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Pseudomonas aeruginosa Lipoamide dehydrogenase (Lpd) binds multiple human plasma proteins for immune escape and to facilitate tissue interaction
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Abstracts / Molecular Immunology 48 (2011) 1666–1733
P106 Pra1 binds and inactivates effector functions of the anaphylatoxin C3a D. Kupka a,∗ , E. Wende b , A. Klos b , P.F. Zipfel a a
HKI Jena, Jena, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover, Germany b
The complement system controls immune homeostasis and attacks invading microbes. However several pathogenic microbes can block and control the host complement by a variety of complement evasion strategies. Here we show that the pH-regulated antigen 1 (Pra1) of the humanpathogenic yeast Candida albicans binds C3a and inhibits C3a receptor-mediated immune effector functions. Pra1 inhibits antifungal activity of both C3a and C3apeptides resulting in an increased survival of C. albicans by up to 70%. The Pra1 mutant shows elevated and pra1 overexpression strain reduced susceptibility to antifungal effects of C3a. In addition, clinical isolates of C. albicans display increased resistance upon C3a challenge and this effect strongly correlated with Pra1-levels at fungal surfaces. In a C3a receptor binding assay with 125-I-C3a, Pra1 prevents binding of C3a to the human C3a receptor of stably transfected HEK cells. Furthermore, Pra1 reduced the C3a receptor-induced calcium signals in HEK cells, IL-8 release and abolishes chemotaxis of human granulocytes in response to C3a as shown in migration assays. In summary, Pra1 binds C3a, inhibits C3a receptor-activated IL-8 release and chemotactic movement of human granulocytes as well as antimicrobial activity. Thus, C3a binding and inhibition identifies a novel molecular mechanism utilized by microbial pathogens to evade complement attack. doi:10.1016/j.molimm.2011.06.326 P107 Pseudomonas aeruginosa Lipoamide dehydrogenase (Lpd) binds multiple human plasma proteins for immune escape and to facilitate tissue interaction T. Hallström a,∗ , D. Barthel a , B. Singh b , M. Mörgelin c , C. Skerka a , K. Riesbeck b , P.F. Zipfel a a
Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany b Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden c Section of Clinical and Experimental Infectious Medicine, Lund, Sweden Introduction: Pseudomonas aeruginosa is a major cause of hospital-acquired infections in immuno-compromised individuals. In order to cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. Methods and results: P. aeruginosa binds the alternative pathway regulators Factor H and FHL-1 and the terminal pathway regulators CFHR1 and vitronectin. Bound regulators protect this Gram-negative pathogen against the complement-mediated attack and increase bacterial survival in human serum. Bacterial survival was strongly reduced when bacteria were cultivated in complement active human serum depleted of the regulators Factor H, FHL-1, CFHR1 and also of vitronectin. Here we identified the 57 kDa Lipoamide dehydrogenase (Lpd) as a new Factor H binding protein. Lpd is a surface exposed protein present in the outer membrane of all 19 P. aeruginosa strains tested. Recombinant Lpd bound Factor H, FHL-1, CFHR1, vitronectin and plasminogen. Lpd binds Factor H via SCRs 6–7 and 18–20 and CFHR1 via SCRs 3–5. By using recom-
binant fragments of human vitronectin and plasminogen, the Lpd binding regions were located within amino acids 353–363 of vitronectin and to kringle domain 4 of plasminogen. Bound to Lpd, the human plasma proteins were functionally active. Factor H and FHL-1 maintained cofactor activity in C3b degradation, Lpd-bound vitronectin inhibited terminal pathway and Lpd-bound plasminogen when converted by uPA to functionally active plasmin cleaved a chromogenic and a natural substrate. Conclusion: Lpd is a new surface exposed virulence factor of P. aeruginosa that binds multiple human plasma proteins for immune escape and tissue interaction. doi:10.1016/j.molimm.2011.06.327 P108 A novel mechanism of immune evasion in Leptospira: The expression of proteases that cleave complement proteins T.R. Fraga a,∗ , S.A. Vasconcellos a , A.S. Barbosa b , L. Isaac a a b
University of São Paulo, São Paulo, Brazil Butantan Institute, São Paulo, Brazil
Introduction: Leptospirosis is a worldwide zoonosis of public health importance. Complement represents a central immune mechanism in blood circulation, but the high ability of pathogenic Leptospira to spread indicates a low efficacy of complement against them. Pathogenic Leptospira bind the host complement regulators FH and C4BP, which control complement activation at the pathogen surface. Here we evaluate the ability of Leptospira to directly cleave complement proteins, which could interfere with effector mechanisms, including opsonization and MAC formation Methods: We analyzed the cleavage of C3, C3b, iC3b, factor B, factor D, properdin and C1q by non-pathogenic and pathogenic Leptospira species. Culture supernatants were incubated with complement proteins and cleavages were detected by Western blot. Results: Pathogenic Leptospira secreted proteases that efficiently cleaved all complement proteins tested, except C1q. The cleavage of C3 and C3b resulted in bands with 65, 43, 38, 36 and 29 kDa. The last three bands were also present after iC3b proteolysis by leptospiral proteases. Factor B was extensively cleaved generating bands with 55, 51, 43, 36, 33, 31 and 14 kDa. The proteolysis of factor D and properdin produced fragments with 12 kDa and 31 kDa, respectively. There was no cleavage of any complement proteins by the non-pathogenic Leptospira biflexa. Conclusion: We describe a novel mechanism of immune evasion in Leptospira: the expression of proteases that cleave complement proteins. These proteases can be considered virulence factors, since they can deactivate immune effector molecules, being potential targets to the development of new treatments and prophylactic approaches in leptospirosis. doi:10.1016/j.molimm.2011.06.328 P109 Human complement regulators interact with leptospiral immunoglobulin-like proteins contributing to immune evasion M.M. Castiblanco-Valencia a,∗ , T.R. Fraga a , L.B. Silva b , D. Monaris b , P.A.E. Abreu b , S. Strobel c , M. Józsi c , L. Isaac a , A.S. Barbosa b a
University of Sao Paulo, SAO PAULO, Brazil Butantan Institute, SAO PAULO, Brazil c Hans Knöll Institute, JENA, Germany b
Introduction: Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular
Abstracts / Molecular Immunology 48 (2011) 1666–1733
P106 Pra1 binds and inactivates effector functions of the anaphylatoxin C3a D. Kupka a,∗ , E. Wende b , A. Klos b , P.F. Zipfel a a
HKI Jena, Jena, Germany Institute of Medical Microbiology and Hospital Epidemiology, Hannover, Germany b
The complement system controls immune homeostasis and attacks invading microbes. However several pathogenic microbes can block and control the host complement by a variety of complement evasion strategies. Here we show that the pH-regulated antigen 1 (Pra1) of the humanpathogenic yeast Candida albicans binds C3a and inhibits C3a receptor-mediated immune effector functions. Pra1 inhibits antifungal activity of both C3a and C3apeptides resulting in an increased survival of C. albicans by up to 70%. The Pra1 mutant shows elevated and pra1 overexpression strain reduced susceptibility to antifungal effects of C3a. In addition, clinical isolates of C. albicans display increased resistance upon C3a challenge and this effect strongly correlated with Pra1-levels at fungal surfaces. In a C3a receptor binding assay with 125-I-C3a, Pra1 prevents binding of C3a to the human C3a receptor of stably transfected HEK cells. Furthermore, Pra1 reduced the C3a receptor-induced calcium signals in HEK cells, IL-8 release and abolishes chemotaxis of human granulocytes in response to C3a as shown in migration assays. In summary, Pra1 binds C3a, inhibits C3a receptor-activated IL-8 release and chemotactic movement of human granulocytes as well as antimicrobial activity. Thus, C3a binding and inhibition identifies a novel molecular mechanism utilized by microbial pathogens to evade complement attack. doi:10.1016/j.molimm.2011.06.326 P107 Pseudomonas aeruginosa Lipoamide dehydrogenase (Lpd) binds multiple human plasma proteins for immune escape and to facilitate tissue interaction T. Hallström a,∗ , D. Barthel a , B. Singh b , M. Mörgelin c , C. Skerka a , K. Riesbeck b , P.F. Zipfel a a
Leibniz Institute for Natural Product Research and Infection Biology, Jena, Germany b Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Malmö, Sweden c Section of Clinical and Experimental Infectious Medicine, Lund, Sweden Introduction: Pseudomonas aeruginosa is a major cause of hospital-acquired infections in immuno-compromised individuals. In order to cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. Methods and results: P. aeruginosa binds the alternative pathway regulators Factor H and FHL-1 and the terminal pathway regulators CFHR1 and vitronectin. Bound regulators protect this Gram-negative pathogen against the complement-mediated attack and increase bacterial survival in human serum. Bacterial survival was strongly reduced when bacteria were cultivated in complement active human serum depleted of the regulators Factor H, FHL-1, CFHR1 and also of vitronectin. Here we identified the 57 kDa Lipoamide dehydrogenase (Lpd) as a new Factor H binding protein. Lpd is a surface exposed protein present in the outer membrane of all 19 P. aeruginosa strains tested. Recombinant Lpd bound Factor H, FHL-1, CFHR1, vitronectin and plasminogen. Lpd binds Factor H via SCRs 6–7 and 18–20 and CFHR1 via SCRs 3–5. By using recom-
binant fragments of human vitronectin and plasminogen, the Lpd binding regions were located within amino acids 353–363 of vitronectin and to kringle domain 4 of plasminogen. Bound to Lpd, the human plasma proteins were functionally active. Factor H and FHL-1 maintained cofactor activity in C3b degradation, Lpd-bound vitronectin inhibited terminal pathway and Lpd-bound plasminogen when converted by uPA to functionally active plasmin cleaved a chromogenic and a natural substrate. Conclusion: Lpd is a new surface exposed virulence factor of P. aeruginosa that binds multiple human plasma proteins for immune escape and tissue interaction. doi:10.1016/j.molimm.2011.06.327 P108 A novel mechanism of immune evasion in Leptospira: The expression of proteases that cleave complement proteins T.R. Fraga a,∗ , S.A. Vasconcellos a , A.S. Barbosa b , L. Isaac a a b
University of São Paulo, São Paulo, Brazil Butantan Institute, São Paulo, Brazil
Introduction: Leptospirosis is a worldwide zoonosis of public health importance. Complement represents a central immune mechanism in blood circulation, but the high ability of pathogenic Leptospira to spread indicates a low efficacy of complement against them. Pathogenic Leptospira bind the host complement regulators FH and C4BP, which control complement activation at the pathogen surface. Here we evaluate the ability of Leptospira to directly cleave complement proteins, which could interfere with effector mechanisms, including opsonization and MAC formation Methods: We analyzed the cleavage of C3, C3b, iC3b, factor B, factor D, properdin and C1q by non-pathogenic and pathogenic Leptospira species. Culture supernatants were incubated with complement proteins and cleavages were detected by Western blot. Results: Pathogenic Leptospira secreted proteases that efficiently cleaved all complement proteins tested, except C1q. The cleavage of C3 and C3b resulted in bands with 65, 43, 38, 36 and 29 kDa. The last three bands were also present after iC3b proteolysis by leptospiral proteases. Factor B was extensively cleaved generating bands with 55, 51, 43, 36, 33, 31 and 14 kDa. The proteolysis of factor D and properdin produced fragments with 12 kDa and 31 kDa, respectively. There was no cleavage of any complement proteins by the non-pathogenic Leptospira biflexa. Conclusion: We describe a novel mechanism of immune evasion in Leptospira: the expression of proteases that cleave complement proteins. These proteases can be considered virulence factors, since they can deactivate immune effector molecules, being potential targets to the development of new treatments and prophylactic approaches in leptospirosis. doi:10.1016/j.molimm.2011.06.328 P109 Human complement regulators interact with leptospiral immunoglobulin-like proteins contributing to immune evasion M.M. Castiblanco-Valencia a,∗ , T.R. Fraga a , L.B. Silva b , D. Monaris b , P.A.E. Abreu b , S. Strobel c , M. Józsi c , L. Isaac a , A.S. Barbosa b a
University of Sao Paulo, SAO PAULO, Brazil Butantan Institute, SAO PAULO, Brazil c Hans Knöll Institute, JENA, Germany b
Introduction: Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular