1. Coxiella burnetii Avoids Macrophage Phagocytosis by Interfering with Spatial Distribution of Complement Receptor 3 By Lionel Williamson

2. Introduction Coxiella burnetii is an obligate intracellular bacteria Coxiella burnetii is an obligate intracellular bacteria Its survival strategy in monocytes/ macrophages is based on growth in acidic phagosomes that do not fuse with lysosomes Its survival strategy in monocytes/ macrophages is based on growth in acidic phagosomes that do not fuse with lysosomes Infection of humans usually occurs by inhalation of these organisms from air that contains airborne barnyard dust contaminated by dried placental material, birth fluids, and excreta of infected herd animals Infection of humans usually occurs by inhalation of these organisms from air that contains airborne barnyard dust contaminated by dried placental material, birth fluids, and excreta of infected herd animals

3. Introduction It causes Q fever It causes Q fever In 1999, Q fever became a reportable disease in the United States In 1999, Q fever became a reportable disease in the United States Most acute cases of Q fever begin with sudden onset of one or more of the following: high fevers (up to 104-105° F), severe headache, general malaise and myalgia, confusion, sore throat, chills, sweats, non-productive cough, nausea, vomiting, diarrhea, abdominal pain, and chest pain Most acute cases of Q fever begin with sudden onset of one or more of the following: high fevers (up to 104-105° F), severe headache, general malaise and myalgia, confusion, sore throat, chills, sweats, non-productive cough, nausea, vomiting, diarrhea, abdominal pain, and chest pain As many as 65% of persons with chronic Q fever may die of the disease. As many as 65% of persons with chronic Q fever may die of the disease.

4. Overview of paper The critical part of infection is C. burnetii getting into the monocyte The critical part of infection is C. burnetii getting into the monocyte Difference between virulent and avirulent strains is this ability to survive in the monocyte Difference between virulent and avirulent strains is this ability to survive in the monocyte

5. Key terms Complement receptor 3 (CD11b:CD18) binds to a pathogen surface that has been opsonized with complement and promotes pathogen phagocytosis Complement receptor 3 (CD11b:CD18) binds to a pathogen surface that has been opsonized with complement and promotes pathogen phagocytosis CD- cluster of differentiation CD- cluster of differentiation Integrin are heterodimeric cell surface proteins involved in cell-cell and cell- matrix interactions they are involved in adhesive interactions and ligand binding Integrin are heterodimeric cell surface proteins involved in cell-cell and cell- matrix interactions they are involved in adhesive interactions and ligand binding

6. Key terms RANTES- is a chemokine that stimulates the migration and activation of cells especially phagocytic cells. It induced psuedopodal extensions that expressed CR3 In THP-1 cells RANTES- is a chemokine that stimulates the migration and activation of cells especially phagocytic cells. It induced psuedopodal extensions that expressed CR3 In THP-1 cells HIV-1 Nef also acts like a chemokine stimulating migration inside the phagocyte. Same as above HIV-1 Nef also acts like a chemokine stimulating migration inside the phagocyte. Same as above

7. What did they know They knew that uptake of the avirulent strain was mediated by άβ integrin and CR3 They knew that uptake of the avirulent strain was mediated by άβ integrin and CR3 Virulent strain entered the monocyte through άβ integrin but interfered with the CR3 which appeared to prevent phagocytosis Virulent strain entered the monocyte through άβ integrin but interfered with the CR3 which appeared to prevent phagocytosis

8. What did they know CR3 must be activated to promote ligand binding and phagocytosis CR3 must be activated to promote ligand binding and phagocytosis Activation required signals from other integrins, chemoattractant receptors or LPS receptors Activation required signals from other integrins, chemoattractant receptors or LPS receptors Ex. ligation of άβ integrin and integrin associated protein (IAP) enhances CR3 binding Ex. ligation of άβ integrin and integrin associated protein (IAP) enhances CR3 binding

9. What did they know CR3 clustering is correlated to increased binding and phagocytosis (Immobile form) CR3 clustering is correlated to increased binding and phagocytosis (Immobile form) The switch from an inactive to an active form is associated with an alteration in the mechanism of interaction CR3 with the cytoskeleton The switch from an inactive to an active form is associated with an alteration in the mechanism of interaction CR3 with the cytoskeleton An immobile subset of CR3 linked to the cytoskeleton is more efficient for phagocytosis than freely diffusing receptors An immobile subset of CR3 linked to the cytoskeleton is more efficient for phagocytosis than freely diffusing receptors

10. What did they know F- actin is part of the cytoskeleton infrastructure F- actin is part of the cytoskeleton infrastructure The virulent but not the avirulent strain has the ability to reorganize the F-actin in THP-1 monocytes The virulent but not the avirulent strain has the ability to reorganize the F-actin in THP-1 monocytes They hypothesized that C. burnetii - mediated impairment of CR3 activity may result from effects of actin reorganization They hypothesized that C. burnetii - mediated impairment of CR3 activity may result from effects of actin reorganization

11. Receptor distribution in C. burnetii- stimulated monocytes THP-1 monocytes were tagged with mAb directed to monocyte receptors involved in C. burnetii recognition THP-1 monocytes were tagged with mAb directed to monocyte receptors involved in C. burnetii recognition Large beads coated with anti mouse IgG Abs and F-actin labeling Large beads coated with anti mouse IgG Abs and F-actin labeling Also beads were attached to monocytes tagged with mAb directed CD11b, CD18, άβ integrin, IAP (no bead phagocytosis was observed) Also beads were attached to monocytes tagged with mAb directed CD11b, CD18, άβ integrin, IAP (no bead phagocytosis was observed)

12. Exp. cont THP-1 were stimulated with virulent C. burnetii THP-1 were stimulated with virulent C. burnetii The stimulation caused F-actin reorganization and psuedopodal extensions in 60% and 40% did not change The stimulation caused F-actin reorganization and psuedopodal extensions in 60% and 40% did not change In the 40% beads bound to Cd11b and CD18, άβ integrin, and IAP were bound randomly as in the control cells In the 40% beads bound to Cd11b and CD18, άβ integrin, and IAP were bound randomly as in the control cells

13. cont In the 60% all the beads attached to CD11 and CD18 were found outside the deformation area In the 60% all the beads attached to CD11 and CD18 were found outside the deformation area This response was seen in as early as 10 min and continued for about 60 min This response was seen in as early as 10 min and continued for about 60 min In contrast the beads bound to άβ integrin were present at the leading edge of the protrusion (1/4 of άβ integrin was found on the protrusion) In contrast the beads bound to άβ integrin were present at the leading edge of the protrusion (1/4 of άβ integrin was found on the protrusion) The pattern of IAP was similar to the integrin The pattern of IAP was similar to the integrin

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15. cont TO prove that the lack of CR3 on the protrusion was not caused by the size of the beads they did the experiment using smaller beads TO prove that the lack of CR3 on the protrusion was not caused by the size of the beads they did the experiment using smaller beads They were able to detect CR3 on the protrusions in <10% compared to 25% of άβ integrin, and IAP were detected on the protrusions They were able to detect CR3 on the protrusions in <10% compared to 25% of άβ integrin, and IAP were detected on the protrusions Confirmed that CR3 was excluded from the extensions Confirmed that CR3 was excluded from the extensions

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17. Cont They repeated the experiment with circulating monocytes with the same results They repeated the experiment with circulating monocytes with the same results Avirulent strain did not induce psuedopodal extension or F-actin reorganization and as a consequence the distribution of CD11b, CD18, άβ integrin, and IAP was similar to control cells Avirulent strain did not induce psuedopodal extension or F-actin reorganization and as a consequence the distribution of CD11b, CD18, άβ integrin, and IAP was similar to control cells

18. Receptor distribution in RANTES- stimulated monocytes They wondered if CR3 rearrangement was specific to C. burnetii or was it indicative of any rearrangements of F-actin They wondered if CR3 rearrangement was specific to C. burnetii or was it indicative of any rearrangements of F-actin Stimulated THP-1 cells with RANTES and was analyzed by transmission electron microscope Stimulated THP-1 cells with RANTES and was analyzed by transmission electron microscope Observed pseudopodia similar to that elicited by C. burnetii Observed pseudopodia similar to that elicited by C. burnetii

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20. cont Using the same technique they found that beads attached to CD11 and CD18 were found in psuedopodal extensions of monocytes stimulated by RANTES(30and 25% respectively) Using the same technique they found that beads attached to CD11 and CD18 were found in psuedopodal extensions of monocytes stimulated by RANTES(30and 25% respectively) The distribution of beads άβ integrin, and IAP were similar to those of C. burnetii The distribution of beads άβ integrin, and IAP were similar to those of C. burnetii This proves that C. burnetii specifically excludes CR3 from pseudopods

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22. RANTES-mediated restoration of C. burnetii phagocytosis Treated some cells with RANTES and the other C. burnetii Treated some cells with RANTES and the other C. burnetii Then incubated the cells with particles which need CR3 for optimal binding to monocytes (IC3b-coated E, zymosan, and Avirulent C. burnetii) Then incubated the cells with particles which need CR3 for optimal binding to monocytes (IC3b-coated E, zymosan, and Avirulent C. burnetii)

23. cont 40% of particles associated with RANTES treated cells were bound 40% of particles associated with RANTES treated cells were bound 20% were associated with C. burnetii treated cells were bound 20% were associated with C. burnetii treated cells were bound

24. cont

25. RANTES effects on C. burnetii treated cells

26. Nef-mediated restoration on C. Burnetii phagocytosis They wondered if expression in THP-1 monocytes interferes with C. burnetii phagocytosis as did RANTES stimulation They wondered if expression in THP-1 monocytes interferes with C. burnetii phagocytosis as did RANTES stimulation Transfected Nef into the cells Transfected Nef into the cells Introduction of C. burnetii to the Nef transfected cells Introduction of C. burnetii to the Nef transfected cells Pseudopodal extensions similar to those observed earlier Pseudopodal extensions similar to those observed earlier

27. cont CD18 bound beads were present in deformation areas of cells that expressed Nef CD18 bound beads were present in deformation areas of cells that expressed Nef The distribution of άβ integrin and IAP were not effected by Nef The distribution of άβ integrin and IAP were not effected by Nef The over expression of Nef up regulated the uptake of virulent C. burnetii The over expression of Nef up regulated the uptake of virulent C. burnetii

28. cont

29. CR3-depended phagocytosis and src kinases Pretreated THP-1 cells with PP1, and inhibitor of src kinase Pretreated THP-1 cells with PP1, and inhibitor of src kinase Stimulated the cells with either RANTES or C. burnetii Stimulated the cells with either RANTES or C. burnetii PP1 did not effect F-actin in RANTES but in C. burnetii cells it significantly inhibited the reorganization of the cells PP1 did not effect F-actin in RANTES but in C. burnetii cells it significantly inhibited the reorganization of the cells This effect was dose dependent This effect was dose dependent PPI up-regulated C. burnetii uptake by 150% PPI up-regulated C. burnetii uptake by 150% This proves that src is involved in cytoskeleton reorganization C. burnetii induced cells This proves that src is involved in cytoskeleton reorganization C. burnetii induced cells

30. CR3 distribution and C. burnetii survival Since RANTES and Nef expression stimulated Cr3 redistribution toward pseudopodal extension and increased phagocytosis of C. burnetii Since RANTES and Nef expression stimulated Cr3 redistribution toward pseudopodal extension and increased phagocytosis of C. burnetii Did this effect the survival Did this effect the survival Pretreated cells with RANTES and Nef and then infected with virulent organisms and cultured for 4 days Pretreated cells with RANTES and Nef and then infected with virulent organisms and cultured for 4 days

31. cont Then researchers did same experiment but added an anti CR3 abs Then researchers did same experiment but added an anti CR3 abs C. burnetii restored its ability to replicate C. burnetii restored its ability to replicate These results show that the availability of CR3 impairs C. burnetii replication but does not result in bacteria elimination These results show that the availability of CR3 impairs C. burnetii replication but does not result in bacteria elimination

32. cont

33. Conclusion Researchers were able to prove that cytoskeleton reorganization and CR3 distribution are distinct and controlled by different factors Researchers were able to prove that cytoskeleton reorganization and CR3 distribution are distinct and controlled by different factors CR3 does not kill the bacteria just decreases ability to replicate CR3 does not kill the bacteria just decreases ability to replicate

34. My question Does C. burnetii inhibit CR3 before or after it gets phagocytosed? Does C. burnetii inhibit CR3 before or after it gets phagocytosed? Do you have any questions for me?

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