Coxiella burnetii Coxiella burnetii Avoids Avoids Macrophage Macrophage

Phagocytosis by Phagocytosis by InterferingInterferingwith Spatial with Spatial

Distribution of Distribution of Complement Receptor Complement Receptor

33By By

Lionel WilliamsonLionel Williamson

Introduction Introduction

Coxiella Coxiella burnetii burnetii is an obligate intracellular is an obligate intracellular bacteriabacteria

Its survival strategy in monocytes/ Its survival strategy in monocytes/ macrophages is based on growth in acidic macrophages is based on growth in acidic phagosomes that do not fuse with phagosomes that do not fuse with lysosomeslysosomes

Infection of humans usually occurs by Infection of humans usually occurs by inhalation of these organisms from air that inhalation of these organisms from air that contains airborne barnyard dust contains airborne barnyard dust contaminated by dried placental material, contaminated by dried placental material, birth fluids, and excreta of infected herd birth fluids, and excreta of infected herd animals animals

Introduction Introduction

It causes Q fever It causes Q fever In 1999, Q fever became a reportable In 1999, Q fever became a reportable

disease in the United Statesdisease in the United States Most acute cases of Q fever begin with Most acute cases of Q fever begin with

sudden onset of one or more of the sudden onset of one or more of the following: high fevers (up to 104-105° F), following: high fevers (up to 104-105° F), severe headache, general malaise and severe headache, general malaise and myalgia, confusion, sore throat, chills, myalgia, confusion, sore throat, chills, sweats, non-productive cough, nausea, sweats, non-productive cough, nausea, vomiting, diarrhea, abdominal pain, and vomiting, diarrhea, abdominal pain, and chest pain chest pain

As many as 65% of persons with chronic Q As many as 65% of persons with chronic Q fever may die of the disease.   fever may die of the disease.  

Overview of paperOverview of paper

The critical part of infection is C. The critical part of infection is C. burnetiiburnetii getting into the monocyte getting into the monocyte

Difference between virulent and Difference between virulent and avirulent strains is this ability to avirulent strains is this ability to survive in the monocytesurvive in the monocyte

Key terms Key terms

Complement receptor 3 (CD11b:CD18) Complement receptor 3 (CD11b:CD18) binds to a pathogen surface that has binds to a pathogen surface that has been opsonized with complement and been opsonized with complement and promotes pathogen phagocytosispromotes pathogen phagocytosis

CD- cluster of differentiationCD- cluster of differentiation Integrin are heterodimeric cell surface Integrin are heterodimeric cell surface

proteins involved in cell-cell and cell-proteins involved in cell-cell and cell-matrix interactions they are involved matrix interactions they are involved in adhesive interactions and ligand in adhesive interactions and ligand bindingbinding

Key termsKey terms

RANTES- is a chemokine that RANTES- is a chemokine that stimulates the migration and stimulates the migration and activation of cells especially activation of cells especially phagocytic cells. It induced phagocytic cells. It induced psuedopodal extensions that psuedopodal extensions that expressed CR3 In THP-1 cellsexpressed CR3 In THP-1 cells

HIV-1 Nef also acts like a chemokine HIV-1 Nef also acts like a chemokine stimulating migration inside the stimulating migration inside the phagocyte. Same as above phagocyte. Same as above

What did they knowWhat did they know

They knew that uptake of the They knew that uptake of the avirulent strain was mediated by avirulent strain was mediated by άβάβ integrin and CR3integrin and CR3

Virulent strain entered the monocyte Virulent strain entered the monocyte through through άβάβ integrin but interfered integrin but interfered with the CR3 which appeared to with the CR3 which appeared to prevent phagocytosisprevent phagocytosis

What did they knowWhat did they know

CR3 must be activated to promote CR3 must be activated to promote ligand binding and phagocytosisligand binding and phagocytosis

Activation required signals from Activation required signals from other integrins, chemoattractant other integrins, chemoattractant receptors or LPS receptorsreceptors or LPS receptors

Ex. ligation of Ex. ligation of άβάβ integrin and integrin and integrin associated protein (IAP) integrin associated protein (IAP) enhances CR3 bindingenhances CR3 binding

What did they knowWhat did they know

CR3 clustering is correlated to increased CR3 clustering is correlated to increased binding and phagocytosis (Immobile form)binding and phagocytosis (Immobile form)

The switch from an inactive to an active The switch from an inactive to an active form is associated with an alteration in the form is associated with an alteration in the mechanism of interaction CR3 with the mechanism of interaction CR3 with the cytoskeletoncytoskeleton

An immobile subset of CR3 linked to the An immobile subset of CR3 linked to the cytoskeleton is more efficient for cytoskeleton is more efficient for phagocytosis than freely diffusing phagocytosis than freely diffusing receptorsreceptors

What did they knowWhat did they know

F- actin is part of the cytoskeleton F- actin is part of the cytoskeleton infrastructureinfrastructure

The virulent but not the avirulent The virulent but not the avirulent strain has the ability to reorganize strain has the ability to reorganize the F-actin in THP-1 monocytesthe F-actin in THP-1 monocytes

They hypothesized that C. They hypothesized that C. burnetii -burnetii -mediated impairment of CR3 activity mediated impairment of CR3 activity may result from effects of actin may result from effects of actin reorganizationreorganization

Receptor distribution in C. Receptor distribution in C. burnetiiburnetii-stimulated monocytes-stimulated monocytes

THP-1 monocytes were tagged with THP-1 monocytes were tagged with mAb directed to monocyte receptors mAb directed to monocyte receptors involved in C. burnetii recognitioninvolved in C. burnetii recognition

Large beads coated with anti mouse Large beads coated with anti mouse IgG Abs and F-actin labelingIgG Abs and F-actin labeling

Also beads were attached to Also beads were attached to monocytes tagged with mAb directed monocytes tagged with mAb directed CD11b, CD18, CD11b, CD18, άβάβ integrin, IAP (no integrin, IAP (no bead phagocytosis was observed)bead phagocytosis was observed)

Exp. contExp. cont

THP-1 were stimulated with virulent THP-1 were stimulated with virulent C. C. burnetii burnetii

The stimulation caused F-actin The stimulation caused F-actin reorganization and psuedopodal reorganization and psuedopodal extensions in 60% and 40% did not extensions in 60% and 40% did not change change

In the 40% beads bound to Cd11b In the 40% beads bound to Cd11b and CD18, and CD18, άβάβ integrin, and IAP were integrin, and IAP were bound randomly as in the control bound randomly as in the control cellscells

contcont

In the 60% all the beads attached to CD11 In the 60% all the beads attached to CD11 and CD18 were found outside the and CD18 were found outside the deformation areadeformation area

This response was seen in as early as 10 This response was seen in as early as 10 min and continued for about 60 minmin and continued for about 60 min

In contrast the beads bound to In contrast the beads bound to άβάβ integrin integrin were present at the leading edge of the were present at the leading edge of the protrusion (1/4 of protrusion (1/4 of άβάβ integrin was found on integrin was found on the protrusion)the protrusion)

The pattern of IAP was similar to the integrinThe pattern of IAP was similar to the integrin

contcont

contcont

TO prove that the lack of CR3 on the TO prove that the lack of CR3 on the protrusion was not caused by the size of protrusion was not caused by the size of the beads they did the experiment using the beads they did the experiment using smaller beadssmaller beads

They were able to detect CR3 on the They were able to detect CR3 on the protrusions in <10% compared to 25% of protrusions in <10% compared to 25% of άβάβ integrin, and IAP were detected on the integrin, and IAP were detected on the protrusionsprotrusions

Confirmed that CR3 was excluded from the Confirmed that CR3 was excluded from the extensions extensions

contcont

ContCont

They repeated the experiment with They repeated the experiment with circulating monocytes with the same circulating monocytes with the same resultsresults

Avirulent strain did not induce Avirulent strain did not induce psuedopodal extension or F-actin psuedopodal extension or F-actin reorganization and as a consequence reorganization and as a consequence the distribution of CD11b, CD18, the distribution of CD11b, CD18, άβάβ integrin, and IAP was similar to integrin, and IAP was similar to control cellscontrol cells

Receptor distribution in RANTES-Receptor distribution in RANTES-stimulated monocytesstimulated monocytes

They wondered if CR3 rearrangement They wondered if CR3 rearrangement was specific to C. was specific to C. burnetii burnetii or was it or was it indicative of any rearrangements of indicative of any rearrangements of F-actinF-actin

Stimulated THP-1 cells with RANTES Stimulated THP-1 cells with RANTES and was analyzed by transmission and was analyzed by transmission electron microscopeelectron microscope

Observed pseudopodia similar to that Observed pseudopodia similar to that elicited by Celicited by C. burnetii. burnetii

contcont

contcont

Using the same technique they found Using the same technique they found that beads attached to CD11 and that beads attached to CD11 and CD18 were found in psuedopodal CD18 were found in psuedopodal extensions of monocytes stimulated extensions of monocytes stimulated by RANTES(30and 25% respectively)by RANTES(30and 25% respectively)

The distribution of beads The distribution of beads άβάβ integrin, integrin, and IAP were similar to those of C. and IAP were similar to those of C. burnetiiburnetii

This proves that C. burnetii specifically This proves that C. burnetii specifically excludes CR3 from pseudopodsexcludes CR3 from pseudopods

contcont

RANTES-mediated restoration of RANTES-mediated restoration of C. C. burnetiiburnetii phagocytosis phagocytosis

Treated some cells with RANTES and Treated some cells with RANTES and the other C. the other C. burnetiiburnetii

Then incubated the cells with Then incubated the cells with particles which need CR3 for optimal particles which need CR3 for optimal binding to monocytes (IC3b-coated E, binding to monocytes (IC3b-coated E, zymosan, and Avirulent C. zymosan, and Avirulent C. burnetiiburnetii) )

contcont

40% of particles associated with 40% of particles associated with RANTES treated cells were boundRANTES treated cells were bound

20% were associated with C. 20% were associated with C. burnetiiburnetii treated cells were boundtreated cells were bound

contcont

RANTES effects on C. burnetii RANTES effects on C. burnetii treated cellstreated cells

Nef-mediated restoration on C. Nef-mediated restoration on C. Burnetii phagocytosisBurnetii phagocytosis

They wondered if expression in THP-1 They wondered if expression in THP-1 monocytes interferes with C. monocytes interferes with C. burnetii burnetii phagocytosis as did RANTES phagocytosis as did RANTES stimulationstimulation

Transfected Nef into the cellsTransfected Nef into the cells Introduction of C. Introduction of C. burnetii burnetii to theto the Nef Nef

transfected cellstransfected cellsPseudopodal extensions similar to Pseudopodal extensions similar to

those observed earlierthose observed earlier

contcont

CD18 bound beads were present in CD18 bound beads were present in deformation areas of cells that deformation areas of cells that expressed Nefexpressed Nef

The distribution of The distribution of άβάβ integrin and integrin and IAP were not effected by NefIAP were not effected by Nef

The over expression of Nef up The over expression of Nef up regulated the uptake of virulent C. regulated the uptake of virulent C. burnetiiburnetii

contcont

CR3-depended phagocytosis and CR3-depended phagocytosis and src kinasessrc kinases

Pretreated THP-1 cells with PP1, and Pretreated THP-1 cells with PP1, and inhibitor of src kinase inhibitor of src kinase

Stimulated the cells with either RANTES or Stimulated the cells with either RANTES or C. C. burnetiiburnetii

PP1 did not effect F-actin in RANTES but in PP1 did not effect F-actin in RANTES but in C. C. burnetii burnetii cells it significantly inhibited the cells it significantly inhibited the reorganization of the cellsreorganization of the cells

This effect was dose dependentThis effect was dose dependent PPI up-regulated C. PPI up-regulated C. burnetii burnetii uptake by 150%uptake by 150% This proves that src is involved in This proves that src is involved in

cytoskeleton reorganization C. cytoskeleton reorganization C. burnetiiburnetii induced cellsinduced cells

CR3 distribution and C. CR3 distribution and C. burnetii burnetii survival survival

Since RANTES and Nef expression Since RANTES and Nef expression stimulated Cr3 redistribution toward stimulated Cr3 redistribution toward pseudopodal extension and pseudopodal extension and increased phagocytosis of C.increased phagocytosis of C. burnetii burnetii

Did this effect the survival Did this effect the survival Pretreated cells with RANTES and Nef Pretreated cells with RANTES and Nef

and then infected with virulent and then infected with virulent organisms and cultured for 4 daysorganisms and cultured for 4 days

contcont

Then researchers did same Then researchers did same experiment but added an anti CR3 experiment but added an anti CR3 abs abs

C. C. burnetiiburnetii restored its ability to restored its ability to replicatereplicate

These results show that the These results show that the availability of CR3 impairs C. burnetii availability of CR3 impairs C. burnetii replication but does not result in replication but does not result in bacteria elimination bacteria elimination

contcont

Conclusion Conclusion

Researchers were able to prove that Researchers were able to prove that cytoskeleton reorganization and CR3 cytoskeleton reorganization and CR3 distribution are distinct and distribution are distinct and controlled by different factorscontrolled by different factors

CR3 does not kill the bacteria just CR3 does not kill the bacteria just decreases ability to replicatedecreases ability to replicate

My questionMy question

Does C. burnetii inhibit CR3 before or Does C. burnetii inhibit CR3 before or after it gets phagocytosed?after it gets phagocytosed?

Do you have any questions for me?Do you have any questions for me?

Well that’s my story and I’m Well that’s my story and I’m sticking to it sticking to it