

and the viable count was then calculated. It was shown that the ability of recombinant murine

properdin to kill Neisseria meningitidis was enhanced by adding 5ug/ml of recombinant

properdin, which indicates that properdin enhanced the functional activity of the alternative

pathway. In this assay heated inactivated serum (HIS) was used as a negative control (the data is


Table 3.12 Statistically significant differences between serum bactericidal assay of mouse serum with or

without recombinant murine properdin against Neisseria meningitidis serogroup B strain MC58 using the

Student’s‎t-test at time point 90 minutes.



WT vs WT + rProperdin YES * (0.0404)


WT vs HIS + rProperdin YES ** (0.0084)

WT + rProperdin vs HIS YES ** (0.0042)

WT + rProperdin vs HIS +

rProperdin YES **(0.0039)



103

To further study the ability of recombinant properdin to enhance the killing of Neisseria

meningitidis, serum bactericidal assays were done against meningococcus using human

serum with or without recombinant human properdin. The results of these assays

showed that the ability of serum to kill Neisseria meningitidis was enhanced by adding

recombinant properdin (see Figures 3.31 and 3.32). The results support the earlier

finding that demonstrated the important role of the alternative pathway in fighting

Neisseria meningitidis.

0 2 0 4 0 6 0 8 0 1 0 0

1

2

3

4

5

6

HIS


N H S

N H S + rP ro p e rd in (5 µg /m l)

M i n u t e s

log

cfu

/ml


properdin against Neisseria meningitidis serogroup A strain Z2491. Bacteria and serum with or

without recombinant human properdin 5µg/ml (20% serum concentration) were incubated at

37°C with shaking. Samples were taken at time points 0, 30, 60 and 90 minutes and plated out

and the viable count was then calculated. It was shown that the ability of recombinant human

properdin to kill Neisseria meningitidis was enhanced by adding 5ug/ml of recombinant

properdin, which indicates that properdin enhanced the functional activity of the alternative

pathway. In this assay heated inactivated serum (HIS) was used as a negative control (the data is



104

Table 3.13 Statistically significant differences between serum bactericidal assay of


meningitidis serogroup‎ A‎ strain‎ Z2491‎ using‎ the‎ Student’s‎ t-test at time point 90

minutes.



NHS vs NHS +

rProperdin YES * (0.0453)

NHS vs HIS YES * (0.0107)

NHS vs HIS + rProperdin YES *(0.0144)

NHS + rProperdin vs HIS YES ** (0.0050)

NHS + rProperdin vs HIS

+ rProperdin YES **(0.0063)



105

0 2 0 4 0 6 0 8 0 1 0 0

2 .0

2 .5

3 .0

3 .5

4 .0

4 .5

5 .0

M i n u t e s

log

cfu

/ml


N H S + rP ro p e rd in (5 µg /m l)

NHS

HIS


properdin against Neisseria meningitidis serogroup B strain MC58. Bacteria and serum

with or without recombinant human properdin 5µg/ml (20% serum concentration) were

incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 and 90

minutes and plated out and the viable count was then calculated. It was shown that the

ability of recombinant human properdin to kill Neisseria meningitidis was enhanced by

adding 5ug/ml of recombinant properdin, which indicates that properdin enhanced the

functional activity of the alternative pathway. In this assay heated inactivated serum

(HIS) was used as a negative control (the data is presented as a mean of all three



106

Table 3.14 Statistically significant differences between serum bactericidal assay of


meningitidis serogroup‎ B‎ strain‎ MC58‎ using‎ the‎ Student’s‎ t-test at time point 90

minutes.



NHS vs NHS + rProperdin YES * (0.0383)

NHS vs HIS YES * (0.0334)

NHS vs HIS + rProperdin NO *(0.0427)

NHS + rProperdin vs HIS YES ** (0.0052)

NHS + rProperdin vs HIS +

rProperdin

YES **(0.0065)



107

3.2 Discussion


fighting against invading microbes including Neisseria meningitidis. This important role

has been illustrated in many published reports (Finne et al., 1987; Estabrook et al.,

1997; Rossi et al., 2001; Jarva et al., 2005). Therefore deficiency in complement

components increases the susceptibility to Neisseria meningitidis infection (Trouw &

Daha, 2011; Rosa et al., 2004; Rossi et al., 2001). For example, increased susceptibility

to Neisseria meningitidis infection is associated with deficiency of terminal pathway

components (C5, C6, C7, C8, or C9) or alternative pathway components such as factor

D and properdin (Morgan and Walport, 1991).

Previous work had shown that MASP-2 deficient serum had a higher bactericidal

activity against Neisseria meningitidis than NHS which correlated with a better survival

of MASP-2 deficient mice in an Neisseria meningitidis infection study compared to

wild-type mice (Hayat 2012). Therefore, this work aimed to explain this finding by

emphasizing the crucial role of the alternative pathway in fighting Neisseria

meningitidis and to define the role of the MASP-3 lectin pathway in activating the

alternative pathway of the complement system and therefore in improving resistance to

Neisseria meningitidis. To achieve this aim, several complement specific in vitro assays

(Chapter 3) were carried out which were followed by in vivo infection experiments

(Chapter 4) in a mouse model of Neisseria meningitidis infection. The mouse model

was a unique mouse strain with a gene-targeted disruption of the murine factor B gene

or MASP-1/3 gene that blocked the activity of the alternative complement pathway

(Takahashi et al., 2010).


108

3.2.1 Binding of Neisseria meningitidis to different complement

recognition molecules

The activation of complement pathways is dependent on initial binding of complement

recognition molecules to the surface of microbes (Carroll and Sim, 2011). Therefore,

ELISA assays were done in order to determine which murine complement recognition

molecules, C1q of the classical pathway and MBL-A, MBL-C, Cl-11 and ficolin-A of

Lectin pathway, bind to the surface of Neisseria meningitidis.

The activation of the classical pathway starts once C1q binds indirectly to the Fc region

of immunoglobulins IgG or IgM or directly to the surface of bacteria (Wallis et al.

2007). The ELISA based binding assays that were done showed that the C1q molecule

had the ability to bind to both strains of Neisseria meningitidis (see Figure 3.1). Thus, in

order to assess the importance of this binding in activating the classical pathway on the

surface of Neisseria meningitidis, a C4 deposition assay using C1q deficient serum and

wild-type serum was used. This assay showed that the absence of C1q and therefore the

classical pathway from the serum had a minimal (but not significant) effect on the

amount of the C4 deposition on the surface of Neisseria meningitidis (see Figures 3.14

and 3.15). This finding could be explained by the source of the serum that was used in

this assay. This serum was collected from mice that had not been exposed to Neisseria

meningitidis before; therefore, it did not have any specific antibodies against Neisseria

meningitidis that could drive the activation of the classical pathway on the surface of

Neisseria meningitidis. This finding is line with previous work (Agarwal et al., 2014)

that there was minimal C4 deposition on the surface of Neisseria meningitidis following

incubation of Neisseria with C1q and C4 only, as the activation of the classical pathway


109

on the surface of Neisseria meningitidis was dependent on the presence of specific

antibodies.

MBL-A and MBL-C are mouse lectin pathway carbohydrate recognition molecules

which could initiate the activation of the lectin pathway by binding to the surface of

Neisseria meningitidis. Therefore in order to assess whether these recognition molecules

have the ability to bind to Neisseria meningitidis, ELISA based binding assays were

done. These assays showed that both murine MBL-A and MBL-C bind to the surface of

the both strains of Neisseria Meningitidis (see Figures 3.2 and 3.3). This result

supported previous work by Van Emmerik et al., (1994) using radiolabelled human

mannan binding protein which showed binding of human MBL to unencapsulated

Neisseria meningitidis, intermediate binding to Neisseria meningitidis serogroup A and

weak binding to other Neisseria meningitides strains. Additionally Jack et al. (2001)

supported this result and showed that MBL has the ability to bind to encapsulated

Neisseria meningitidis serogroup C. Another study carried out by Kuipers et al. (2003)

showed high binding of MBL to Neisseria meningitidis serogroup B and C.

Ficolins are lectin carbohydrate recognition molecules mostly present in serum that

have ability to bind to N-acetyl glucosamine sugars and lipoteichoic acids of bacteria

(Garred et al., 2009; Endo et al., 2005). While three types of ficolin exist in humans,

mice have only two types of ficolin: ficolin-A and ficolin-B. Although both ficolins

have almost similar structure, ficolin-A, is the only ficolin in mice that has the ability to

activate the lectin pathway by binding to MASP-2 (Runza et al., 2008). Thus, in order

to assess whether mouse ficolin-A can bind to Neisseria meningitidis a binding assay


110

based on ELISA showed that ficolin-A has no ability to bind to either strains of

Neisseria meningitidis; thus, it is unable to activate the lectin pathway on the surface of

Neisseria meningitidis (see Figure 3.5).

Collectin 11 is one of the lectin recognition molecules that can bind to D-mannose and

L-fucose terminal saccharides on the surface of microbes. Collectin 11 has the ability to

activate the lectin pathway by interacting with MASP-1 and MASP-3 (Hansen et al.,

2010). Therefore, an ELISA based binding assay was used to assess the ability of

Collectin 11 to bind to the surface of Neisseria meningitidis. This assay showed that

Collectin 11 had the ability to bind to both strains of Neisseria meningitidis (see Figure

3.4).

Following the results of the binding assays of lectin pathway recognition molecules a

C4 deposition assay was used to assess the role of the independent lectin pathway in the

activation of the complement system on the surface of strains of Neisseria meningitidis.

Mouse serum was diluted in MBL binding buffer in order to dissociate the C1q complex

and inhibit the classical pathway. The result of this study showed high levels of C4

deposition on the surface of tested strains of Neisseria meningitidis (see Figure 3.13).

This result is compatible with previously published results, which reported that a

significant amount of C4 was found on the surface of Neisseria meningitidis following

incubation with whole blood. This deposition of C4 can be reduced by using

monoclonal anti-MBL antibodies. The inhibition of the amount of C4 deposited on the

surface of Neisseria meningitidis confirms that C4 deposition occurred only via the

lectin pathway (Sprong et al., 2003).


111


meningitidis requires a close cooperation between the lectin and

alternative pathway

The ability of Neisseria meningitidis to activate the complement system was studied

using several in vitro assays, which showed that both the strains of Neisseria

meningitidis used throughout this study had the ability to activate the complement

system mainly through the lectin and alternative pathways (see Figures 3.6 and 3.7).

This finding is line with previous reports (Jarvis, 1994; Vogel et al., 1997) that show an

ability of Neisseria meningitidis to activate the complement system as determined by

measuring the C3 deposition on the surface of Neisseria meningitidis.

In order to investigate the role of the different complement pathways in complement

activation by Neisseria meningitidis, wild-type serum and MASp-2 deficient serum

lacking the lectin pathway was used to assess the role of the lectin pathway leading to

C3 deposition on the surface of Neisseria meningitidis. This assay was carried out under

conditions (i.e. high serum dilution in Barbital buffer saline buffer) that allowed the

activation to occur only through the classical and lectin pathways. The assay showed

impairment of the ability of MASP-2 deficient serum to allow C3 deposition on the

surface of both tested strains of Neisseria meningitidis, which suggested that there is an

important role of the lectin pathway C3 deposition on the surface of Neisseria

meningitidis (see Figure 3.8) Following this result the same assay was repeated with a

high serum concentration in BBS buffer which allows functional activity of all three

complement pathways. Surprisingly, the results showed an increase in the amount of C3


112

deposition on Neisseria meningitidis with MASP-2 deficient serum than with wild-type

serum (see Figures 3.9 and 3.10). This observation suggested that alternative pathway

may provide compensation for the absence of the lectin pathway (MASP-2) through the

remaining lectin pathway molecules MASP-1 and/or MASP-3 that could also drive the

alternative pathway and therefore form C3 deposition on the surface of Neisseria

meningitidis. This result is in line with previous work by Iwaki et al., (2011) who

suggested that MASP-3 of lectin pathway has the ability to induce the activity of the

alternative pathway on the surface of Staphylococcus aureus by cleaving Factor D and

therefore form C3 convertase via the alternative pathway.

To further analyse these results, C3 deposition assays were done on both tested strains

of Neisseria meningitidis using different mice sera (wild-type, C1q deficient, FB

deficient and MASP-2 deficient) under conditions (high serum concentration in BBS

buffer) that allow all three complement pathways to work. These assays showed

different levels of C3 deposition on the surface of both strains of Neisseria meningitidis.

The lowest level of the C3 deposition on both strains came from factor B deficient

serum presumably because it lacks a functionally active alternative pathway which

could then have contributed to both classical and lectin pathway activation. It also

suggests that the alternative pathway plays an important role in inducing C3 deposition

on the surface of the Neisseria meningitidis strains tested. On the other hand, C3

deposition on the surface of Neisseria meningitidis was minimally affected compared to

wild-type serum by the use of C1q deficient serum lacking classical pathway functional

activity suggesting that the classical pathway plays a minimal role in forming the

initiation of C3 deposition on the surface of Neisseria meningitidis. Additionally, it


113

emphasized the important role of the other two complement pathways, alternative and

lectin pathways, which contributed to the formation of the C3 deposition on the surface

Neisseria meningitidis. The minimal role of the classical pathway could be explained by

the source of the serum, which came from mice that had not been exposed to Neisseria

meningitidis before; therefore, they did not have any specific antibodies against

Neisseria meningitidis that could drive the activation of the classical pathway on the

surface of Neisseria meningitidis. This finding is in line with previous work (Agarwal et

al., 2014) that showed minimal activation of classical pathway on the surface Neisseria

meningitidis when incubating the Neisseria with C1q and C4 only, as the activation of

the classical pathway on the surface of Neisseria meningitidis is dependent on

antibodies.

However the highest level of the C3 deposition was seen with MASP-2 deficient serum,

which lacks the action of the lectin pathway and may have enhanced activation of the

alternative pathway. The results using the MASP-2 deficient serum suggest that the

remaining lectin pathway molecules, i.e. MASP-1and/or MASP-3, could drive the

alternative pathway and therefore form C3 deposition on the surface of Neisseria

meningitidis. This result supports the previous work by Bjerre et al., (2011) who studied

the activation of the complement system on the surface of Neisseria meningitidis using

a whole blood model and he reported that the activation of the complement system on

the surface of Neisseria meningitidis was mainly dependent on the lectin and alternative

pathways (see Figures 3.11 and 3.12).


114

To conduct further analysis, serum bactericidal assays were used to measure the lytic

activity of serum against Neisseria meningitidis. In these assays different mice sera

(wild-type, C1q deficient, factor B deficient, Masp-1/3 deficient and MASP-2 deficient)

were used under conditions that did not block any of the three complement pathways.

The results of these assays were consistent (and in agreement) with the previous results

of C3 deposition assays and demonstrated that the absences of the classical pathway, i.e.

C1q deficiency, had minimal effect on the lytic activity of C1q deficient serum due to

the presence of the other two (alternative and lectin) complement pathways (see Figures

3.16 and 3.17). However, in the absence of the lectin pathway, i.e. MASP-2 deficiency,

there was an increase in the lytic activity of the MASP-2 deficient serum (see Figures

3.20 and 3.21), which could be explained by the presence of the lectin pathway

molecules MASP-1 and/or MASP-3 that could also drive activation of the alternative

pathway (Iwaki et al., 2011). In addition to that it demonstrates that the MASP-2 lectin

pathway does not seem to play a major role in the lysis of Neisseria meningitides and

confirms previous work carried out in our lab (Hayat, 2012).

The important role of the alternative pathway was highlighted by using different

alternative pathway deficient sera (factor B deficient and MASP-1/3 deficient) which

showed that even in the presence of the other two complement pathways, classical and

lectin pathways, the lytic activity of the sera was inefficient due to the deficiency of the

alternative pathway (see Figures 3.18 and 3.19). This result is in agreement with

previous reports (Biesma et al., 2001; Hiemstra et al., 1989; Sprong et al., 2006) that

showed an association between alternative pathway deficiency and Neisseria

meningitidis infection. To further study whether the impairment of alternative pathway


115

lytic activity was due to deficiency of MASP-3, experiments were undertaken to restore

alternative pathway by the addition of truncated recombinant MASP-3 to the MASP-1/3

deficient sera. Serum bactericidal assays using MASP-1/3 deficient serum with and

without truncated recombinant murine MASP-3 showed that the lytic activity of

alternative pathway against both strains of Neisseria meningitidis was completely

restored following the addition of truncated recombinant murine MASP-3 (see Figures

3.22 and 3.23). In addition to this, it highlighted the importance of the alternative

pathway in fighting Neisseria meningitidis infection. This result supported previous

work (Takahshi et al., 2010) which showed that MASP-1/3 deficient mice lack

alternative pathway activity. This is also consistent with Iwaki et al., (2011) who

showed that MASP-3 of lectin pathway has the ability to induce the activity of the

alternative pathway by cleaving C4b bound factor B on the surface of Staphylococcus

aureus.

To further analyse this, the bacteriolytic activity of human serum from a patient with

Carnevale, Mingarelli, Malpuech and Michels syndrome (3MC) - these patients have

MASP-1 but not MASP-3 (Rooryck et al., 2011). The mutation in MASP-3 makes it

dysfunctional. This serum showed lytic activity toward Neisseria meningitidis

serogroup B strain MC58, but not to serogroup A strain Z2491, which could be due to

the presence of antibodies against Neisseria meningitidis in the serum, which binds to

the surface and then activates the classical pathway. In addition to this, restoring the

activity of the alternative pathway by adding truncated recombinant MASP-3 enhanced

the lytic activity of the serum (see Figures 3.24 and 3.25). This finding supports the

previous results which showed that MASP-3 was crucial in driving the activity of the


116

alternative pathway and therefore enhancing the lytic activity of the serum toward the

Neisseria meningitidis, as well as the role of alternative pathway in fighting Neisseria

meningitidis.

Additionally, other in vitro assays demonstrated the importance of the lectin pathway

molecule MBL in fighting Neisseria meningitidis. This was demonstrated when testing

the bacteriolytic activity of MBL deficient serum against Neisseria meningitidis, which

showed that the ability of mouse MBL deficient serum to kill Neisseria meningitidis

was impaired compared to mouse MBL sufficient serum (see Figures 3.20 and 3.21).

The same observation was also seen with human serum, where the ability of human

MBL deficient serum to lyse Neisseria meningitidis was impaired compared to normal

human serum (see Figures 3.24 and 3.25). These results are in agreement with previous

results (Hibberd et al., 1999) which showed the important role of MBL in fighting

Neisseria meningitidis infection.

Taken together, the results strongly suggest that both lectin pathway molecules, i.e.

MBL and MASP-3, play crucial roles in mediating the lytic activity toward Neisseria

meningitidis. This observation is in line with Iwaski et al.’s‎(2011)‎report‎which‎showed‎

the complex of MASP-3 and MBL was able to trigger the activation of the alternative

pathway on the surface of Staphylococcus aureus by cleaving the proenzymes

C3(H2O)B or C3bB to their enzymaticly active forms. Moreover, the findings presented

in this chapter and in previously publisheded work (Schwaeble et al., 2011)

demonstrates that the two effector arms of lectin pathway, which are:


117

1) Lectin pathway MASP-2 drives the activation of the lectin pathway throughout the

formation of C3 and C5 convertase (Schwaeble et al., 2011)

2) Lectin pathway MASP-3 drives the activation of the alternative pathway throughout the

cleavage of the alternative pathway C3b bound factor B which forms alternative

pathway C3 convertase.

Figure 3.33 Lectin pathway effector arms. (Figure courtesy of Prof. Wilhelm

Schwaeble, University of Leicester, Leicester, UK).


118

3.2.3 Recombinant properdin enhances the serum bacteriolytic

activity against Neisseria meningitidis

Properdin is a glycoprotein known as positive regulator of complement activation. It

works to stabilize the C3 convertase C3bBb on the pathogen through increasing its half-

life. This function (stabilizing C3 convertase) can be done in two ways. One of them is

by binding to the C3 convertase on the surface of the pathogen. The other way is

through binding to the surface of the pathogen and then starting the formation of the

alternative pathway C3 convertase (Schwaeble and Reid, 1999). Kemper et al., (2008)

claimed that properdin could initiate alternative pathway activation by working as a

pattern recognition molecule. However, Harboe et al., (2012) studied the binding of

properdin to the pathogen surface and found that the binding between properdin and the

pathogen surface could not be found in the absence of C3 deposition indicating that this

binding does not initiate the alternative pathway. The association between properdin

deficiency and Neisseria meningitidis infection has been reported previously,

illustrating the important role of properdin in fighting Neisseria meningitidis infection

(Fijen et al., 1995).

Therefore, in order to assess the effect of properdin on the formation of C3 deposition

and thus the lytic activity of the serum, the effect of adding recombinant properdin to

serum on the formation of C3 deposition was examined by measuring the amount of the

C3 deposition on the surface of Neisseria meningitidis using both ELISA assay and

FACS analysis. The results of these assays showed an increased amount of C3

deposition on the surface of the tested Neisseria meningitidis strains following the

addition of recombinant properdin (see Figures 3.26, 3.27 and 3.28). This finding is


119

consistent with that of Agarwal et al., (2010) who reported an increase in C3 deposition

on the surface of Neisseria meningitidis that were pre-incubated with properdin,

followed by the addition of properdin depleted serum.

To further analyse and confirm the important role of properdin in enhancing the serum

bactericidal activity against Neisseria meningitidis. It was shown that serum bactericidal

activity of human or mouse was enhanced significantly by adding recombinant

properdin compared to normal serum alone (See Figures 3.29, 3.30, 3.31 and 3.32). This

result further demonstrated the important role of the alternative pathway in fighting


In conclusion, the in vitro study demonstrated the important roles of the lectin and the

alternative pathways in fighting Neisseria meningitidis infection. It also highlighted the

major role of the lectin pathway complex molecule i.e. MASP-3 and MBL, in driving

the activation of the alternative pathway on the surface of Neisseria meningitidis

through the cleavage of the alternative pathway zymogen pro-factor D to Factor D

which is essential to allow C3bB complexes to be converted into the alternative

pathway C3 convertase complex C3bBb in a factor D dependent fashion. The

amplification loop that the alternative pathway provides to generate optimal activation

rates for the formation of MAC complexes appears to be critical in allowing an effective

lysis of Neisseria meningitidis. This is in line with the observation that serum

bactericidal activity against Neisseria meningitidis can be significantly increased

through the addition of highly active, high grade polymerised recombinant properdin

which in turn increases the half-live of the alternative pathway C3 and C5 convertase


120

complexes by protecting C3b within these complexes from the decay activity of serum

factor H and other cofactors, like DAF or MCP. Thus the promotion of alternative

pathway functional activity was shown to enhance the ability of mice to fight Neisseria

meningitidesinfection.

Chapter 4: In vivo study

121


The current in vitro study of the alternative pathway against Neisseria meningitidis

shows that a limited level of C3 deposition was observed following the use of

alternative pathway deficient serum compared to wild-type serum. Furthermore, the

serum bactericidal assay of sera from factor B deficient mice and MASP-1/3 deficient

mice showed no killing of Neisseria meningitidis compared to the wild-type serum.

These results led to do in vivo infection experiments in a mice model of Neisseria

meningitidis infection in Factor B deficient mice and MASP-1/3 deficient mice which

provide a mice model with a total defect in the alternative pathway.

4.1 Results:

4.1.1 Genotyping of factor B deficient mice and MASP-1/3 deficient

mice

All gene-targeted complement deficient mouse lines, i.e. factor B deficient mice and

MASP-1/3 deficient mice that have been used throughout this study were bred and

housed in the Central Research Facility at the University of Leicester which allows to

breed mice in a high quality pathogen free environment.

The alternative pathway Factor B molecule is a single glycoprotein that upon activation

is attached to C3b cleaved by Factor D into two fragments, the major Bb fragment that

is composed of a von Willebrand factor domain and the serine protease domain and the


122

Ba fragment (composed of 3 CCP domains and a spacer fragment (alpha-L). While the

Bb fragment remains bound to C3b to form the alternative pathway C3 convertase

C3bBb, the Ba fragment is released and does not contribute to further complement

activation processes. (Mole et al. 1984, Williams et al. 1999). This molecule is encoded

by the complement Factor B gene which consists of 18 exons. Targeting exons 4-9

disrupted the Factor B gene resulting a complete deficiency of Factor B (Matsumoto et

al., 1997).

The results of the PCR show the amplified products which are a single band at

approximately 986bp identified as wild-type allele (wild-type mouse FB gene), a single

band at approximately 650bp identified as the gene targeted allele (factor B deficient

allele). If both bands of approximately 986bp and 650bp were amplified on DNA of our

FB line, they indicate that these mice are heterozygous containing both the WT and the

targeted FB alleles (heterozygous mice) (see Figure 4.1).


123

Figure 4.1 Genotyping results for the FB targeted mouse line showed the amplified products

which are: single band at approximately 986bp which identifies the wild-type allele and a single

band at approximately 650bp which identifies the Factor B targeted allele. Dtecting both bands

of approximately 234bp and 453bp indicated that the DNA was isolated from heterozygous

mice. The DNA ladder, 1Kbp plus, was run with the PCR samples in a 1% agarose TAE-gel.

The lectin pathway serine proteases MASP-1 and MASP-3 and third a gene

product without a serine protease domain, called MAp-44 are encoded by the

MASP1 gene located on mouse chromosome 16. This gene is composed of 18

exons that encode the heavy and light chains of MASP-1 and MASP-3. Exons 1-

11 are encoding the heavy chain shared by MASP-1, MASP-3 and Map-44. While

exon 12 encodes the light chain of MASP-3 (the serine protease domain of MASP-

3), the exons 13-18 encode the light chain or serine protease domain of MASP-1.

In order to target the MASP1 gene of the MASP-1/3 deficient mouse line used in


124

my study, exon 2 of the MASP-1 gene was replaced with a neomycin cassette

which disrupted the open reading frame shared between all three gene products

resulting a complete deficiency of MASP-1, MASP-3 and MAp-44 (Takada et al.

1995; Takahashi et al. 2008). The results of the PCR showed the amplified

products, specifically a single band at approximately 539bp identified as wild-type

allele (wild-type mice), a single band at approximately 639bp identified as the

gene targeted allele (in MASP-1/3 deficient mice) and amplifying two bands of

approximately 539bp and 639bp indicated that the DNA used as a template for the

genotyping came from a heterozygous mouse bearing both a WT and a disrupted

allele (see Figure 4.2).

Figure 4.2 Genotyping results for MASP1/3 mice showed the amplified products which are: a

single band at approximately 539bp identified the wild-type allele, a single band at

approximately 639bp identified the MASP-1/3 targeting allele. Two bands at approximately

539bp and 639bp identified the template DNA to come from a heterozygous mouse. The DNA

ladder, 1Kbp, was run with samples in a 1% agarose TAE-gel.


125

4.1.2 The optimal infective dose

In order to optimize the infective dose of Neisseria meningitidis serogroup B strain

MC58 for infection experiments, three groups of mice (all on C57BL/6 background)

aged between 10-13 weeks were challenged with different doses via intra-peritoneal

injection. The infective doses were added after adding iron to a final concentration of

400mg/kg body weight 12 hours before the infection dose (see Figure 4.3).

0 1 2 2 4 3 6 4 8 6 0 7 2 8 4

0

2 0

4 0

6 0

8 0

1 0 0

T im e p o s t in fe c t io n ( h r s )

% S

urv

iva

l

1 1 05 C FU /m ou se



Figure 4.3 Survival of C57BL/6 wild-type mice following an i.p. injection with different

doses of N.meningitidis serogroup B strain MC58 to evaluate the optimal dose. The infective

doses were given after pre-dosing the mice with iron (given at a final concentration of

400mg/kg body weight). The experiment included five mice per group.


126

4.1.3 Survival of factor B deficient mice and factor B sufficient mice

following experimental Neisseria meningitidis infection

The host immune response to Neisseria meningitidis infection in terms of the role of

alternative pathway was assessed using Factor B (FB) deficient mice and Factor B (FB)

sufficient mice (WT). The two groups of mice aged between 10-13 weeks, i.e. Factor B

deficient and Factor B sufficient, were challenged with a low dose (1×105 colony

forming unit (CFU)/mouse) of Neisseria meningitidis serogroup B strain MC58, via an

intra-peritoneal route, as a higher dose would kill the wild-type control mice. Mice

received iron dextran (400mg/kg body weight) 12 hours before the administration of the

infection dose. Mice were constantly monitored for the development or onset of the

signs of illness throughout the experiment.

Figures 4.4, 4.5 and 4.6 show the survival of Factor B deficient mice, illness scores and

the recovery of viable bacterial counts from blood in FB deficient compared to FB

sufficient WT mice.

At 12 hours post infection, all FB deficient mice started developing clear signs of illness

and at the time point 24 hours post infection 50% of the FB deficient mice had to be

euthanized before they progressed to a lethargic stage. At the same time points, Factor

B sufficient WT mice looked healthy with no signs of illness. The rest of the Factor B

deficient mice continued developing signs of illness, and the experiment had to be

terminated as the remaining mice approached a lethargic end stage with a mortality rate

of 100% at 48 hours post infection (see Figure 4.4), the Factor B sufficient mice showed


127

only minor signs of illness at 36 hours post infection and later they started to recover

(see Figure 4.5). These results showed that there was a dramatically increased

susceptibility of Factor B deficient mice to Neisseria meningitidis infection compared

with the Factor B sufficient WT control mice which showed significantly lower signs of

illness in terms of disease severity.

The difference between the survival of Factor B deficient mice and Factor B sufficient

mice has been statistically analysed using the Log-rank (Mantel-Cox) test, which

showed a significant difference between the two groups of mice, emphasizing the

important role of the alternative pathway in terms of the host immune response to


0 1 2 2 4 3 6 4 8 6 0 7 2

0

2 0

4 0

6 0

8 0

1 0 0


% S

urv

iva

l

W T

F B- / -

* * * * p < 0 .0 0 0 1

Figure 4.4 Survival of wild type (on C57/BL6 background) and Factor B deficient mice (on

C57/BL6 background) following an i.p. injection with a low dose (1×105) of Neisseria

meningitidis serogroup B-MC58. The infective dose was added after adding iron to a final

concentration of 400mg/kg body weight. It was shown that Factor B deficient mice were

significantly more susceptible to Neisseria meningitidis infection compared to the Factor B

sufficient mice. ****p < 0.0001 Log-rank (Mantel-Cox) test; n=10/group.


128

612

24

36

48

72

0

1

2

3

4

5


Av

era

ge

ill

ne

ss

sc

ore

W T

FB- / -

* * * *

* * * ** * * *

* * * *

Figure 4.5 Average illness score of wild type and Factor B deficient mice following an i.p.

injection with a low dose (1×105) of Neisseria meningitidis serogroup B-MC58. The data are

presented as mean with SEM. ****p < 0.0001, Student t test.

4.1.3.1 The viable bacterial load of Neisseria meningitidis in the blood of infected

mice

Throughout the course of the infection experiment of Factor B deficient mice and their

sex and age matched Factor B sufficient WT control mice (WT) infected with a low

dose (1×105 CFU/mouse) of Neisseria meningitidis serogroup B strain MC58, the mice

were monitored and blood was taken at constant time intervals via a tail bleed route to

assess the viable bacterial load in the blood at defined time points 12, 24, 36 and 48

hours post infection. This showed that the bacterial load in the blood of FB deficient


129

mice was significantly higher compared to the bacterial load in the blood of Factor B

sufficient WT control mice, indicating the development of bacteraemia (see Figure 4.6).

At 48 hours post infection, the Factor B sufficient mice started to clear the bacteria from

the blood, while the survivors of the FB deficient mice group had developed higher

bacteraemia, showing signs of terminal disease; thus, they had to be euthanized.

12.

24.

36.

48.

0

1

2

3

4

5

6

T im e p o s t in f e c t io n ( h r s )

Lo

g C

FU

/ml

of

blo

od W T

F B- /-

*

*

* *

* *

Figure 4.6 Bacterial load in the blood of Factor B deficient mice and wild-type mice given an

i.p. injection of a low dose (1x105

CFU/mouse) of Neisseria meningitidis serogroup B strain

MC58 at time points 12, 24, 36 and 48 hours post infection. The data are presented as mean

with SEM. *p < 0.0156, **p < 0.0063, Student t test.


130

4.1.4 Survival of MASP-1/3 deficient mice and MASP-1/3 sufficient

mice following experimental Neisseria meningitidis infection

The lectin pathway molecule MASP-1 was recently shown to play an important role in

maintaining alternative pathway functional activity (Takahashi et al. 2010). I therefore

assessed if MASP-1/3 deficient mice are similarly compromised in their ability to

Neisseria meningitidis infections like FB deficient mice. Thus, in order to assess this

role of MASP-1/3 deficiency in vivo, two groups of mice, MASP-1/3 deficient mice and

MASP-1/3 sufficient mice (WT), aged between 10-13 weeks (all on C57/BL6

background), were challenged with a low dose of Neisseria meningitidis serogroup B

strain MC58 (1×105

CFU/mouse) via an intra-peritoneal route after receiving iron

dextran (400mg/kg body weight) 12 hours prior to challenge. The mice were

subsequently checked, and the development of meningitis was monitored throughout the

experiment. Figures 4.7, 4.8 and 4.9 show the survival of MASP-1/3 deficient mice,

their illness scores and the recoverable viable bacterial load from blood samples taken at

different time points after infection were compared to MASP-1/3 sufficient WT mice.

The MASP-1/3 deficient mice started developing the signs of illness at 12 hours post

infection. These signs progressed, leading to the death of 40% of these mice at 24 hours

post infection. At the same time, the wild-type mice remained healthy with no sign of

illness. At 54 hours post infection, the remaining MASP-1/3 deficient mice showed

signs of terminal disease and had to be euthanized (see Figure 4.7) while the MASP-1/3

sufficient mice were have started to recover with only minor signs of illness that

developed at 36 hours post infection (see Figure 4.8). These results highlighted the

importance of the lectin pathway molecule MASP-1/3 in driving the alternative pathway

to clear the bacteria. In contrast MASP-1/3 deficiency increases the susceptibility of


131

mice to Neisseria meningitidis infection, unlike in MASP-1/3 sufficient WT mice,

which show significantly lower signs of illness in terms of disease severity when

compared to MASP-1/3 deficient mice.

The difference between the survival of MASP-1/3 deficient mice and MASP-1/3

sufficient WT mice was statistically analysed using the Log-rank (Mantel-Cox) test,

which showed a significant difference between the two groups of mice emphasizing the

important role of MASP-1/3 in driving the alternative pathway activation in terms of

host immune response to Neisseria meningitidis infection.

0 1 2 2 4 3 6 4 8 6 0 7 2

0

2 0

4 0

6 0

8 0

1 0 0


% S

urv

iva

l

W T

M A S P -1 /3- / -

****p < 0 .0 0 0 1

Figure 4.7 Survival of WT and MASP-1/3 deficient mice (both on C57/BL6 background)

following an i.p. injection with a low dose (1×105) of Neisseria meningitidis serogroup B-

MC58. The infective dose was added after adding iron to a final concentration of 400mg/kg

body weight. . It was shown that MASP-1/3 deficient mice were more susceptible to

Neisseria meningitidis infection compared to the MASP-1/3 sufficient mice. ****p < 0.0001

Log-rank (Mantel-Cox) test; n=10/group.


132

612

24

36

48

54

72

0

1

2

3

4

5


Av

era

ge

ill

ne

ss

sc

ore

W T

M A S P -1 /3- / -

* * * *

* * * ** * * *

* * * * * * * *

Figure 4.8 Average illness score of wild type and MASP-1/3 deficient mice following an i.p.

injection with a low dose (1×105) of Neisseria meningitidis serogroup B-MC58. The data are

presented as mean with SEM. ****p < 0.0001, Student t test.


mice

Two groups of mice, MASP-1/3 deficient mice and MASP-1/3 sufficient WT mice,

were challenged with a low dose (1×105 CFU/mouse) of Neisseria meningitidis

serogroup B strain MC58, and monitored. Blood was taken via a tail bleeding route to

assess the viable bacterial load in blood at different time points 12, 24, 36, 48 and 54

hours post infection. The results showed that the bacterial load in blood of MASP-1/3

deficient mice was significantly higher compared to the bacterial load in blood of

MASP-1/3 sufficient mice, which reflects the development of signs of illness (see

Figure 4.9). At 48 hours post infection, the MASP-1/3 sufficient mice started to clear


133

the bacteria from the blood while the remaining MASP-1/3 deficient mice had

developed higher bacteraemia, leading to the development of the signs of terminal

illness; thus, these animals had to be euthanized.

12.

24.

36.

48.

54

0

1

2

3

4

5

6

T im e p o s t in f e c t io n ( h r s )

Lo

g C

FU

/ml

of

blo

od

W T

M A S P - 1 /3- /-

*

*

* *

* ** *

Figure 4.9 Bacterial load in blood of MASP-1/3 deficient mice and wild-type mice given an

i.p. injection of a low dose (1x105 CFU/mouse) of Neisseria meningitidis serogroup B strain

MC58 at time points 12, 24, 36, 48 and 54 hours post infection. The data are presented as mean

with SEM. *p < 0.0161, **p < 0.0047, Student t test.


134

4.1.5 Effect of full length recombinant MASP-3 on reconstituting the

absence of the alternative pathway functional activity in MASP-

1/3 deficient mice

In order to assess whether full length recombinant murine MASP-3 had the ability to

reconstitute the functional activity of the alternative pathway in MASP-1/3 deficient

mice and therefore the bacteriolytic activity toward the Neisseria meningitidis, MASP-

1/3 sufficient mice and MASP-1/3 deficient mice were divided into four groups. The

first group are MASP-1/3 deficient mice used as a control. The second groups are

MASP-1/3 deficient mice treated with a single dose of full-length recombinant murine

MASP-3 (20 µg/mouse) given i.v. 96 hours prior to the final bleed. The third groups

comprised the MASP-1/3 deficient mice treated twice with full length recombinant

murine MASP-3 (20 µg/mouse) given in two separate doses at 96 hours before infection

and 24 hours prior to the final bleed. The fourth groups is made up of sex and age

matched MASP-1/3 sufficient WT mice used as a reference for mice with full

alternative pathway functional activity. These mice were monitored and bled every day

until the end of the experiment (day 4).


135

Table 4.1 The design of the MASP-3 reconstitution experiment

Mice group

Dosage

Full length recombinant

MASP-3

Days of injection

Group one MASP-1/3 deficient mice

i.v.‎20‎μg/mice Day zero

Group two MASP-1/3 deficient mice

i.v.‎20‎μg/mice

i.v.‎20‎μg/mice

Day zero

Day three

Group three MASP-1/3 deficient mice

Saline only Day zero

Group four MASP-1/3 sufficient mice (WT)

Saline only Day zero

The ability of full length recombinant murine MASP-3 to reconstitute the alternative

pathway functional activity was assessed by measuring C3 deposition of alternative

pathway. This assay showed that the functional activity of MASP-1/3 deficient mice

was restored by 35% at 24 hours post injection. The functional activity of alternative

pathway was nearly fully restored at 72 hours post injection. However, at 96 hours post

injection, the alternative pathway activity started to decline again in MASP-1/3

deficient mice as shown in the group of mice that was treated only once, while the

second group (treated twice) kept the level of the alternative pathway activity steady as

it was treated twice (see Figure 4.10).


136

24

48

72

96

0 .0

0 .3

0 .6

0 .9

1 .2

T im e p o s t in je c t io n (d y s )

OD

At

40

5n

m

W T

M A S P -1 /3- / -

M A S P -1 /3- / -

+ rM A S P -3 (4 0 u g /m o u s e )

M A S P -1 /3- / -

+ rM A S P -3 (2 0 u g /m o u s e )

Figure 4.10 C3 deposition of MASP-1/3 deficient mice treated with full length recombinant

murine MASP-3. Mice were treated once or twice with rMASP-3 (20µg/mouse) by i.v.

injection. Mice were monitored and bled 24, 48, 72 and 96 hours post injection. The C3

deposition assay was used to assess the functional activity of the alternative pathway. Mouse

serum was diluted 13% with EGTA buffer. Zymosan was used to coat the plates. The binding of

the C3 cleavage product (C3c) was detected by using rabbit anti-human C3c polyclonal

antibody. It was shown that the full functional activity of the alternative pathway was restored

gradually by 72 hours post injection. This restored functionality started to decline in the group

that was treated once while in the group that was treated twice, it remained unchanged. (Data

presented as a mean of two independent experiments with duplicate for each +/- SEM).


137

Table 4.2 Statistically significant differences for the C3 deposition assay of different mouse

sera (MASP-1/3-/-

+ full-length rMASP-3 (40µg/mouse), MASP-1/3-/-


(20µg/mouse), MASP-1/3-/-,‎WT‎and‎HIS)‎using‎the‎Student’s‎t-test at time point 96 hours.

Student’s‎t-test at time point 96 hours


WT vs MASP-1/3-/-

YES ** (0.0031)

WT vs MASP-1/3-/-

+rMASP-3

(20µg/mouse) YES

* (0.0126)

WT vs MASP-1/3-/-

+rMASP-3

(40µg/mouse) YES

* (0.0276)

MASP-1/3-/-

vs MASP-1/3-/-

+ rMASP-

3 (20µg/mouse) YES

* (0.0050)

MASP-1/3-/-

vs MASP-1/3-/-

+ rMASP-

3 (40µg/mouse) YES

** (0.0015)

MASP-1/3-/-

+ rMASP-3 (20µg/mouse)

vs MASP-1/3-/-

+ rMASP-3

(40µg/mouse)

YES

* (0.0356)


138

To study this reconstitution of a deficient AP functional activity by recombinant MASP-

3 further, serum bactericidal assays were performed to assess the effect of this

reconstitution on serum bactericidal activity (SBA) of mouse serum towards Neisseria

meningitidis after collecting blood from the mice by cardiac punctures. The results from

these assays showed that the killing of meningococcus was restored through

reconstitution of MASP-1/3 deficient mice by the full length recombinant murine

MASP-3 indicating that the activity of alternative pathway towards Neisseria

meningitidis had been restored (see Figures 4.11 and 4.12). The SBA of the mice treated

with a single dose of full length recombinant murine MASP-3 was nearly half of that

seen in mice treated with two doses of full length recombinant murine MASP-3.


139

0 2 0 4 0 6 0 8 0 1 0 0

4 .2

4 .4

4 .6

4 .8

5 .0

M i n u t e s

log

cfu

/ml

HIS W T

M A S P -1 /3- /-

+ rM A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

+ rM A S P -3 (2 0 g /m o u s e )

M A S P -1 /3- /-


+ full length

rMASP-3 (treated with two doses of 20µg/mouse each given 96 and 24 hours), MASP-1/3-/-

+

full length rMASP-3 (20µg/mouse given 96 hours prior to bleeding), MASP-1/3-/-

(untreated)

and WT mouse blood and heat-inactivated WT mouse blood, i.e. HIS) against Neisseria

meningitidis serogroup A strain Z2491. Bacteria and sera (30% serum concentration) were

incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 And 90 minutes,

plated out and the recoverable viable bacterial count was calculated. It was shown that the

killing of meningococci was restored through reconstitution by giving full length recombinant

murine MASP-3 96 and 24 hours prior to bleeding. The serum bacteriolytic activity of the mice

treated with a single dose of full length recombinant murine MASP-3 was nearly half of the

mice that were treated with two doses of full length recombinant murine MASP-3. In this assay

heated inactivated serum (HIS) was used as a negative control (the data is presented as a mean



140

Table 4.3 Statistically significant difference assessed between serum bactericidal assay of


+ full length rMASP-3 (treated with two doses of

20µg/mouse each given 96 and 24 hours), MASP-1/3-/-

+ full length rMASP-3 (20µg/mouse

given 96 hours prior to bleeding), MASP-1/3-/-

(untreated), WT and HIS) against Neisseria

meningitidis serogroup‎A‎strain‎Z2491‎using‎the‎Student’s‎t-test at time point 90 min.



WT vs MASP-1/3-/-

YES ** (0.0032)

WT vs MASP-1/3-/-

+rMASP-3

(20µg/mouse) YES

** (0.0081)

WT vs MASP-1/3-/-

+rMASP-3

(40µg/mouse) YES

* (0.0335)

MASP-1/3-/-

vsMASP-1/3 -/-

+

rMASP-3 (20µg/mouse) YES

* (0.0237)

MASP-1/3-/-

vsMASP-1/3-/-

+


** (0.0080)

MASP-1/3-/-

+ rMASP-3

(20µg/mouse) vs MASP-1/3-/-

+

rMASP-3 (40µg/mouse)

YES

* (0.0379)


141

0 2 0 4 0 6 0 8 0 1 0 0

4 .0

4 .5

M i n u t e s

log

cfu

/ml

HISW T

M A S P -1 /3- /-

+ rM A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

+ rM A S P -3 (2 0 g /m o u s e )

M A S P -1 /3- /-


+ full length

rMASP-3 (treated with two doses of 20µg/mouse each given 96 and 24 hours), MASP-1/3-/-

+

full length rMASP-3 (20µg/mouse given 96 hours prior to bleeding), MASP-1/3-/-

(untreated)

and WT mouse blood and heat-inactivated WT mouse blood, i.e. HIS) against Neisseria

meningitidis serogroup B strain MC58. Bacteria and sera (30% serum concentration) were

incubated at 37°C with shaking. Samples were taken at time points 0, 30, 60 And 90 minutes,

plated out and the recoverable viable bacterial count was calculated. It was shown that the

killing of meningococci was restored through reconstitution by giving full length recombinant

murine MASP-3 96 and 24 hours prior to bleeding. The serum bacteriolytic activity of the mice

treated with a single dose of full length recombinant murine MASP-3 was nearly half of the

mice that were treated with two doses of full length recombinant murine MASP-3. In this assay

heated inactivated serum (HIS) was used as a negative control (the data is presented as a mean



142

Table 4.4 Statistically significant difference assessed between serum bactericidal assay of


+ full length rMASP-3 (treated with two doses of

20µg/mouse each given 96 and 24 hours), MASP-1/3-/-

+ full length rMASP-3 (20µg/mouse

given 96 hours prior to bleeding), MASP-1/3-/-

(untreated), WT and HIS) against Neisseria

meningitidis serogroup‎B‎strain‎MC58‎using‎the‎Student’s‎t-test at time point 90 min



WT vs MASP-1/3-/- YES ** (0.0083)

WT vs MASP-1/3-/-

+rMASP-3

(20µg/mouse) YES

** (0.0095)

WT vs MASP-1/3-/-

+rMASP-3

(40µg/mouse) YES

* (0.0356)

MASP-1/3-/-

vsMASP-1/3-/-

+

rMASP-3 (20µg/mouse) YES * (0.0128)

MASP-1/3-/-

vsMASP-1/3-/-

+


** (0.0091)

MASP-1/3-/-

+ rMASP-3

(20µg/mouse) vs MASP-1/3-/-

+

rMASP-3 (40µg/mouse)

YES * (0.0475)


143

4.1.6 Effect of full length recombinant MASP-3 administration on

mortality in a mouse model of Neisseria meningitidis infection

MASP-1, a lectin pathway specific serine protease, has previously been reported to play

a role in driving the alternative pathway activation on the surface of pathogens by

cleaving pro-Factor D (FD) to Factor D (FD) to allow the alternative pathway C3

convertase zymogen C3bB to be converted into its active form C3bBb (Takahashi et al.,

2010). My work demonstrates that it is in fact the absence of MASP-3 that leads to the

loss of AP functional activity and not the absence of MASP-1. Reconstitution

experiments of the MASP-1/3 deficient mouse line with recombinant MASP-1 failed to

restore the AP deficient phenotype (Takahashi et al., 2010). My work shows that the

administration of MASP-3 in MASP-1/3 deficient mice restores the functional activity

of the alternative pathway and also restores the resistance of reconstituted MASP-1/3

deficient mice against Neisseria meningitidis both in vitro and as you see below also in

vivo.

In order to assess the potential benefits of the therapeutic administration of full length

recombinant MASP-3 in MASP-1/3 deficient mice in fighting the Neisseria

meningitidis infections, three groups of mice aged between 11-13 weeks were

challenged with a low dose (1×105 CFU/mouse) of

Neisseria meningitidis serogroup B

strain MC58, via the intra-peritoneal route, following injection the mice with or without

full length recombinant murine MASP-3 via an intravenous route. Prior to infection, all

mice received iron (400mg/kg body weight) 12 hours prior to the infection dose. The

mouse groups were: i) group A MASP-1/3 deficient mice, ii) group B composed of


144

MASP-1/3 deficient mice treated twice with full length recombinant murine MASP-3,

96 hours and 24 hours prior to infection, and iii) group C composed of MASP-1/3

sufficient mice (WT) used as a positive control. Following the infection dose, the mice

were checked and continuously monitored for the development of signs of illness

throughout the experiment. The MASP-1/3 deficient mice, with or without being given

full-length recombinant MASP-3, were compared in terms of survival, illness scores

and bacterial viable load of blood with the MASP-1/3 sufficient mice (see Figures 4.13,

4.14 and 4.15) .

While both groups of MASP-1/3 deficient mice developed signs of illness, the MASP-

1/3 deficient mice that were not treated with full-length recombinant MASP-3

developed more severe signs compared to the treated ones. At the same time the MASP-

1/3 sufficient mice were healthy with no sign of illness. The severity of disease in

MASP-1/3 deficient mice that were not reconstituted with MASP-3 continued to

progress, and at time point 24 hours post infection 30% of this group had to be

euthanized because of the severity of disease signs. At time point 54 hours post

infection the rest of the MASP-1/3 deficient mice progressed to terminal disease

symptoms and had to be euthanized. In comparison, at this time point (i.e. 54 hours post

infection) only 30% of the MASP-3 reconstituted MASP-1/3 deficient mice developed

signs of terminal disease and had to be euthanized. The remaining mice in this group

recovered from the infection with an overall survival of 70% in the treatment group,

compared to 0% survival in the non-treated MASP-1/3 deficient littermates. MASP-1/3

sufficient WT mice developed minor signs of illness, but all of them recovered fully and

survived. At the end of experiment i.e. 96 hours post infection, we observed no

significant difference in illness scores when comparing the MASP-3 reconstituted


145

MASP-1/3 deficient mice with their age and sex-matched MASP-1/3 sufficient WT

controls (See Figures 4.13 and 4.14).

In this survival experiment, the results showed that the MASP-3 reconstituted MASP-

1/3 deficient mice re-gained a significant degree of resistance to Neisseria meningitidis

infection compared to their non-reconstituted MASP-1/3 deficient littermate controls

(see Figure 4.13).

0 1 2 2 4 3 6 4 8 6 0 7 2 8 4 9 6

0

2 0

4 0

6 0

8 0

1 0 0


% S

urv

iva

l

W T

M A S P -1 /3- / -

+ rM A S P -3

(4 0 µ g /m o u s e )

M A S P -1 /3- / -

***p <0 .0 0 0 2

n s

Figure 4.13 Survival of wild type mice, MASP-3 reconstituted MASP-1/3 deficient mice

(receiving 2 doses of full length recombinant MASP-3 (20µg/mouse at time points 96 hours and

time point 24 hours prior to infection) and non-treated MASP-1/3 deficient mice (all on

C57/BL6 background) were infected by i.p. injections with a low dose (1×105) of Neisseria

meningitidis serogroup B-MC58. The infective dose was added after giving the mice an i.p.

injection with iron to a final concentration of 400mg/kg body weight. The outcome of this

experiment demonstrates that MASP-3 reconstitution effectively increases resistance of MASP-


146

1/3 deficient mice against Neisseria meningitidis infection compared to the non-treated MASP-

1/3 deficient mice. ***p < 0.0002 Log-rank (Mantel-Cox) test; n=10/group.

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

1 2 h o u r s p o s t in f e c t io n

Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

***

***

0

1

2

3

4


Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

****

***

0

1

2

3

4

5


Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

****

****

0

1

2

3

4


Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

***

***

0 .0

0 .5

1 .0

1 .5


Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

**

a ll d e a d

0 .0

0 .2

0 .4

0 .6

0 .8

1 .0


Av

era

ge

ill

ne

ss

sc

ore

W T M A S P -1 /3- /-M A S P -1 /3

- /-+

r M A S P -3 (4 0 g /m o u s e )

n s

a ll d e a d

Figure 4.14 Average illness score for wild type, treated MASP-1/3 deficient mice and non-

treated MASP-1/3 deficient mice following an i.p. injection with a low dose (1×105) of

Neisseria meningitidis serogroup B-MC58. The data are presented as mean with SEM. ***p <

0.0001, **p < 0.0041, Student t test.


147


mice

Three groups of mice, i.e. non-treated MASP-1/3 deficient mice, treated MASP-1/3

deficient mice and MASP-1/3 sufficient mice (WT), were challenged with a low dose

(1×105 CFU/mouse) of Neisseria meningitidis serogroup B strain MC58. The mice were

monitored and bled via a tail vein route was collected to assess the viable bacterial load

in blood at different time points 12, 24, 36, 54, 72 and 96 hours post infection. This

assessment showed that the bacterial load in blood of MASP-1/3 deficient mice was

significantly higher compared to the bacterial load in blood of treated MASP-1/3

deficient mice, which indicates that the treated mice had better ability to clear the

bacteria compared to non-treated mice. (See Figure 4.15). At 54 hours post infection,

the MASP-1/3 sufficient mice started to clear the bacteria from the blood while the rest

of the treated MASP-1/3 deficient mice started to clear the bacteria from the blood 60

hours post infection. At 96 hours post infection, the difference in the bacterial load of

blood between the treated MASP-1/3 deficient mice and MASP-1/3 sufficient mice was

not significant. However at an earlier stage, non-treated MASP-1/3 deficient mice had

developed a greater bacteremia, indicating signs of terminal illness; thus, these mice had

to be euthanized.


148

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5L

og

CF

U/m

l o

f b

loo

d

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

* *

*

*


0

1

2

3

4

Lo

g C

FU

/ml

of

blo

od

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

* **

*


0

1

2

3

4

5

Lo

g C

FU

/ml

of

blo

od

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

* *

*

*

3 6 h o u r s p o s t in fe c t io n

0

1

2

3

4

5L

og

CF

U/m

l o

f b

loo

d

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

* **

*


0 .0

0 .5

1 .0

1 .5

2 .0

Lo

g C

FU

/ml

of

blo

od

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

*


0 .0

0 .2

0 .4

0 .6

0 .8

1 .0

Lo

g C

FU

/ml

of

blo

od

W T M A S P -1 /3+ /+

+

r M A S P -3 (4 0 g /m o u s e )

M A S P -1 /3- /-

n s


Figure 4.15 Bacterial load in blood of wild-type mice, treated MASP-1/3 deficient mice and

non-treated MASP-1/3 deficient mice given an i.p. injection of a low dose (1x105 CFU/mouse)

of Neisseria meningitidis serogroup B strain MC58 at time points 12, 24, 36, 54, 72 and 96

hours post infection. The data are presented as mean with SEM. *p < 0.0161, **p < 0.0047,

Student t test.


149

4.2 Discussion

4.2.1 Mice deficient in the alternative pathway functional activity

show dramatically higher susceptibility to Neisseria meningitidis

infection

In Chapter 3, in vitro assays showed that lectin pathway deficient sera (MASP-2

deficient sera) showed greater C3 deposition than MASP-2 sufficient sera in conditions

that allow all three complement activation pathways to be active. When the assay

conditions were changed to allow only the classical or the lectin pathway to be active,

the ability of the MASP-2 deficient sera to allow C3 deposition was impaired, while

MASP-2 sufficient sera showed a higher degree of C3 deposition on the surface of


In further analyses, the bacteriolytic activity of different sera was tested against

Neisseria meningitidis, showing that the killing activity of MASP-2 deficient serum was

higher than the killing activity the MASP-2 sufficient serum. These results from in vitro

assays suggested that the alternative pathway had an important role in mediating the

complement system for killing Neisseria meningitidis.

The alternative pathway is the part of the complement system that maintains a

continuous activation of complement pathways. It is initiated when C3 is spontaneously

hydrolysed to form C3(H2O) that binds to Factor B (FB) in the presence of Mg+2

,


150

leading to the creation of a zymogen complex C3(H2O)B. C3(H2O)B is then cleaved by

factor D (FD) into two parts: Ba and Bb. While the fragment Ba is released from the

complex, the other fragment Bb stays attached to the complex, leading to the formation

of the alternative pathway C3 convertase C3(H2O)Bb. This C3 convertase cleaves C3

into C3a and C3b, which can bind to a pathogen surface. Factor B will bind to the newly

generated C3b to form new C3 convertase C3bBb after cleaving Factor B via Factor D

(Thurman & Holers, 2006). This binding is stabilized by Properdin, which is one of the

components of the alternative pathway, and acts as positive regulator of the alternative

pathway that works to stabilize the C3 convertase C3bBb on the pathogen by increasing

its half-life 5-10 folds (Schwaeble and Reid, 1999).


shows that a limited level of C3 deposition was observed following the use of factor B

deficient serum (i.e. alternative pathway deficient serum) compared to wild-type serum.

Furthermore, the serum bactericidal assay of factor B deficient serum showed no killing

of Neisseria meningitidis compared to the wild-type serum. These results also suggest

the importance of the alternative pathway in fighting Neisseria meningitidis. To further

analyse these results in vivo infection experiments were used in a mice model of

Neisseria meningitidis infection in Factor B deficient mice, mice with gene-targeted

disruption of Factor B gene, providing a mouse model with a total defect in the

alternative pathway.


151

The infective dose for the infection experiment was a low dose (1×105

CFU/mouse) of

Neisseria meningitidis serogroup B strain MC58 administrated via an intra-peritoneal

route. The virulence of the Neisseria meningitidis infection was enhanced by

administrating iron into the murine model of infection (Holbein, 1981; Perkins-Balding

et al., 2004). Therefore in all the infection experiments mice received iron (400mg/kg

body weight) intra-peritoneally 12 hours before the infection dose.

Although the infective dose was low, there was a significant difference in the survival

of the two groups of mice (i.e. Factor B deficient mice and Factor B sufficient control

mice). The Factor B deficient mice were more susceptible to Neisseria meningitidis

infection with 100 % mortality compared to the Factor B sufficient mice that were more

resistance to Neisseria meningitidis infection with a survival rate of 100%. The illness

scores of the mice reflected this observation. While Factor B sufficient mice showed

only minor signs of illness, the Factor B deficient mice showed significantly higher

signs of illness within 24 hours post infection, leading some mice to reach a lethargic

end stage. These mice were euthanized according to the home office regulations.

Although none of the Factor B sufficient mice progressed to a lethargic end stage, all

the Factor B deficient mice showed higher signs of illness and reached the lethargic end

stage by 48 hours post infection (see Figures 4.4 and 4.5).

The presence of culturable Neisseria meningitidis in blood gave a better illustration of

disease progression during the infection experiment. Although the presence of the

bacteria in the blood started to appear in both groups at 12 hours post infection, the


152

viable count of bacteria in the blood of Factor B deficient mice was higher than the

viable count of bacteria in the blood of Factor B sufficient mice at each time post

infection. The results also showed that Factor B sufficient mice were able to

significantly reduce and clear the bacteria from the blood, which was in contrast to the

Factor B deficient mice (see Figure 4.6). The results of this infection experiment are in

line with previous reports showing an association between an alternative pathway

deficiency and Neisseria meningitidis infection (Biesma et al., 2001; Hiemstra et al.,

1989; Sprong et al., 2006).

MASP-3 is a component of the lectin pathway of MBL associated serine proteases

discovered by Dahl et al. (2001) and is produced by the same gene that produces the

MASP-1(MASP-1 gene) located on chromosome 16. Although they have the same

heavy chain, the linker regions are different (the last 15 C-terminal residues). Unlike the

other MBL serine protease (MASP-1 and MASP-2) MASP-3 is not an auto activated

molecule and requires the action of MASP-1 or C1r to be activated (Megyeri et al.,

2014; Wijeyewickrema et al., 2013). MASP-3 was firstly thought to act as a negative

regulator of the lectin pathway by competing with the binding site of MASP-1 and

MASP-2 to MBL (Dahl et al., 2001). However, Takahashi et al (2010) showed that

MASP-1/3 deficient mice lack the full function of an alternative pathway.

Therefore in order to assess role of MASP-1/3 in fighting Neisseria meningitidis

infection, an infection experiment using MASP-1/3 deficient mice and MASP-1/3

sufficient mice was performed and showed that MASP-1/3 deficient mice were more


153

susceptible to Neisseria meningitidis than MASP-1/3 sufficient mice. While all of the

MASP-1/3 deficient mice developed signs of illness 6 hours post infection that led to all

of the mice reaching a lethargic end stage and were euthanized by 53 hours post

infection, the MASP-1/3 sufficient mice were healthy with only minor signs of illness

and recovered by 72 hours post infection. The viable count of Neisseria meningitidis in

the blood of mice showed the ability of MASP-1/3 sufficient mice to fight and clear the

bacteria, while the MASP-1/3 deficient mice showed a significantly higher count of

bacteria in their blood. The results of this infection study suggest that MASP-1/3 has an

important role in fighting Neisseria meningitidis infection (see Figures 4.7, 4.8 and 4.9).


dramatically improves the survival of MASP-1/3 deficient mice

from Neisseria meningitidis infection

Recently published work by Iwaki et al. (2011) postulated that on the surface of

Staphylococcus aureus the lectin pathway components MASP-3 and MBL may actually

allow alternative pathway activation to be initiated through the direct cleavage of

zymogen C3(H2O)B or C3bB and convert these zymogen complexes into their active

form by converting C3bB to C3Bb. In order to assess whether MASP-3 has an ability to

drive activation of the alternative pathway and therefore increase the lysis of Neisseria

meningitidis, an in vivo MASP-3 reconstitution experiment in MASP-1/3 deficient mice

was performed. These mice were divided into two groups. The first group received one

dose of full-length recombinant MASP-3 (20µg/mouse) 96 hours before terminal


154

bleeding. The second group received two doses of full length recombinant MASP-3

(20µg/mouse) 96 and 24 hours before the terminal bleeding. The results of this

reconstitution experiment showed gradual restoration of the functional activity of the

alternative pathway. The levels of this restoration were monitored and measured by

assessing the formation of the alternative pathway C3 deposition at 24, 48, 72 and 96

hours post MASP-3 injection.

This experiment showed that while the restoration levels were nearly 35% in both

groups at 24 hours post injection, this increased to 60% at 48 hours post injection

compared to the wild-type mice serum. At 72 hours post injection alternative pathway

functional activity was nearly fully restored in both groups of mice. However, at 96

hours post injection the level of the alternative pathway function was reduced in the

group of mice that received only one dose of full-length recombinant MASP-3

compared to the second groups of mice that received two doses of full-length

recombinant MASP-3 (see Figure 4.10). My results clearly show that it is MASP-3 and

not MASP-1 (as previously claimed by Takahashi et al. 2010) that is responsible for the

loss of alternative pathway functional activity in the blood of MASP-1 and MASP-3

double deficient mice.

Serum bactericidal assays were also done to assess the cytolytic activity of the sera

taken from both groups of mice at 96 hours post injection to Neisseria meningitidis. The

results of these assays showed that both sera had restored the ability to kill the Neisseria

meningitidis, which is in line with findings by Iwaki et al. (2011). However in this


155

experiment, it was observed that even though both sera had the ability to lyse the

Neisseria meningitidis, the sera of the group that received two doses of full length

recombinant MASP-3 showed significantly higher lysis than the group receiving only

one dose of full length recombinant MASP-3. This may be due to the instability and

subsequent breakdown of the administrated recombinant MASP-3 (see Figures 4.11 and

4.12).

Current results of the reconstitution experiment suggest that reconstitution of full-length

recombinant MASP-3 in MASP-1/3 deficient mice reduced the mortality in MASP-1/3

deficient mice in a model of Neisseria meningitidis infection. Therefore an experiment

to assess this hypothesis was performed using three groups of mice: MASP-1/3

sufficient mice, MASP-1/3 deficient mice and MASP-1/3 deficient mice that received

two doses of full length recombinant MASP-3 at 96 and 24 hours before the Neisseria

meningitidis dose. The infective dose was a relatively low dose (1×105 CFU/mouse) of

Neisseria meningitidis serogroup B strain MC58 and this was used after adding iron to a

final concentration of 400mg/kg body weight.

The result of this infection experiment showed a significant difference in the survival of

the mouse groups showing that MASP-1/3 deficient mice were more susceptible to

Neisseria meningitidis infection with 100 % mortality compared to the wild-type mice

which were more resistant to Neisseria meningitidis infection with a survival rate of

100%. Interestingly the survival rate of the treated MASP-1/3 deficient mice was 70%

compared to the non-treated MASP-1/3 deficient mice. This result shows the enhanced


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ability of the treated MASP-1/3 to fight Neisseria meningitidis infection. The illness

scores of the mice also reflected this observation. While MASP-1/3 sufficient mice

showed minor signs of illness and none of them progressed to the lethargic end stage,

the MASP-1/3 deficient mice show significantly higher signs of illness that lead some

of the mice to reach a lethargic end stage at 36 hours post infection and had to be

euthanized. The remaining MASP-1/3 deficient mice developed greater signs of illness

and reached the lethargic end stage by 54 hours post infection. In contrast even though

the treated MASP-1/3 deficient mice showed signs of illness that lead some of the mice

to the lethargic end stage at 54 hours post infection, the other mice recovered and

returned to a normal health status at 96 hours post infection ( see Figures 4.13 and 4.14).

In line with the survival and illness scores results, the viable bacterial count in blood

showed a significantly higher bacterial count in the blood of none treated MASP-1/3

deficient mice than the treated MASP-1/3 deficient and MASP-1/3 sufficient mice.

Although the presence of the bacteria in the blood started to appear in all the groups at

12 hours post infection, the viable count of bacteria in the blood of non-treated MASP-

1/3 deficient mice was higher than the viable count of bacteria in blood of treated

MASP-1/3 deficient mice at each time point post infection, which indicated that the

treated MASP-1/3 deficient mice were able to more significantly reduce and clear the

bacteria of the blood than the MASP-1/3 deficient mice. Interestingly, even though the

count of bacteria in the blood of treated MASP-1/3 deficient mice was significant higher

than the bacterial count in the MASP-1/3 sufficient mice at each time point post

infection, the difference between the two groups become non-significant at the end of

the experiment (see Figure 4.15).


157

In summary, results of the survival, illness scores and viable bacterial count

demonstrated that the lectin pathway molecule MASP-3 was able to drive

the activation of the complement system (alternative pathway) which

resulted in a reduction and clearance of the bacteria and led to a dramatic

increase in the survival rate of MASP-3 reconstituted MASP-1/3 deficient

mice.

Chapter 5: Conclusion and future work

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5.1 Conclusion

The complement system is a vital part of the immune system, composed of many

proteins that interact with each other to form a complex interacting network. This

network of proteins is activated by three different pathways designated as the classical,

the alternative and the lectin pathways. Regardless of the pathways involved, the

complement system plays several vital physiological roles especially in the process of

protection against pathogens and links the non-specific (innate) immune system with the

specific (adaptive) immune system. Also waste products such as apoptotic cells and

debris are removed by the complement system by facilitating their uptake by phagocytic

cells (Schwaeble et al., 2002; Walport, 2001).

Neisseria meningitidis is the main cause of bacterial meningitis throughout the world

and causes a significant rate of mortality. While there has been considerable progress in

its diagnosis, vaccination and treatment in recent years meningococcal disease is still a

major health threat with a mortality rate by septicaemia of up to 10%. Neisseria

meningitidis colonisation starts on the surface of the mucosal epithelium and

intraepithelially on the host nasopharynx. It is the invasion of the blood stream that

causes the well-known clinical symptoms ranging from mild meningococcal infection to


159

sepsis and to full-blown meningococcal septicemia and meningitis (Schneider et al.,

2007; Connolly and Noah, 1999).

The association between Neisseria meningitidis and the complement system has been

extensively studied and many published reports (Finne et al., 1987; Estabrook et al.,

1997; Rossi et al., 2001; Rosa et al., 2004; Jarva et al., 2005; Trouw & Daha, 2011)

indicate the important role of the complement system in fighting Neisseria meningitidis

infection. In this study, I have focussed on the crucial role of the alternative pathway in

fighting Neisseria meningitidis infection and managed to define the so far unknown role

of the lectin pathway component, serine protease MASP-3, in directing alternative

pathway functional activity to the surface of Neisseria meningitidis to enable the

complement system to lyse these pathogens, a mechanism that is vital in fighting off

infection and clearing the bacteria from the blood stream. To define the role of the lectin

pathway dependent enzyme MASP-3 in more detail, several complement-specific in

vitro assays were established and the lessons learned from these were tested in

subsequent in vivo infection studies using mouse models of Neisseria meningitidis

infection. The mouse models of infection included wild-type C57/BL6 mice as well as

gene-targeted mouse strains of the same genetic background with deficiencies of

essential alternative pathway effector enzymes [factor B gene (FB-/-

mice)] or mice with

a disrupted MASP1 gene which were deficient in the MASP1 gene products MASP-1.

MASP-3 and MAp44 (MASP-1/3-/-

mice) that have been previously reported to present

with a deficiency in alternative pathway functional activity (Takahashi et al., 2010).


160

The results showed that, the in vitro study has newly demonstrated the important role of

a new link between the lectin and the alternative pathways in fighting Neisseria

meningitidis infection. It has also highlighted the major role of the lectin pathway

complex molecules MASP-3 and MBL, in driving the activation of the alternative

pathway on the surface of Neisseria meningitidis through the cleavage of C3b bound

Factor B leading to the formation of the alternative pathway C3 convertase and

subsequently to the formation of the MAC complex and thus lysis of Neisseria

meningitidis. The findings emphasize the ability of recombinant properdin to enhance

the lytic activity of serum and this knowledge could be used to develop a therapeutic

approach to fight Neisseria meningitidis infection. The in vivo study emphasizes the

importance of the alternative pathway in fighting Neisseria meningitidis infection. Also,

it has highlighted the important role of the lectin pathway component MASP-3 in

driving the activation of the alternative pathway and fighting Neisseria meningitidis

infection.

5.1.1 Binding of complement system recognition molecules to Neisseria

meningitidis

As complement activation on the surface of Neisseria meningitidis may be dependent

on the binding of the classical pathway recognition molecule C1q and/or lectin pathway

recognition molecules such as Ficolin-A, MBL-A, MBL-C and CL-11, the binding of

C1q and other lectin pathway recognition molecules to different serotypes of Neisseria

meningitidis strains (serotype A strain Z2491 and serotype B strain MC58) was studied.


161

The results showed that Neisseria meningitidis serotype A strain Z2491 and serotype B

strain MC58 were recognised by the classical pathway recognition molecule C1q as

shown by the binding of C1q from sera of non-immune mice to the N. meningitidis

strains tested (see Figure 3.1). However, this direct binding of C1q to the tested strains

of Neisseria meningitidis did not induce classical pathway dependent complement

activation, as shown by the lack of CP sufficient serum to deposit C3 activation

products when serum of MBL-null mice or serum of non-immune MBL-deficient blood

donors was used.

Then the binding of the recognition molecules of the lectin pathway such as ficolin-A,

MBL-A, MBL-C and CL-11 to Neisseria meningitidis were assessed. The murine lectin

pathway recognition molecule ficolin-A showed no binding to either of the two

different serotypes of meningococcus strains tested, i.e. serotype A strain Z2491 and

serotype B strain MC58, while both murine MBL molecules MBL-A and MBL-C as

well as the MBL-related collectin CL-11 all showed binding to both Neisseria

meningitidis serotypes (see Figures 3.2 to 3.5).


162


meningitidis requires a close cooperation between the lectin and

alternative pathways

The ability of Neisseria meningitidis to activate the complement system was studied

using several in vitro assays which showed that both strains of Neisseria meningitidis

used throughout this study had the ability to activate complement (see Figures 3.6 and

3.7). These results encouraged me to investigate the specific contributions of each of the

different complement pathways towards this activation. The results revealed different

levels of C3 deposition on the surface of both strains of Neisseria meningitidis

depending on which sera were used to measure the relative amount of C3 deposition on

the surface of strains tested. The lowest level of the C3 deposition on both strains was

observed when using serum from factor B deficient mice (see Figures 3.11 and 3.12).

This serum was totally deficient of alternative pathway functional activity while both

the classical and lectin pathways were unaffected. This result suggests that there is a

predominant role of the alternative pathway in driving C3 deposition and complement

activation on the surface of the strains tested. C3 deposition on the surface of Neisseria

meningitidis was either not or only minimally affected when using C1q deficient non-

immune serum (which totally lacks classical pathway functional activity) in comparison

to wild-type serum (see Figures 3.11 and 3.12). This result suggests that the classical

pathway plays a minimal (and rather insignificant) role in mediating C3 deposition on

the surface of Neisseria meningitidis. Additionally, it emphasized the important role of

the other two complement pathways, the alternative and lectin pathways, which

contributed to the majority of C3 deposition on the surface of Neisseria meningitidis in

non-immune sera. Surprisingly, the highest level of the C3 deposition was seen in sera


163

of MASP-2 deficient mice, which is deficient in its ability to form the lectin pathway

C3 convertase C4aC2b (see Figures 3.11 and 3.12). This finding again underlined the

important contribution of the alternative pathway in driving complement activation on

the surface of this pathogen. The high bactericidal activity of MASP-2 deficient serum

and the finding that MBL is required to trigger this bactericidal activity in non-immune

serum implies that a lectin pathway enzyme other than MASP-2 may support alternative

pathway function and contribute to C3 deposition and initiation of serum killing of


Serum bactericidal assays to quantify the lytic activity against Neisseria meningitidis

included both human and different mouse sera (wild-type, C1q deficient, factor B

deficient, MASP-1/3 deficient and MASP-2 deficient mouse sera) were performed.

These assays were run under conditions that did allow activation of each of the three

complement activation pathways. The results of these assays were consistent (i.e. in full

agreement) with the results obtained using the C3 deposition assays. Like the C3

deposition results, these results showed that in the serum bactericidal assays, in the

absence of classical pathway activity, i.e. in C1q deficient serum, there was no, or only

a minimal contribution of the C1q driven classical pathway towards the bacteriolytic

activity of the non-immune serum. The other two complement activation pathways, i.e.

the alternative and the lectin pathways, fully compensated for the loss of classical

pathway functional activity in non-immune serum (see Figures 3.16 and 3.17). Similar

to my observations using the C3 deposition assays, MASP-2 deficient serum showed a

significantly increased lytic activity (see Figures 3.20 and 3.21) which could be

explained by the previously postulated phenomenon that either of the two remaining


164

lectin pathway serine proteases, MASP-1 or MASP-3, could be involved in driving the

alternative pathway (Takahashi et. 2010; Iwaki et al., 2011). Notably, these findings

demonstrate that the MASP-2 dependent effector arm of the lectin pathway does not

play a major role in the lysis of Neisseria meningitidis and that the therapeutic

inhibition of MASP-2 functional activity does not increase the predisposition of patients

treated with therapeutic MASP-2 inhibitors to suffer more frequent or more severe

meningococcal infections.

The important role of the alternative pathway was highlighted when using different

alternative pathway deficient sera (Factor B deficient and MASP-1/3 deficient). In both

sera highly compromised and lacking in bactericidal activity was observed even in the

presence of the other two complement activation pathways, classical and the MASP-2

dependent lectin pathway activation route (see Figures 3.18 and 3.19).

To further analyse whether the loss of bacteriolytic activity in MASP-1 and MASP-3

deficient mouse sera or MASP-1 and MASP-3 deficient human 3MC serum was due to

the deficiency of either MASP-1 or MASP-3 or both, the sera were reconstituted with

functionally active recombinant MASP-3, a recombinant fragment composed of the

MASP-3 domains CCP-1/CCP-2 and the serine protease domain, termed rMASP-3

(catalytic fragment, cf). Bactericidal assays were performed using MASP-1/3 deficient

serum and human 3MC serum, with or without rMASP-3 (cf). The results of these

assays showed that the alternative pathway dependent lytic activity against both strains

of Neisseria meningitidis was restored by the addition of rMASP-3 (cf). In addition the


165

results highlighted the importance of the alternative pathway in fighting Neisseria

meningitidis infection (see Figures 3.22 to 3.25).

In vitro assays also demonstrated the importance of the lectin pathway recognition

molecule MBL in mediating bacteriolytic activity towards Neisseria meningitidis.

Human and murine MBL deficient sera were not able to kill Neisseria meningitidis

when tested in a serum bactericidal assay. This observation was consistent in both

human and murine MBL deficient sera (see Figures 3.20, 3.21, 3.24 and 3.25).

In context with the new essential role that this work has identified for the lectin pathway

serine protease MASP-3, the requirement of both MASP-3 and the lectin pathway

recognition subcomponent MBL implies that the MASP-3 dependent initiation of

alternative pathway mediated killing of Neisseria meningitidis is dependent on lectin

pathway activation/function that takes place on the surface of Neisseria meningitidis

since this event requires both the recognition component, MBL (that binds to the

surface of the pathogen), and the lectin pathway enzyme MASP-3 that converts pro-

Factor D to enzymatically active Factor D which in turn is required to convert the

alternative pathway C3 convertase zymogen complex C3bB into its enzymatically

active form (i.e. C3bBb). These results on the role of MBL in mediating serum

bacteriolytic activity towards Neisseria meningitidis are in full agreement with a

previously published report which implied that MBL had an important role in fighting

Neisseria meningitidis infection (Hibberd et al., 1999).


166

Taken together, the results strongly suggest that both lectin pathway molecules, i.e.

MBL and MASP-3, play crucial roles in mediating the lytic activity toward Neisseria

meningitidis.

5.1.3 Recombinant properdin enhances the serum bacteriolytic

activity against Neisseria meningitidis

Properdin is a glycoprotein known as a positive regulator of complement activation. It

works to stabilize the C3 convertase C3bBb on the pathogen through increasing its half-

life. The association between properdin deficiency and Neisseria meningitidis infection

has been reported previously, illustrating the important role of properdin in fighting

Neisseria meningitidis infection (Fijen et al., 1995).

Therefore, in order to assess the effect of properdin on the formation of C3 deposition

(and thus the lytic activity) on the surface of Neisseria meningitidis was measured

using ELISA assay and FACS analysis. The results of these assays showed increased

amounts of C3 deposition on the surface of Neisseria meningitidis strains following the

addition of recombinant properdin (see Figures 3.26, 3.27 and 3.28).

To further analyse and confirm the important role of properdin in enhancing the serum

bactericidal activity against Neisseria meningitidis it was shown that the bactericidal

activity of human or mouse serum was enhanced significantly by adding recombinant

properdin compared to normal serum alone (see Figures 3.29 to 3.32).


167

5.1.4 Mice deficient in the alternative pathway functional activity

show dramatically higher susceptibility to Neisseria meningitidis

infection


shows that a limited level of C3 deposition was observed following use of Factor B

deficient serum, (alternative pathway deficient serum) compared to wild-type serum.

Further, the serum bactericidal assay of Factor B deficient serum showed no killing of

Neisseria meningitidis compared to the wild-type serum. These results also suggest the

importance of the alternative pathway in fighting Neisseria meningitidis. To further

analyse these results, an in vivo infection experiment in a mouse model of Neisseria

meningitidis infection in Factor B deficient mice (i.e. mice with a gene-targeted

disruption of Factor B gene) which provides a mouse model with a total defect in the

alternative pathway, was performed.

Although the infective dose was low, there was a significant difference in the survival

of the Factor B deficient mice and the Factor B sufficient control mice. The Factor B

deficient mice were more susceptible to Neisseria meningitidis infection, with 100%

mortality, compared to the Factor B sufficient mice which were more resistant to

Neisseria meningitidis infection with a survival rate of 100%. The illness scores of the

mice reflected this observation. While Factor B sufficient mice showed only minor

signs of illness, the Factor B deficient mice showed significantly higher signs of illness.

Additionally, the results showed that Factor B sufficient mice were able to significantly


168

reduce and clear the bacteria from the blood, which was in contrast to the Factor B

deficient mice (see Figures 4.4 to 4.6).

MASP-3 is one of the lectin pathway MBL associated serine proteases which was firstly

thought to act as a negative regulator of the lectin pathway by competing with the

binding site of MASP-1 and MASP-2 to MBL (Dahl et al., 2001). However, Takahashi

et al. (2010) showed that MASP-1/3 deficient mice are lacking in the full function of an

alternative pathway. Thus, in order to assess its role in fighting Neisseria meningitidis

infection, an infection experiment using two groups of mice; MASP-1/3 deficient mice

and MASP-1/3 sufficient mice was performed. The results from this infection study

showed that MASP-1/3 deficient mice were more susceptible to Neisseria meningitidis

than MASP-1/3 sufficient mice. Illness scores of mice showed higher signs of illness in

MASP-1/3 deficient mice compared to MASP-1/3 sufficient mice that were healthy

with only minor signs of illness. Also, the viable count of Neisseria meningitidis in the

blood of mice showed the ability of MASP-1/3 sufficient mice to fight and clear the

bacteria while the MASP-1/3 deficient mice showed a significantly higher count of

bacteria in their blood (see Figures 4.7 to 4.9).


169


dramatically improves the survival of MASP-1/3 deficient mice

from Neisseria meningitidis infection

Recently published work by Iwaki et al. (2011) showed that the activation of an

alternative pathway on the surface of Staphylococcus aureus through the lectin pathway

complex between the MASP-3 and MBL which cleaves the proenzymes C3(H2O)B or

C3bB to their enzymatic active form. Therefore, in order to assess whether MASP-3 had

the ability to drive the activation of the alternative pathway and therefore increase the

lysis of Neisseria meningitidis, an in vivo MASP-3 reconstitution experiment in MASP-

1/3 deficient mice using recombinant murine full-length MASP-3 zymogen (rMASP-3

(flz)),‎ produced‎ by‎ Dr.‎ SadamYaseen‎ in‎ Professor‎ Schwaeble’s‎ laboratory‎ were‎

performed. rMASP-3 (flz) (20 micrograms) was given i.v. to MASP-1/3-/-

mice and

reconstitution of alternative pathway functional activity was measured in blood taken at

24, 36, 48 and 96 hours after injection. After 24 hours alternative pathway functional

activity was restored to approximately 35% of that of wild-type mice, after 48 hours to

about 60% and after 72 hours to more than 90%. The results of this reconstitution

experiment clearly demonstrated the ability of the lectin pathway serine protease

MASP-3 to restore the severely compromised alternative pathway functional activity in

MASP-1/3-/-

mice (see Figures 4.10) and the subsequent experiment that was conducted

by infecting MASP-3 reconstituted MASP-1/3-/-

mice with Neisseria meningitidis

demonstrated the essential role of MASP-3 in the innate immune response to Neisseria

meningitidis infection.


170

The results of the reconstitution experiment demonstrated for the first time that

reconstitution of MASP-1/3-/-

mice with full length recombinant MASP-3 dramatically

reduced the mortality in the MASP-1/3 deficient mice in a model of Neisseria

meningitidis infection. Therefore this infection experiment was repeated to confirm this

findings using three groups of mice: MASP-1/3 sufficient WT mice, MASP-1/3

deficient mice and MASP-1/3 deficient mice that received two doses of full length

recombinant MASP-3 at 96 and 24 hours prior to intraperitoneal Neisseria meningitidis

infection. The results of this infection experiment showed that the survival rate of the

treated MASP-1/3 deficient mice was 70%, compared to the survival rate of non-treated

MASP-1/3 deficient mice which was 0% with 100% mortality. The wild-type mice were

resistant to the relatively low infectious dose of 1x 105 Neisseria meningitidis serogroup

B strain MC58 given i.p. leading to a survival rate of 100%. The illness scores

established for the different experimental mouse groups were fully in line with the

overall survival results. While MASP-1/3 sufficient mice showed minor signs of illness,

the MASP-1/3 deficient mice show significantly higher signs of illness. In contrast,

even though the treated MASP-1/3 deficient mice showed signs of illness that led 30%

of the mice to the lethargic end stage, the remaining treated MASP-1/3 deficient mice

recovered and returned to a normal health status. In line with the survival and illness

scores results, the viable bacterial count in blood showed a significantly higher bacterial

count in the blood of non-treated MASP-1/3 deficient mice than the treated MASP-1/3

deficient and MASP-1/3 sufficient mice. Interestingly, even though the count of bacteria

in the blood of treated MASP-1/3 deficient mice was significantly higher than the

bacterial count in the MASP-1/3 sufficient mice at each time point post-infection, the


171

difference between the two groups was non-significant at the end of the infection

experiment (see Figures 4.11 to 4.15).

In summary, results of the survival, illness scores and viable bacterial count

demonstrated that the lectin pathway molecule MASP-3 was able to drive the activation

of the complement system (alternative pathway) and resulted in the reduction and

clearance of the bacteria and led to an increase in the survival rate of treated MASP-1/3

deficient mice.


172

5.2 Future work

A number of interesting aspects of the research arose during this work, but due to time

constraints these could not be followed up.

5.2.1 Assess the therapeutic benefit of recombinant MASP-3 in

fighting other microbial infection

My work has highlighted the major role of the lectin pathway molecule i.e. MASP-3, in

driving the alternative pathway, and thus fighting the Neisseria meningitidis infection.

Therefore, it would be interesting to assess the therapeutic benefit of recombinant

MASP-3 in fighting other microbial infection.

5.2.2 Assess the ability of recombinant properdin in restoring the

killing of properdin-deficient sera

This study has shown the important role of properdin in enhancing the serum

bactericidal activity against Neisseria meningitidis. It was shown that serum bactericidal

activity of human or mouse was enhanced significantly by adding recombinant

properdin compared to normal serum alone. Therefore the ability of recombinant

properdin in restoring the killing activity of properdin-deficient sera should be tested to

assess to what extent the administration of properdin can be used as a therapy in

properdin-deficient individuals.

Chapter 6: Bibliography

173


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meningococcal long-term carriage among university students and their

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