University of Groningen

Breaking walls: combined peptidic activities against Gram-negative human pathogensLi, Qian

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Chapter

1General Introduction

Qian Li1, Manuel Montalban-Lopez1,2, Oscar P. Kuipers1

1Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands

2 Department of Microbiology, Faculty of Sciences, University of Granada, Spain

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1. Overview of lanthipeptides

Ribosomally synthesized and post-translationally modified peptides (RiPPs) have been identified in the 21st century as the fifth major group of natural products, besides terpenoids, alkaloids, polyketides and non-ribosomal peptides [1]. Lanthipeptides are a class of polycyclic RiPPs containing meso-lanthionine (Lan) and 3-methyllanthionine (MeLan) residues [1, 2]. Lanthipeptides can display diverse activities, including morphogenetic [3], antiviral [4], antimicrobial [5] or antial-lodynic effect [6]. Lanthipeptides possessing antimicrobial activity are called lantibiotics (lanthionine-containing antibiotics). Lanthionine (Lan) is composed of two alanine residues whose beta carbons are crosslinked via a thioether bridge; while methyllanthionine (MeLan) contains one additional methyl group compared to lanthionine [2]. These (methyl)lanthionine bonds are critical for their activities [7, 8] as well as their thermostability, proteolytic resistance and are important features in pharmaceutical applications [9–11].

1.1. Classification of lanthipeptidesNatural lanthipeptides are ribosomally synthesized as precursor pep-tides and the linear precursor peptide contains a leader peptide and a core peptide. The core peptide can become the mature compound through the insertion of posttranslational modifications (PTMs) car-ried out by PTM enzymes and their transport and activation by the specific leader protease. These processes are mainly guided by the leader peptide [5, 12, 13]. The Lan and MeLan residues are introduced to the precursor peptides by two enzymatic steps mediated by one or more enzymes. Serine and threonine are dehydrated to become dehydroal-anine (Dha) and dehydrobutyrine (Dhb), respectively, which then can be coupled to a cysteine via a Michael-type addition to form a thioether link. Based on the PTM enzymes involved in the maturation process of core peptides, lanthipeptides can be divided into four distinct classes (Class I, II, III and IV) [2] (Figure 1).

In class I lanthipeptides (e.g. nisin, gallidermin), the (methyl) lan-thionine residues are formed by a dehydratase (LanB) and a cyclase (LanC) (Figure 1). Subsequently, the fully modified peptides are ex-ported by a transmembrane ATP-binding cassette (ABC) transporter

12

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1 (LanT) and the leader peptides can be cleaved by a protease (generally LanP) [14]. The elongated and flexible secondary structure of class I lanthipeptides plays a key role in the antimicrobial effect of them to bind to lipid II and/or form pores in most cases [15, 16].

In class II lanthipeptides (e.g. mersacidin, lacticin 481, halodura-cin), both the dehydration and cyclization reactions are catalyzed by a bifunctional modification enzyme, called LanM. The N-terminal dehydratase domain of LanM does not share similarities with Lan B [17] but the C-terminus shows about 25 % sequence homology to LanC, including the conserved zinc-binding residues [2, 18] (Figure 1). A single, multifunctional protein LanT, with a conserved N-terminal protease domain, is responsible for secretion and leader processing in class II lanthipeptides [2, 14]. It is notable that there are various two-component lantibiotics within class II lanthipeptides, including lacticin 3147 [19], haloduracin [20], lichenicidin [21], plantaricin W [22] and some others. The two peptides work synergistically to exert antimicrobial activity. They are encoded by their own structural genes and modified by individual LanM enzymes but transported by a single LanT, which will cleave off the leader peptides.

Class III lanthipeptides (e.g. SapB, SapT, labyrinthopeptins), which perform morphogenetic and signal functions instead of antimicrobial activity, are modified by a single trifunctional enzyme termed LanKC. LanKC contains an N-terminal lyase domain, a central kinase domain and a putative C-terminal cyclase domain [23]. The cyclase domain bears limited homology to LanC and LanM, but is lacking the con-served zinc ligands [24] (Figure 1). In addition, labyrinthopeptins, known as a class III lanthipeptides, can also form a so-called labionin structure [6]. The labionin (Lab) structure is synthesized from two serine residues and one cysteine residue. It refers to a carbocyclic structure formed by two steps, including 1) the generation of an eno-late intermediate by the addition of a cysteine thiol to Dha and 2) the addition of a second Dha to the intermediate [25, 26].

Class IV lanthipeptides have been established in 2010 after the inden-tification of venezuelin, in Streptomyces venezuelae [27]. The synthetase, LanL, resembles the LanKC, but differs at the C-terminal domain. The C-terminal cyclase domain of LanL shows homology to LanC and contains the characteristic zinc-binding motif [27, 28] (Figure 1).

13

1

1.2. Activity of lanthipeptidesThe mechanism by which lantibiotics exert their antimicrobial activities has been fully investigated only in a few cases, showing that the modes of action of lantibiotics are mainly based on the inhibition of cell wall biosynthesis, disruption of membrane integrity through pore forma-tion or a combination of both [2]. In some cases lipid II, the essential precursor for cell wall biosynthesis, serves as the target of lantibiotics to inhibit the growth of bacteria (Figure 2). Nisin binds to the pyro-phosphate moiety of lipid II via the N-terminal ring A and ring B and forms a pyrophosphate cage. Then nisin bends, inserts its C-terminus into the membrane and forms transmembrane pores [29]. Thus, nisin exerts two killing mechanisms: it permeabilizes the membrane and inhibits cell wall synthesis [16, 30, 31].

Since the ring pattern of rings A and B are quite conserved in some lantibiotics other than nisin, including microbisporicin, mutacin 1140, gallidermin and epidermin, the same binding motif can probably also be formed for these antibiotics [2, 32, 33]. However, unlike nisin, the

Figure 1. Schematic representation of the four classes of lanthipeptides, based on the lanthi-

onine introducing modification enzymes (adapted from Knerr et al. 2012 [2]). The dark lines

in LanC and LanC-like cyclase domain represent the conserved Zn-ligands. LanB, lanthipeptide

dehydratase; LanC, lanthipeptide cyclase; LanM, class II lanthipeptide synthetase; LanKC, class

III lanthipeptide synthetase; LanL, class IV lanthipeptide synthetase.

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C-terminal part of epidermin and gallidermin is shorter and thus the compound is unable to translocate over the cell membrane to form pores [32]. Lipid II is also a target for class II lantibiotics such as mersacidin, which inhibits transglycosylation, but does not form pores [34, 35]. Ring C is essential for this interaction and it is conserved in mersacidin-like peptides, which suggests a similar reaction for those peptides [35, 36]. For the two-component lantibiotics, the two peptides work synergisti-cally, in such a way that the α peptide binds to lipid II resembling the mersacidin-binding motif (Figure 2) and the β peptide is involved in

Figure 2. Structures of representative lanthipeptides (adapted from Knerr et.al. 2012 [2]

and Dischinger et.al. 2014 [14]). The canonical nisin- and mersacidin-lipid II binding motifs

are highlighted with green or red dashed circles, respectively. The rings of nisin, haloduracin,

SapT are labelled. Dehydrated amino acids are shown in green. Dha denotes dehydroalanine

and Dhb is dehydrobutyrine. Lan are shown in pink, MeLan are shown in blue. Disulfide bridges

are shown is yellow.

15

1pore formation upon binding to the complex lipid II-α-peptide [37, 38]. Some other activities were also reported, such as for Pep5 and epilancin K7, which do not use lipid II as target, but still form pores [15]. Cinnamycin-like peptides were found to inhibit phospholipase A2

[39]. They bind to phosphatidylethanolamine in the cell wall, induce transbilayer lipid movement and may confer toxic effects [40].

1.3. Additional post-translational modifications (PTMs) of lantibiotics

Lan/MeLan residues define and play vital roles in the biological activity and stability of the lanthipeptides. At present, many other different post-translational modifications (PTMs) have been documented in lantibiotics [2, 41, 42]. The PTMs have greatly enhanced the diversity of the lantibiotics, which initially is limited to 20 canonical amino acids [43]. During the process of Lan/MeLan formation, cysteine residues are involved and bound to unsaturated amino acids dehydroalanine (Dha) and dehydrobutyrine (Dhb). Moreover, the C-terminal cysteine residues can be enzymatically oxidized and decarboxylated render-ing S-aminovinyl-D-cysteine (AviCys) or S-aminovinyl-3-methyl-D- cysteine (AviMeCys) structures, as found in epidermin and mersacidin, respectively [42]. These structures are introduced by EpiD and MrsD, both of which reveal a conserved Rossman fold typically found in flavo-doxin-like proteins [44, 45]. AviCys and AviMeCys can protect the pep-tide from carboxypeptidases and contribute to the full activity [46, 47].

Spontaneous hydrolysis of N-terminally exposed Dha and Dhb resi-dues has also been reported during the maturation of Pep5, epicidin 280 and epilancin 15X [48–50]. Dha and Dhb residues become exposed after leader processing and are subsequently hydrolyzed to yield 2-oxopro-pionyl (OPr) and 2-oxobutyryl (OBu) [51]. OPr can be further reduced by a LanO enzyme to form a 2-hydroxypropionyl (Hop) residue [49, 50]. Acetylation of the N-terminus of mature lantibiotics is observed in paenibacillin, isolated from Paenibacillus polymyxa OSY-DF [52, 53]. This N-terminal capping is likely to protect the compound from aminopeptidases [42].

Moreover, LanJ can convert L-serine (L-Ser) to D-alanine (D-Ala) with Dha as an intermediate in lactocin S, carnolysin and lacticin 3147 [54–56]. The mechanism by which L-Ser is converted to D-Ala

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1 residues in lacticin 3147 is a two-step process involving dehydration by the enzyme LtnM and stereospecific hydrogenation by LtnJ [57]. Cinnamycin and duramycin contain a lysinoalanine bridge (catalyzed by Cinorf7) and a hydroxylated aspartic acid (catalyzed by Cinx) [58, 59]. Microbisporicin exhibits two unique PTMs, a chlorinated trypto-phan (Cl-Trp) and a hydroxylated proline (HPro) [2, 60]. The disulfide ring presented in haloduracin and plantaricin W can protect the com-pounds from proteolytic degradation by proteases [61].

2. Nisin

Nisin, produced by Lactococcus lactis, is one of the oldest and most widely used antimicrobials and was first reported in 1928 [62]. Nisin is a cationic, amphipathic peptide, which can effectively kill Gram- positive bacteria including Bacillus cereus, Listeria monocytogenes, Staphylococci and Streptococci [12, 63, 64]. It is generally recognized as a safe additive in food preservation and recent studies show that it could also be applied as pharmaceutical [30, 64–66].

2.1. Biosynthesis of nisinA two-component system, involving a histidine protein kinase NisK and a response regulator protein NisR, is required for the nisin biosyn-thesis regulation [12, 67] (Figure 3). In response to the external signal, which is fully mature nisin, the sensor kinase NisK phosphorylates itself and transfers a phosphoryl group to a conserved aspartic acid of NisR [12, 67]. NisR triggers the binding of the response regulator to nisA and nisF operators and then activates the transcription of the operons nisABTCIP and nisFEG [63, 68, 69]. The nisA gene encodes precursor nisin that consists of a leader peptide and a core peptide part. After ribosomal synthesis, prenisin can be dehydrated by NisB and the dehydrated residues are coupled to cysteine by NisC to form (methyl)lanthionine rings [12, 70]. Subsequently, the modified peptide is transported out of the cell by the ABC-transporter NisT and then the protease NisP can cut off the N-terminal leader peptide and liber-ate active nisin [71–74]. NisI and NisFEG are immunity proteins that protect the host from the antimicrobial action of nisin [12, 67, 75, 76].

17

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The dehydration, cyclization and transport, which are performed by NisB, NisC and NisT, respectively, are three crucial steps during nisin biosynthesis. It has been reported that a wide range of clinical relevant non-lantibiotic peptides (e.g. enkephalin, angiotensin-(1–7) and an erythropoietin-mimicking-peptide) can be successfully de-hydrated and secreted by a L. lactis strain containing nisBTC genes [77]. In vivo experiments with NisB, NisC and NisT were performed and the results showed that nisin modification enzymes have very relaxed substrate specificities [71, 77, 78]. Therefore, it suggests that the nisin modification system is very useful for efficient biotechno-logical production of various non-lantibiotic peptides with enhanced stability and/or modulated bioactivities.

Figure 3. Mode of biosynthesis of nisin in Lactococcus lactis (based on Oscar P. Kuipers,

et al, 1995 [67], Chan-Ick Cheigh and Yu-Ryang Pyun, 2005 [63]). The extracellular mature

nisin can act as an antimicrobial and the producing cells are protected against the nisin activity

via the immunity system consisting of NisI and NisFEG. Mature nisin can also activate the

biosynthesis of prenisin via NisR and NisK. Promoters marked with star (P*) are controlled by

the two-component system NisRK.

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1 The nisin inducible system (NICE), which employs the auto- induction mechanism of nisin for gene expression, was developed by Kuipers et al. [67, 79]. This induction system has been used to pro-duce a set of lanthionine-containing designed substrates that helped the characterization of the enzyme promiscuity and provided novel antimicrobial molecules. Initially, a pNZ-based vector where nisin mutants or fusions of peptides linked to the nisin leader peptide, un-der the control of PnisA, were constructed and expressed in a ΔnisA derivative of NZ9700. This renders mature molecules without the leader peptide in the culture supernatant, which eventually could give immunity problems. Thus, a two plasmids system was created to overcome the immunity issue when the designed peptides have antimicrobial activity. One contains nisB/C/T (or variants thereof) and the other one contains a polylinker, by which easy cloning of diverse structural genes fused to the nisin leader peptide's sequence can be achieved. Both the enzymes and the polylinker are under the control of the inducible promoter PnisA. In any case, when a gene of interest is placed behind the promoter PnisA on a plasmid [80] or on the chromosome [81–83], the expression of the gene can be triggered by nisin and the modification will be inserted. The modified precursor peptides can be isolated from the supernatant or cytoplasm, depending on whether NisT is included in the expression system or not. The NICE system has been extensively and successfully used for high expression of proteins from different origins for various applications [77, 84–88].

2.2. Protein engineering of nisin Five natural nisin variants have been described so far: nisin A, nisin Z, nisin Q, nisin U1 and nisin U2 [12]. Among them, both nisin A and nisin Z are produced by L. lactis and have only one amino acid differ-ence in position 27 (histidine in Nisin A and asparagine in Nisin Z) [42, 63]. The biological activities of nisin A and nisin Z are reported to be similar [89]. These variants highlight the tolerance of certain residues and domains within the molecule to change.

The engineering of nisin can help us to understand the mode of action, substrate specificity, biosynthesis regulation and biosynthesis machinery as well as to obtain new variants of nisin with altered bio-logical activities [12].

19

1A variety of nisin mutants have been created and reported (Table 1) since the first mutation made for nisin Z in 1992 [90]. Another expres-sion system was reported recently [91], via which nisin can produced with leader and then be processed in vitro later on for downstream applications. This well-established approach enabled the generation of interesting and improved mutants [71, 92].

Among all the mutants made so far, only two mutants with alteration of amino acids connected to the sulfur atoms forming lanthionine bonds were reported (S3T and T13C) [93, 94]. This resulted in the replace-ment of lanthionine by methyllanthionine and vice versa. Both of them strongly reduced the activity of nisin Z against Gram-positive bacteria and their activity on liposomes is also decreased. Thus, the specific ring structure of nisin is vital to the activity of nisin. However, the change of other amino acid residues within the (methyl)lanthionine ring had less impact on the activity (I4, S5, G10, M17 and G18) [90, 92–95]. The mutation I4K/S5F/L6I can even increase the activity of nisin and the stability of S5T was improved compared to that of wide type nisin Z. The lysine at position 12, which is between ring B and ring C, was proved to be a quite tolerant position for substitutions. K12A, K12S, K12P, K12V and K12T displayed slightly enhanced antimicrobial activities relative to nisin and K12A was 2–4 fold more active than nisin against all the nine strains tested [96]. The hinge region between rings A/B/C and rings D/E, which is postulated to be vital to confer flexibility for pore formation, has been quite intensively investigated by amino acids alterations [93, 97–99]. It has been reported that the introduction of aromatic residues or negatively charged residues at any position in the hinge had a negative impact on nisin bioactivity [97]. The introduction of positively charged residues is preferred and generally tolerated. The mutants display an activity similar to wild type nisin, but there are still structural considerations (the bulkier arginine residue showed the most reduced activity) [97]. Mutants N20P, M21V, M21G, M21A, K22G, K22A, K22T and K22S exerted enhanced bioactivity against Gram-positive bacteria including L. lactis, Listeria monocytogenes and/or Staphylococcus aureus [97]. Mutants N20K and M21K have a higher solubility than wide type nisin Z and displayed some antimicrobial activity against Gram- negative bacteria including Shigella flexneri, Pseudomonas aeruginosa and Salmonella enterica [98]. These and other

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1 results indicated a preference for small, positively charged and chiral amino acids within the hinge region. The deletion of amino acids at position 20 and 21, which showed strongly reduced activity, illustrated that the length is also important for the hinge region functionality [93]. In 2012, Des Field and his co-authors reported a series of variants with changes at the position 29 of nisin A [100]. S29G and S29A were found to have enhanced efficacy against Staphylococcus aureus SA113, as well as Escherichia coli, Cronobacter sakazakii and Salmonella enterica [100]. Mutants with truncated nisin A/Z were also reported, and the results illustrated the importance of the ring structures for the activity [92, 93, 101, 102]. The mutant of nisin A with 32 amino acids of nisin A and an amidated C-terminus, kept a similar activity compared to nisin A, while the other mutants lost most of their antimicrobial activity against L. lactis and Micrococcus luteus or other strains (e.g. NisA1-31, NisA1-29, NisA1-20, NisA1-22 and NisA1-29) [92, 101, 102]. Moreover, the introduc-tion of fluorescent labels and tryptophan or its analogues all increase the fluorescent properties of nisin but decrease the activity of the mutants [95, 102, 103]. When a tail, which can facilitate the compounds to pass the outer-membrane of Gram-negative species, was added to nisin or truncated nisin, the activity of fusions against Gram-negative pathogens can be improved [104, 105]. These noticeable achievements by engi-neering nisin encourage the further investigation and application of lantibiotic compounds and provides a novel technology for molecular improvement.

3. Status of antibiotic use

‘Antibiotic’ was firstly used as a designation by Selman Waksman in 1941 to describe any small molecule which was made by a microbe to antago-nize the growth of other microbes [109]. Antibiotic discovery and clinical use is undoubtedly one of the landmark medical advances of modern medicine. Since Alexander Fleming found penicillin in 1928, the earliest use of antibiotics had a dramatic impact on the decrease of mortality of life-threatening bacterial infections. The period from 1945 to 1955, with the development of penicillin, streptomycin, chloramphenicol, and tetracycline, can be regarded as the golden age for antibiotics [109]. The industrial production made antibiotics available for common treatments.

21

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tus o

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se

Tabl

e 1.

Nis

in A

/Z m

utan

ts a

nd th

eir c

hara

cter

istic

s.

IDM

utat

ion

Gen

e na

me

Biol

ogic

al a

ctiv

ity (r

elat

ive

to

the

wild

type

)Ph

ysic

al p

rope

rtie

s (re

lativ

e to

the

wild

type

)C

hara

cter

istic

sR

efer

ence

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

1I1

Wni

sZSi

mila

r act

ivity

aN

DFl

uore

scen

t lab

el[1

03]

2I1

Wni

sARe

duce

d ac

tivity

bN

DIn

trod

ucin

g tr

ypto

phan

[95]

3I1

–5FW

nisA

Redu

ced

activ

ity b

ND

Intr

oduc

ing

tryp

toph

an a

nalo

gue

[95]

4I1

–5H

Wni

sASt

rong

ly re

duce

d ac

tivity

bN

DIn

trod

ucin

g tr

ypto

phan

ana

logu

e[9

5]5

T2S

nisZ

Incr

ease

d ac

tivity

a,c

Redu

ced

activ

ity o

n lip

osom

eD

ha p

rese

nt in

the

final

pro

duct

inst

ead

of D

hb[9

3]6

T2A

nisZ

Sim

ilar a

ctiv

ity a,

cRe

duce

d ac

tivity

on

lipos

ome

Alte

ring

dehy

drat

ed re

sidue

s[9

3]7

T2V

nisZ

Sim

ilar a

ctiv

ity a,

cRe

duce

d ac

tivity

on

lipos

ome

Alte

ring

dehy

drat

ed re

sidue

s[9

3]8

S3T

nisZ

Stro

ngly

redu

ced

activ

ity a,

cRe

duce

d ac

tivity

on

lipos

ome

Dhb

pre

sent

in th

e fin

al p

rodu

ct in

stea

d of

Dha

[93]

9I4

–5FW

nisA

Redu

ced

activ

ity b

ND

Intr

oduc

ing

tryp

toph

an a

nalo

gue

[95]

10I4

K/L

6Ini

sASi

mila

r act

ivity

d,e

ND

Alte

ring

resid

ues i

n rin

g A

of n

isin

A[9

2]11

I4K

/S5F

/L6I

nisA

Incr

ease

d ac

tivity

d,e

ND

Alte

ring

resid

ues i

n rin

g A

of n

isin

A[9

2]12

I4V

/S5F

/L6G

nisA

redu

ced

activ

ity d,

eN

DA

lterin

g re

sidue

s in

ring

A o

f nisi

n A

[92]

13S5

Cni

sZSt

rong

ly re

duce

d ac

tivity

b,c

ND

Alte

ratio

n of

deh

ydra

tabl

e re

sidue

whi

ch ta

kes p

art i

n th

e rin

g fo

rmat

ion

[94]

14S5

Tni

sZRe

duce

d ac

tivity

a,c

Incr

ease

stab

ility

Dhb

pre

sent

in th

e fin

al p

rodu

ct in

stea

d of

Dha

[90]

15D

ha5D

hbni

sZSt

rong

ly re

duce

d ac

tivity

a,c

Incr

ease

stab

ility

D

hb p

rese

nt in

the

final

pro

duct

inst

ead

of D

ha[8

9]16

S5A

nisZ

Redu

ced

activ

ityN

DA

lterin

g de

hydr

ated

resid

ues

[106

]17

S5A

/S33

Ani

sASt

rong

ly re

duce

d ac

tivity

ND

Alte

ring

dehy

drat

ed re

sidue

s[1

06]

18G

10T

nisA

Stro

ngly

redu

ced

activ

ity d,

eN

DA

lterin

g re

sidue

s to

dehy

drat

ed re

sidue

in ri

ng B

of n

isin

A[9

2]19

K12

Hni

sARe

duce

d ac

tivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

20K

12R

nisA

Redu

ced

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]21

K12

Tni

sAIn

crea

sed

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]

22

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

22K

12S

nisA

Incr

ease

d ac

tivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

23K

12N

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]24

K12

Qni

sASi

mila

r act

ivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

25K

12Y

nisA

Redu

ced

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]26

K12

Ani

sAIn

crea

sed

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]27

K12

Pni

sAIn

crea

sed

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]28

K12

Vni

sAIn

crea

sed

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]29

K12

Mni

sASi

mila

r act

ivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

30K

12C

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]31

K12

Lni

sASi

mila

r act

ivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

32K

12I

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]33

K12

Gni

sARe

duce

d ac

tivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

34K

12W

nisA

Redu

ced

activ

ity b

ND

Alte

ring

resid

ue b

etw

een

ring

A a

nd ri

ng B

/C[9

6]35

K12

Fni

sARe

duce

d ac

tivity

bN

DA

lterin

g re

sidue

bet

wee

n rin

g A

and

ring

B/C

[96]

36K

12P

nisZ

Sim

ilar a

ctiv

ity a

ND

Posit

ive

char

ge re

duct

ion

[103

]37

T13C

nisZ

Stro

ngly

redu

ced

activ

ity a,

cSt

rong

ly re

duce

d ac

tivity

on

lipos

ome

Alte

ratio

n of

deh

ydra

tabl

e re

sidue

whi

ch ta

kes p

art i

n th

e rin

g fo

rmat

ion

[93]

38M

17W

nisA

Stro

ngly

redu

ced

activ

ity b

ND

Intr

oduc

ing

tryp

toph

an

[95]

39M

17–5

HW

nisA

Stro

ngly

redu

ced

activ

ity b

ND

Intr

oduc

ing

tryp

toph

an a

nalo

gue

[95]

40M

17W

nisZ

Redu

ced

activ

ity a,

cSi

mila

r act

ivity

on

lipos

ome

Fluo

resc

ent l

abel

[107

]41

M17

Kni

sZRe

duce

d ac

tivity

a,c

Incr

ease

solu

bilit

yPo

sitiv

e ch

arge

intr

oduc

tion

[93]

42M

17C

nisZ

Stro

ngly

redu

ced

activ

ity b,

cN

DIn

trod

uctio

n of

deh

ydra

tabl

e re

sidue

whi

ch ta

kes p

art i

n th

e rin

g fo

rmat

ion

[94]

43M

17Q

/G18

Tni

sZSi

mila

r act

ivity

a,c

ND

Alte

ring

resid

ues i

n rin

g C

of n

isin

Z[9

0]

23

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

44M

17Q

/G18

Dhb

nisZ

Sim

ilar a

ctiv

ity a,

cN

DA

lterin

g re

sidue

s in

ring

C o

f nisi

n Z

[90]

45N

20C

nisA

Stro

ngly

redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

46N

20A

nisA

Redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

47N

20S

nisA

Sim

ilar a

ctiv

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

48N

20T

nisA

Sim

ilar a

ctiv

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

49N

20V

nisA

Redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

50N

20L

nisA

Redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

51N

20I

nisA

Redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

52N

20P

nisA

Incr

ease

d ac

tivity

fN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]St

rong

ly re

duce

d ac

tivity

g53

N20

Fni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]54

N20

Yni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]55

N20

Wni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]56

N20

Dni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]57

N20

Rni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]58

N20

Hni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]59

M21

Nni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]60

M21

Qni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]61

M21

Cni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]62

M21

Gni

sAIn

crea

sed

activ

ity f ,

gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]63

M21

Ani

sAIn

crea

sed

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

64M

21S

nisA

Sim

ilar a

ctiv

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

65M

21T

nisA

Sim

ilar a

ctiv

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

CH

APT

ER 1

: Sta

tus o

f ant

ibio

tic u

se

24

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

66M

21V

nisA

Incr

ease

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]67

M21

Lni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]68

M21

Ini

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]69

M21

Pni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]70

M21

Fni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]71

M21

Yni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]72

M21

Wni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]73

M21

Eni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]74

M21

Rni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]75

M21

Kni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]76

K22

Qni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]77

K22

Gni

sAIn

crea

sed

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

78K

22A

nisA

Incr

ease

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]79

K22

Sni

sAIn

crea

sed

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

80K

22T

nisA

Incr

ease

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]81

K22

Vni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]82

K22

Lni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]83

K22

Pni

sASi

mila

r act

ivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]84

K22

Mni

sARe

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]85

K22

Fni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]86

K22

Wni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]87

K22

Dni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]88

K22

Eni

sASt

rong

ly re

duce

d ac

tivity

f ,gN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

7]

25

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

89K

22R

nisA

Stro

ngly

redu

ced

activ

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

90K

22H

nisA

Sim

ilar a

ctiv

ity f ,g

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[97]

91N

20A

/M21

A/K

22A

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[99]

Stro

ngly

redu

ced

activ

ity f

92N

20A

/M21

Ani

sASt

rong

ly re

duce

d ac

tivity

b,f

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[99]

93N

20S/

M21

A/K

22A

nisA

Stro

ngly

redu

ced

activ

ity b,

fN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

9]94

N20

S/M

21L/

K22

Sni

sASt

rong

ly re

duce

d ac

tivity

b,f

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[99]

95M

21A

/K22

Ini

sASi

mila

r act

ivity

bSt

rong

ly re

duce

d ac

tivity

fN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

9]

96N

20E

nisZ

Stro

ngly

redu

ced

activ

ity a,

cN

DA

lterin

g re

sidue

s in

hing

e re

gion

[9

8]97

N20

Fni

sZSi

mila

r act

ivity

a,c

Sim

ilar s

tabi

lity

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

98N

20H

nisZ

Sim

ilar a

ctiv

ity a,

cSi

mila

r sta

bilit

yA

lterin

g re

sidue

s in

hing

e re

gion

[9

8]99

N20

Kni

sZIn

crea

sed

activ

ity h,

iIn

crea

sed

solu

bilit

yA

lterin

g re

sidue

s in

hing

e re

gion

by

intr

oduc

ing

posit

ive

char

ge[9

8]Si

mila

r act

ivity

a,c

100

N20

Qni

sZSi

mila

r act

ivity

a,c

Impr

oved

stab

ility

in h

ighe

r te

mpe

ratu

re a

nd p

HA

lterin

g re

sidue

s in

hing

e re

gion

[9

8]

101

N20

Vni

sZSt

rong

ly re

duce

d ac

tivity

a,c

Sim

ilar s

tabi

lity

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

102

M21

Eni

sZSt

rong

ly re

duce

d ac

tivity

a,c

ND

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

103

M21

Gni

sZSi

mila

r act

ivity

a,c

Impr

oved

stab

ility

in h

ighe

r te

mpe

ratu

re a

nd p

HA

lterin

g re

sidue

s in

hing

e re

gion

[9

8]

104

M21

Hni

sZSi

mila

r act

ivity

a,c

Sim

ilar s

tabi

lity

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

105

M21

Kni

sZIn

crea

sed

activ

ity h,

iIn

crea

sed

solu

bilit

yA

lterin

g re

sidue

s in

hing

e re

gion

by

intr

oduc

ing

posit

ive

char

ge[9

8]Si

mila

r act

ivity

a,c

106

K22

Gni

sZSi

mila

r act

ivity

a,c

Sim

ilar s

tabi

lity

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

107

K22

Hni

sZSi

mila

r act

ivity

a,c

Sim

ilar s

tabi

lity

Alte

ring

resid

ues i

n hi

nge

regi

on

[98]

CH

APT

ER 1

: Sta

tus o

f ant

ibio

tic u

se

26

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

108

N20

K/M

21K

nisZ

Sim

ilar a

ctiv

ity a,

cSi

mila

r sta

bilit

yD

oubl

e m

utat

ion

of a

spar

agin

e 20

and

met

hion

ine

21 to

lysin

es[9

8]

109

N20

F/M

21L/

K22

Qni

sZSi

mila

r act

ivity

a,c

Sim

ilar s

tabi

lity

Hin

ge re

gion

of n

isinZ

to h

inge

regi

on o

f sub

tilin

[98]

110

N20

A/M

21K

/Dhb

/K

22G

nisZ

Stro

ngly

redu

ced

activ

ity a,

cSi

mila

r sta

bilit

yH

inge

regi

on o

f nisi

nZ to

hin

gere

gion

of e

pide

rmin

[98]

111

N20

P/M

21P

nisZ

Stro

ngly

redu

ced

activ

ity a,

cSt

rong

ly re

duce

d ac

tivity

on

lipos

ome

Alte

ring

resid

ues i

n hi

nge

regi

on

[93]

112

M21

Gni

sZSt

rong

ly re

duce

d ac

tivity

a,c

Stro

ngly

redu

ced

activ

ity o

n lip

osom

eA

lterin

g re

sidue

s in

hing

e re

gion

[9

3]

113

N27

Kni

sZSi

mila

r act

ivity

a,b,c

Incr

ease

d so

lubi

lity

Cha

rge

alte

ratio

n[8

9]11

4S2

9Tni

sARe

duce

d ac

tivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

115

S29Q

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]11

6S2

9Nni

sASi

mila

r act

ivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

117

S29Y

nisA

Redu

ced

activ

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]11

8S2

9Dni

sAIn

crea

sed

activ

ity b,

kN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

Sim

ilar a

ctiv

ity f ,j

119

S29E

nisA

Incr

ease

d ac

tivity

b,h,j

,lN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

Sim

ilar a

ctiv

ity f

120

S29R

nisA

Redu

ced

activ

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]12

1S2

9Hni

sARe

duce

d ac

tivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

122

S29K

nisA

Redu

ced

activ

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]12

3S2

9Ani

sAIn

crea

sed

activ

ity b,

f ,h,j ,k

,lN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

124

S29V

nisA

Redu

ced

activ

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]12

5S2

9Gni

sAIn

crea

sed

activ

ity b,

f ,h,j ,k

,lN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

126

S29C

nisA

Redu

ced

activ

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]

27

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

I Va

rian

ts w

ith fu

ll le

ngth

of n

isin

A/Z

127

S29L

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]12

8S2

9Ini

sARe

duce

d ac

tivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

129

S29W

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]13

0S2

9Fni

sARe

duce

d ac

tivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

131

S29M

nisA

Sim

ilar a

ctiv

ity b

ND

Alte

ring

the

resid

ue at

pos

ition

29

[100

]13

2S2

9Pni

sASi

mila

r act

ivity

bN

DA

lterin

g th

e re

sidue

at p

ositi

on 2

9 [1

00]

133

I30W

nisA

Sim

ilar a

ctiv

ity b

ND

Fluo

resc

ent l

abel

[108

]13

4H

31K

nisZ

Sim

ilar a

ctiv

ity a,

cIn

crea

sed

solu

bilit

yC

harg

e al

tera

tion

[89]

Stro

ngly

redu

ced

activ

ity b

135

V32

Wni

sZRe

duce

d ac

tivity

a,c

ND

Fluo

resc

ent l

abel

[102

]13

6V

32K

nisZ

Redu

ced

activ

ity a,

cN

DPo

sitiv

e ch

arge

intr

oduc

tion

[102

]13

7V

32E

nisZ

Stro

ngly

redu

ced

activ

ity a,

cN

DN

egat

ive

char

ge in

trod

uctio

n[1

02]

138

S33A

nisA

Stro

ngly

redu

ced

activ

ity

ND

Alte

ring

dehy

drat

ed re

sidue

s[1

06]

Part

II V

aria

nts w

ith tr

unca

ted

nisi

n A

/Z13

9ΔN

20/Δ

M21

nisZ

Stro

ngly

redu

ced

activ

ity a,

cSt

rong

ly re

duce

d ac

tivity

on

lipos

ome

Alte

ring

hing

e re

gion

[93]

140

NisZ

1-32 V

32E

nisZ

Stro

ngly

redu

ced

activ

ity a,

cN

DIn

fluen

ce o

f the

C-t

erm

inal

[102

]14

1N

isA1-3

2 am

ide

nisA

Sim

ilar a

ctiv

ity b,

mN

DPr

oteo

lytic

ally

clea

ved,

all

lant

hion

ine

ring

pres

ent

[101

]14

2N

isA1-3

1ni

sASt

rong

ly re

duce

d ac

tivity

bN

DPr

oteo

lytic

ally

clea

ved,

all

lant

hion

ine

ring

pres

ent

[101

]14

3N

isA1-2

9ni

sASt

rong

ly re

duce

d ac

tivity

b,m

ND

Prot

eoly

tical

ly cl

eave

d, a

ll la

nthi

onin

e rin

g pr

esen

t[1

01]

144

NisA

1-20

nisA

Stro

ngly

redu

ced

activ

ity b,

mN

DPr

oteo

lytic

ally

clea

ved,

ring

D a

nd E

rem

oved

[101

]14

5N

isA1-1

2ni

sASt

rong

ly re

duce

d ac

tivity

b,m

ND

Prot

eoly

tical

ly cl

eave

d, ri

ng C

, D a

nd E

rem

oved

[101

]14

6N

isA1-2

2ni

sASt

rong

ly re

duce

d ac

tivity

bN

Drin

g D

and

E re

mov

ed[9

2]14

7N

isA1-2

2 G10

Tni

sASt

rong

ly re

duce

d ac

tivity

bN

Drin

g D

and

E re

mov

ed, A

lterin

g re

sidue

s in

ring

B of

nisi

n A

[92]

CHAPTER 1: Status of antibiotic use

CH

APT

ER 1

: Sta

tus o

f ant

ibio

tic u

se

28

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1ID

Mut

atio

nG

ene

nam

eBi

olog

ical

act

ivity

(rel

ativ

e to

th

e w

ild ty

pe)

Phys

ical

pro

pert

ies (

rela

tive

to th

e w

ild ty

pe)

Cha

ract

eris

tics

Ref

eren

ce

Part

II V

aria

nts w

ith tr

unca

ted

nisi

n A

/Z14

8N

isA1-2

2 I4K

/L6I

nisA

Stro

ngly

redu

ced

activ

ity b

ND

ring

D a

nd E

rem

oved

, Alte

ring

resid

ues i

n rin

g A

of n

isin

A[9

2]14

9N

isA1-2

2I4

K/S

5F/L

6Ini

sASt

rong

ly re

duce

d ac

tivity

bN

Drin

g D

and

E re

mov

ed, A

lterin

g re

sidue

s in

ring

A o

f nisi

n A

[92]

150

NisA

1-22 I

4V/S

5F/L

6Gni

sASt

rong

ly re

duce

d ac

tivity

bN

Drin

g D

and

E re

mov

ed, A

lterin

g re

sidue

s in

ring

A o

f nisi

n A

[92]

Part

III V

aria

nts w

ith n

isin

A a

nd ta

ils15

1N

isA1-3

4PR

PPH

PRL

nisA

Incr

ease

d ac

tivity

lN

D“P

RPPH

PRL”

wer

e ad

ded

after

nisi

n A

[105

]St

rong

ly re

duce

d ac

tivity

b15

2N

isA1-3

4N

GV

QPK

Yni

sAIn

crea

sed

activ

ity l,i

,n,o

ND

“NG

VQ

PKY”

wer

e ad

ded

after

nisi

n A

[104

]St

rong

ly re

duce

d ac

tivity

b15

3N

isA1-2

8SV

NG

VQ

PKYK

nisA

Incr

ease

d ac

tivity

l,i,n,

pN

D“S

VN

GV

QPK

YK” w

ere

adde

d aft

er ri

ng A

BCD

E of

nisi

n A

[104

]St

rong

ly re

duce

d ac

tivity

b15

4N

isA1-2

8SV

KIA

KVA

LKA

LKni

sAIn

crea

sed

activ

ity l,i

,n,o,p

ND

“SV

KIA

KVA

LKA

LK” w

ere

adde

d aft

er ri

ng A

BCD

E of

nisi

n A

[104

]St

rong

ly re

duce

d ac

tivity

b15

5N

isA1-2

8SV

PRPP

HPR

LKni

sAIn

crea

sed

activ

ity l,i

,n,o,p

ND

“SV

PRPP

HPR

LK” w

ere

adde

d aft

er ri

ng A

BCD

E of

nisi

n A

[104

]St

rong

ly re

duce

d ac

tivity

b

Not

e: N

D, n

ot d

eter

min

ed.

(1) I

ncre

ased

act

ivity

, >12

0 % co

mpa

red

to th

e ac

tivity

of w

ild ty

pe n

isin

A/Z

; Sim

ilar a

ctiv

ity, 8

0 %-1

00 %

; Red

uced

act

ivity

, 20–

80 %

; Str

ongl

y re

duce

d ac

tivity

, <20

%.

(2) C

olum

ns a

re la

belle

d ac

cord

ing

to th

e bi

olog

ical

act

iviti

es o

f the

mut

ants

. Mut

ants

with

incr

ease

d bi

olog

ical

act

ivity

are

labe

lled

as b

old

and

dark

gre

y; m

utan

ts w

ith si

mila

r bi

olog

ical

act

ivity

are

labe

lled

as li

ght g

ray.

(3) 5

FW, 5

-fluo

rotr

ypto

phan

; 5H

W, 5

-hyd

roxy

tryp

toph

an. H

inge

regi

on, a

min

o ac

id re

sidue

s bet

wee

n rin

g A

/B/C

and

ring

D/E

; ΔN

20/Δ

M21

, del

etio

n of

asp

arag

ine

in p

ositi

on 2

0 an

d m

ethi

onin

e in

pos

ition

21; N

umbe

r in

supe

rscr

ipt,

amin

o ac

id p

ositi

on.

(4) B

iolo

gica

l act

ivity

(rel

ativ

e to

the

wild

type

), le

tter i

n su

pers

crip

t for

indi

cato

r str

ains

: a, M

icroc

occu

s flav

us; b

, Lac

toco

ccus

lact

is; c,

Stre

ptoc

occu

s the

rmop

hiles

; d, P

edio

cocc

us

pent

osac

eus;

e, Le

ucon

osto

c mes

ente

roid

es; f

, Sta

phyl

ococ

cus a

ureu

s; g,

Stre

ptoc

occu

s aga

lact

iae,

h, S

alm

onell

a en

teric

a; i,

Pse

udom

onas

aer

ugin

osa;

j, B

acill

us ce

reus

; k, C

rono

bact

er

saka

zaki

i; l,

Esch

erich

ia co

li; m

, Micr

ococ

cus l

uteu

s; n,

Kleb

siella

pne

umon

iae;

o, A

cinet

obac

ter b

aum

anni

i; p,

Ent

erob

acte

r aer

ogen

es.

29

1

CH

APT

ER 1

: Bac

teria

l cel

l env

elop

e an

d an

tibio

tics a

ctin

g at

the

cell

enve

lope

According to a report for 71 selected countries, between 2000 and 2010, the consumption of antibiotic drugs substantially increased by 35 % (from 52,057,163,835 standard units in 2000 to 70,440,786,553 standard units in 2010, where standard unit means a single dose unit, i.e. pill/cap-sule/or ampoule). Among these selected countries, India was revealed to be the biggest consumer of antibiotics in 2010 with the consumption of 12.9 × 109 units in total and 10.7 units per person, followed by China (approximate 10.0 × 109 units in total, 7.5 units per person) and the USA (6.8 × 109 units in total, 22.0 units per person, with a moderate decrease from 2000 to 2010 actually) [110]. Moderately high consumption of an-tibiotics was also reported for Australia and New Zealand. The antibiotic consumption increased substantially in developing countries, and the highest rates are found in BRICS countries (Brazil, Russia, India, China, and South Africa) and French West Africa. An increased consumption of glycopeptides, carbapenems, polymixins, and monobactams was observed and reported in many countries [110].

The widespread excessive or sometimes abusive use of the antibiot-ics in agriculture, veterinary and human medicine is one of the main reasons for the dramatic increase of bacterial resistance [111, 112]. In order to fight the increased resistance of bacteria to existing antibiotics, there is a rather urgent need for discovering and developing new anti-biotics. However, the time-consuming and costly clinical development process and unpredictable economic benefit of antibiotics at the generic market has lost its attractiveness to pharmaceutical industry [113]. As a consequence, only a few antibiotics reached the market in recent decades [114–116]. Figure 4 shows the novel antibiotics and the total number of molecules approved by US Food and Drug Administration (FDA) for each year from 2003 to 2017 [114].

4. Bacterial cell envelope and antibiotics acting at the cell envelope

4.1. Bacterial cell envelope Bacteria face various environments, which are usually unpredictable and hostile. To survive, bacteria have evolved a sophisticated and complex cell envelop that acts as a barrier to the environment and

30

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1 protects them [117, 118]. The cell envelope comprises membrane(s) and other structures that surround and protect the cytoplasm. Besides protection, the cell envelope allows the selective transport of nutrients from the outside and waste products from the inside of the cells, and it is also indispensable for division, growth, and morphogenesis [117].

The cell envelope can fall in two major categories, Gram-negative and Gram-positive (Figure 5), distinguished by Gram staining [117]. There are two membranes in Gram-negative bacteria, the outer membrane (OM), a thin peptidoglycan layer in between, and the cytoplasmic or inner membrane (IM). The OM is composed of glycolipids, princi-pally lipopolysaccharide (LPS) [119] and it is responsible for the low penetrability and high resistance to some antibiotics [120, 121]. LPS is critical to the barrier function of OM and is responsible for the endo-toxic shock associated with the septicemia caused by Gram-negative organisms [117, 122]. The proteins of the OM can be divided into two categories, lipoproteins (LP) and β-barrel proteins, which are also called outer membrane proteins (OMP). The thin peptidoglycan layer cannot retain the crystal violet stain upon decoloration with ethanol during Gram staining. The Gram-positive bacteria lack a defined periplasmic space and OM and the cytoplasmic membrane is surrounded by a very thick peptidoglycan (PG) layer (30–100 nm) with other molecules, e.g. teichoic acids, attached to it [117]. The PG is essential for morphol-ogy and responsible for the retention of the crystal violet dye during Gram staining procedure [123]. The PG is composed of a disaccharide- peptide repeat coupled through glycosidic bonds to form linear glycan strands and peptide bonds to link the glycan strands. It differs among different Gram-positive bacteria [117, 118, 124].

4.2. Outer membrane (OM) permeabilityThe outer-membrane (OM) of Gram-negative bacteria is crucial for bacterial survival in harsh environment and serves as a selective and low penetrable barrier for the exchange of materials [125, 126]. The OM is mostly an asymmetric and highly hydrophobic bilayer com-posed of glycerol phospholipids and LPS, as well as pore-forming proteins of specific size-exclusion properties. A typical LPS molecule consists of three parts: 1) lipid A, a glucosamine-based phospholipid; 2) a core oligosaccharide and 3) a distal polysaccharide (O-antigen)

https://en.wikipedia.org/wiki/Gram_staining

31

1

Figure 4. Number of novel FDA-approved drugs by year (Based on Stefan et al. 2018 [114]).

Figure 5. Schematic overview of the Gram-positive and Gram-negative cell envelope (adapted

from Thomas J. Silhavy et al. 2010 [117] and Samuel I. Miller 2016 [125]). Peptidoglycan

(PG) layer is much thinner in Gram-negative bacteria than in Gram-positive bacteria. WTA, wall

teichoic acid; CAP, covalently attached protein; LTA, lipoteichoic acid; IMP, inner membrane

protein; LPS, lipopolysaccharide; LP, lipoprotein; OMP, outer membrane protein.

CH

APT

ER 1

: Bac

teria

l cel

l env

elop

e an

d an

tibio

tics a

ctin

g at

the

cell

enve

lope

32

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1 [122]. The length of the core oligosaccharide and O-antigen varies in different strains of E. coli since these structures are not essential for the species growth. Some of the core oligosaccharide and O-antigen sugars contain phosphate groups that mediate the interaction with divalent metal ions, e.g. Mg2+, and this contributes to the tightly assembled structure of LPS. This well-packed structure creates an extremely or-dered network with sugar chains on the cell face. This well-assembled and low fluidity surface, hydrophobicity of LPS, as well as the diverse and widely distributed efflux pumps [127], are directly responsible for the low penetration of OM for some compounds [126, 128].

Although the composition and tightly-packed surface of the OM prevent the access of antibiotics and other molecules to the cytoplasm, this barrier also presents opportunities for the uptake of some com-ponents [125, 129]. There are two pathways for antibiotics to traverse the OM other than disruption and permeabilization of the OM barri-er with polymyxins and other cationic antimicrobial peptides. Some small hydrophobic antibiotics, such as chloramphenicol, macrolides (erythromycin), rifamycins, novobiocin, fusidic acid and aminoglyco-sides (gentamycin, kanamycin), are able to diffuse through the lipid components of the OM. Specific β-barrel proteins can form porins or selective channels and allow hydrophilic compounds, e.g. penicillin and other β-lactam-based antibiotics, to pass through the OM [128, 130].

Notably, it was reported that both the presence of porins (OmpF) and the manipulations that disrupt the OM can sensitize drug flux and susceptibility of quinolones [131, 132]. What is more, there is an equilibrium of charged and uncharged species of quinolones de-pending on the pH. The quinolone molecules with negative charge are prone to pass through porin channels, while the uncharged quinolone molecules prefer the lipid-mediated pathway [133]. Porin-deficient mutants of E. coli were more resistant to tetracycline than the wild-type (increased minimal inhibitory concentration of tetracycline against E. coli) [133–135] and uncharged tetracycline was observed to enter the cell via diffusion through the lipid layer of OM [133]. Thus, quinolones and tetracycline can utilize both pathways to pass through the OM depending on their protonation status.

P. aeruginosa is less susceptible to most antibiotics than other Gram-negative microorganisms and this phenomenon was initially

33

1believed to be due to active efflux pumps [128, 129, 136, 137]. It was recently shown that the triclosan resistance of P. aeruginosa PAO1 is due to the carriage of an insensitive allele of fabI which encodes an enoyl-ACP reductase enzyme (the target for triclosan in sensitive species) [130, 138]. S. typhimurium was found to rapidly regulate membrane permeability via alteration of OM porins in peroxide treatment [139].

Daptomycin and vancomycin are both active against Gram-positive bacteria, but not effective against Gram-negative bacteria. However, the causes of their inefficiency against Gram-negatives are different. The antibacterial mechanism of action of daptomycin is the Ca2+- mediated insertion into the cytoplasmic membrane causing depolar-ization and the loss of intracellular contents [140, 141]. Nevertheless, the lower proportion of anionic phospholipids in the cytoplasmic membrane in Gram-negative bacteria reduced the efficiency of dap-tomycin insertion [140]. As for vancomycin, its target is D-Ala-D-Ala peptides in lipid II and then inhibits the crosslinking of peptidoglycan. However, vancomycin cannot pass through the OM and reach its target in the periplasm [130, 142].

Moreover, the OM of Gram-negative bacteria is hard to penetrate, but there are still some reports of peptides which can pass through the membrane and inhibit the growth of the bacteria [104, 143, 144].

4.3. Antibiotics acting at peptidoglycan (PG)The cell envelope is one of the main targets for numerous antibiotics, including some with high clinical relevance [30, 145, 146]. Antibiot-ics either inhibit the activity of enzymes or sequester the substrates [118, 146]. The first committed step of peptidoglycan (PG) synthesis is inhibited by fosfomycin, of which MurA is the target. Fosfomycin inactivates the MurA-catalyzed reaction acting as a structural analog of the cosubstrate of the reaction [118, 147]. D-Cycloserine can in-hibit both D-alanine racemase and D-alanine/D-alanine ligase, which finally prevents the crosslinking of the peptidoglycan network [148, 149]. Lipid II has been recognized as the target for lots of antibiotics including lantibiotics, ramoplanin, vancomycin or bacitracin [2, 12, 30, 66]. Nisin links to the pyrophosphate and forms a pyrophosphate cage [12, 30]. Vancomycin and other glycopeptide antibiotics, such as

CH

APT

ER 1

: Bac

teria

l cel

l env

elop

e an

d an

tibio

tics a

ctin

g at

the

cell

enve

lope

34

CH

APT

ER 1

: Gen

eral

Intr

oduc

tion

1 teicoplanin, also bind to lipid II, but to the D-alanine dipeptide termi-nus. Thus, they will block the glycan polymerization and cross-linking [150, 151]. Ramoplanin is produced by non-ribosomal peptide syn-thesis, and binds to lipid II on the external surface of the membrane as well [152, 153]. Bacitracin, a cyclic nonribosomally synthesized do-decylepeptide antibiotic, binds tightly to undecaprenyl pyrophosphate and then prevents the cycling of the lipid carrier by dephosphorylation [154, 155]. Penicillin and some other β-lactams inhibit the formation of peptidoglycan cross links through covalently modify the active site of transpeptidases, which are also called penicillin-binding proteins (PBPs). The β-lactam antibiotics are analogues of the D-alanyl-D- alanine terminus of the pentapeptide side chain, and the amount of PBPs and the affinities of PBPs binding β-lactams vary among bacterial species [118, 145, 151].

5. Antibiotic resistance (AR)

Antibiotic resistance (AR) refers to the ability of microorganisms to resist the effect of an antibiotic, which was once successfully used to fight the microbe [156, 157]. AR has been an issue since the introduc-tion of the first agents into clinical use in the 1940s and became one of the most serious global public health threats in this century [111, 157]. The development of AR is a natural ecological phenomenon and AR has been found in the microorganism from pristine sites, e.g. isolated caves and permafrost [158, 159]. AR has brought enormous damages to human health and economy throughout the world [160]. What is even worse, in recent times, the development of bacterial resistance to several antibiotic classes has resulted in quite dangerous multidrug- resistant (MDR) bacterial strains such as methicillin-resistant Staph-ylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) [112], carbapenem-resistant Enterobacteriaceae (CRE) [161], multidrug-resistant Acinetobacter baumannii (MRAB) [162], third generation penicillin-resistant Enterobacter aerogenes and Klebsiella pneumoniae strains [112, 161], or MDR Salmonella typhimurium phage type DT10 [157]. It was reported in Europe in 2007 that the number of infections by MDR bacteria was 400,000. The cost associated with these

35

1

CH

APT

ER 1

: Ant

ibio

tic re

sista

nce

(AR)

infections in terms of extra hospital expense and productivity losses exceed €1.5 billion annually [163]. In the United States, antibiotic- resistant infections render $20 billion per year in excess health care costs per year and 23,000 deaths as a direct result [163, 164].

5.1. Intrinsic resistancesBacteria can be intrinsically resistant to certain antibiotics, which can be explained by their inherent structural or functional characteristics. As discussed in section 4.2, the protective OM of Gram-negative bacte-ria acts as an efficient barrier to prevent several antibiotics (vancomy-cin, teicoplanin, nisin, gallidermin, epidermin, mersacidin and other lantibiotics) from reaching their targets at the cytoplasmic membrane and/or the cytoplasm, which complicates treatments towards (multi-drug-resistance (MDR)) Gram-negative pathogens [165, 166]. The intrinsic difference of the cytoplasmic membrane of Gram-negative bacteria and Gram-positive bacteria affects the Ca2+ mediated inser-tion of daptomycin as well as the antibiotic efficiency of daptomycin [140]. Like OM, biofilms in P. aeruginosa, E. coli and S. epidermidis behave as an impenetratable barrier to the diffusion of antibiotics and reducing the efficiency of antibiotics [167, 168]. Besides, efflux pumps are capable of transporting antibiotics out of the bacterial cell and then exhibit resistance to certain compounds [127, 169]. In Enterobacteri-aceae, Pseudomonas spp. and Acinetobacter spp., reduction of porin expression was proved to contribute to the resistance to carbapenems and cephalosporins [170–172]. These approaches for AR are all a re-sult of the absence of susceptible targets of specific antibiotics or the difficulty to reach them.

Recently, many genes have been identified to be responsible for in-trinsic resistance to antibiotics [141, 173]. Isolates of Gram-negative bacteria such as K. pneumoniae, E. coli, P. aeruginosa and A. baumannii have emerged to be resistant to all β-lactam antibiotics as a result of β-lactamases production in the strains [174–176]. It was reported that various phenotypes of E. coli can be generated from genes knockouts and the susceptibility of these strains displayed significantly increased sensitivity to at least one of the antibiotics (e.g. triclosan, rifampin, nitrofurantoin, aminoglycosides and β-lactams) [173].

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1 5.2. Acquired resistancesOne of the main intrinsic capacities for Gram-negative bacteria to protect themselves against antibiotics is prevention of access to the antibiotic target. However, it can also be achieved by a new acquisition. The exposure to carbapenems exerted a selective pressure and accu-mulated emergence of mutations in porin genes as well as the genes that regulate porin expression in Enterobacter spp.. The alteration in porin expression, including the shift of porin expression and lack of porins, contributed to the reduced permeability and the strain’s adaptive response to carbapenems treatment [171, 177, 178]. It was reported that an IncH1 plasmid, isolated from a Citrobacter freundii strain, was shown to carry genes coding a tripartite resistance nodu-lation division (RND) pump [179]. Thus, this resistance mechanism became transmissible.

The second strategy for bacterial acquisition of AR is the alteration or modification of the targets. AR can be acquired by alteration of target proteins, e.g. methylation of the ribosome [180, 181], or genetic exchange of the targets, including mutations of one or more genes [182], transformation by plasmids [183], transduction of plasmids [157], conjugation of plasmids [184], transposons [185] and integrons [157], both between and within species [157]. The chloramphenicol- florfenicol resistance (cfr) methyltransferase can specifically methyl-ate A2503 in the 23S rRNA, which then confer resistance to various antibiotics that have targets near this site [186]. Uptake of DNA from the environment leads to the formation of mosaic genes and confers antibiotic resistance by target protein modification. A mosaic penA, which encodes a penicillin-binding protein in N. gonorrhoeae, was found to exhibit high-level resistance to cefixime and ceftriaxone [187]. In methicillin-resistant S. aureus (MRSA), mecC and mecA are two allele genes, which encode the β-lactam-insensitive protein. The isolates of MRSA carrying mecC are more sensitive to oxacillin than the ones carrying mecA [188].

In addition, bacteria also exhibit resistance to antibiotics via inac-tivation of antibiotics via hydrolysis or transfer of a chemical group that blocks their action. Diverse enzymes have been identified that can degrade and modify antibiotics since the discovery of penicilli-nase in 1940 [176, 189–191]. Gram-negative bacteria carrying diverse

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extended-spectrum β-lactamases (ESBLs) and carbapenemases were found to be resistant to all β-lactam antibiotics [174–176]. Aminogly-coside antibiotics are normally large molecules with exposed hydroxyl and amide groups and they are prone to be modified by three amino-glycoside-modifying enzymes: acetyltransferases, phosphotransferases and nucleotidyltransferases. This modification was reported to cause resistance of Campylobacter coli to several aminoglycoside antibiotics including gentamicin, neomycin, streptomycin and kanamycin [192].

5.3. Concluding remarksThe World Health Organization (WHO) published a report to give a global priority list of antibiotic-resistant bacteria to guide research, discovery and development of antibiotics [193]. Remarkably, 9 of these 12 “superbugs” are Gram-negative pathogens while the 3 “critical” bacteria are all Gram-negative pathogens (Acinetobacter baumannii (carbapenem-resistant), Pseudomonas aeruginosa (carbapenem-resis-tant) and Enterobacteriaceae (carbapenem-resistant, 3rd generation cephalosporin-resistant) [193]. Therefore searching new antibiotics or new therapeutic strategies against Gram-negative organisms is important and urgent.

Understanding the molecular mechanism of intrinsic bacterial resis-tance as well as the spectrum of activities of antibiotics, can therefore identify and guide a novel drug combinations and a design of modi-fication of antibiotics. In vitro synergism has been identified between combinations of antibiotics or antibiotics with other compounds, which can be used to target specific problematic pathogens [194–198].

6. Outline of this thesis

The research work described in this thesis focuses on further engineer-ing of nisin-like lantibiotics and the use of nisin/vancomycin in synergy with other peptides. The NICE system is used in various hosts to func-tionally express different biologically important proteins, including the introduction of Melan/Lan to clinically relevant peptides to improve their stability and pharmacodynamic properties [9, 11, 77, 87, 88, 199, 200]. Nisin and vancomycin are quite active against Gram-positive

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1 bacteria but not against Gram-negative bacteria because of the low penetrating capacity of the outer-membrane. If nisin/vancomycin can reach the inner-membrane of Gram-negative bacteria, the activity of nisin/vancomycin can be highly improved [104].

There are 6 chapters in this thesis. Chapter 1 contains a general introduction of 1) antimicrobial classes, PTMs and mode of action of lanthipeptides; 2) biosynthesis and engineering of nisin; 3) current status of antibiotic use;4) differences and antibiotic targets of cell en-velopes of bacteria. 5) antibiotic resistances. The NICE system is also introduced in chapter 1.

Chapter 2: The NICE and NisBTC systems were applied to intro-ducing a lanthionine bridge into vasopressin. Thus, the first cysteine of wild type vasopressin was changed to serine, and then expressed in the NICE