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Pra1 from Candida albicans has a metalloprotease function to cleave the central complement component C3
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Abstracts / Molecular Immunology 56 (2013) 240–316
Conclusion: We have generated tools to study the role of CfHR proteins in the complement system and on the survival of N. meningitidis in human serum and found indications of direct interactions between fHbp and CfHR proteins. Our data show for the first time, to our knowledge, possible binding of CfHR proteins to meningococcal fHbp. http://dx.doi.org/10.1016/j.molimm.2013.05.205 P IEPM 9 Sequence variations and expression of the Candida albicans immune evasion proteins Gpm1 and Pra1 in clinical isolates contribute to virulence S. Luo 1,2,∗ , U.-C. Hipler 3 , C. Münzberg 2 , C. Skerka 2 , A. Verschoor 1 , P.F. Zipfel 2,4 1 Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, München, Germany 2 Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Department of Infection Biology, Jena, Germany 3 Friedrich-Schiller-University Hospital, Clinic of Dermatology and Allergology, Jena, Germany 4 Friedrich-Schiller-University, Jena, Germany
Candida albicans is an important human fungal pathogen, that utilizes multiple evasion strategies to control, modulate or inhibit host immune responses. As clinical C. albicans strains vary in pathogenicity, we analyzed sequence polymorphisms and variations in expression of two central fungal immune evasion proteins: 1) phosphoglycerate mutase (Gpm1), and 2) pH-regulated antigen 1 (Pra1). In 13 clinical isolates, derived from different patient tissues, four synonymous nucleotide exchanges were identified in the 747-bp-long GPM1 gene. For the 900-bp-long PRA1 gene, 16 nucleotide exchanges were identified, representing both synonymous, as well as non-synonymous exchanges. For the PRA1 gene, a homozygous nucleotide exchange at position 73 was identified in all 13 clinical isolates tested and each expresses the Val 25 variant. Surface expression of Gpm1 and Pra1 varied 8.2 and 3.3 fold respectively, and influenced fungal immune fitness. The expression level of Gpm1 and Pra1 at C. albicans strains correlates positively with complement regulator protein binding, survival in complement active, Factor H depleted human serum, adhesion to human endothelial cells, and negatively with C3b/iC3b surface deposition. Thus, coding variations and in particular the levels of expression of two complement evasion proteins Gpm1 and Pra1 influence immune fitness and contribute to fungal virulence. http://dx.doi.org/10.1016/j.molimm.2013.05.206 P IEPM 10 Pra1 from Candida albicans has a metalloprotease function to cleave the central complement component C3 C. Pilz 1,∗ , S. Luo 1 , A. Hartmann 1 , C. Skerka 1 , P.F. Zipfel 1,2 1 2
Hans Knöll Institut Jena, Infection Biology, Jena, Germany Friedrich Schiller University, Jena, Germany
Complement as a first-line defense of human innate immunity is an immediately acting defense system that recognizes, opsonizes and eliminates infectious microbes. The opportunistic human pathogenic fungus Candida albicans regulates the host complement and expresses several immune evasion proteins. The pH regulated antigen 1 (Pra1) of C. albicans is one important fungal complement evasion protein. Secreted Pra1 binds to C3, blocks C3 activation and conversion by the C3 convertase. Attached to
the surface of C. albicans, Pra1 acquires the complement regulator Factor H and prevents C3b opsonization. Sequence analysis of Pra1 revealed a metalloprotease-like motif (H178 RXXH182 ). However, the metalloprotease proteins M35 like Deuterolysin (Aspergillus oryzae), Mep20 (A. fumigatus) and Penicillolysin (Penicillium citrinum) have a HEXXH motif at their catalytic site, which is also relevant for zinc binding. The Pra1 sequence has arginine (R) instead of the glutamic acid residue (E). The two histidine residues in the metalloprotease motif of the M35 family and in Candida Pra1 are conserved. Based on the presence of this putative metalloprotease domain we first analyzed whether Candida Pra1 displays proteolytical activity and assayed cleavage of complement protein C3. Pra1 when incubated with C3, cleaved C3 and two fragments were generated, a C3a-like (C3a-L) and a C3b-like (C3b-L) fragment. C3a-L has a higher mobility as typical C3a. N-terminal sequencing analysis of C3b-L localized the Pra1 cleavage site in the ␣-chain of C3 six amino acids prior to the cleavage site of the endogenous C3 convertase. C3b-L generated by Candida Pra1 is further processed and degraded by the host protease Factor I in the presence of the cofactor Factor H. To confirm that Candida Pra1 is a metalloprotease, the proteolytic activity of Pra1 was blocked by the metalloprotease inhibitor EDTA. The C3a-L fragment is further processed by Pra1, but not C3a, generated by the conventional C3 convertase. The cleavage of C3 by Pra1 inactivates the central complement component C3, blocks C3 central effector functions of the inflammatory and antimicrobial C3a-L activation fragment. Thus Candida Pra1 is a protease of the metalloprotease group that by cleaving host complement C3 contributes the fungal immune evasion. http://dx.doi.org/10.1016/j.molimm.2013.05.247 P IEPM 12 Genetic manipulation of the relapsing fever spirochete Borrelia hermsii permits study of the role of factor H binding and a novel complement evasion mechanism D.P. Miller ∗ , L. Fine, R.T. Marconi Virginia Commonwealth University, Microbiology and Immunology, Richmond, United States Tick-borne relapsing fever (TBRF) is the most common cause of febrile disease, surpassing even malaria, in certain regions of Africa. TBRF is also endemic to regions of North America, Central and South America, the Mediterranean, and Central Asia. The disease is caused by several different Borrelia species that are transmitted to humans through bites of infected Ornithodoros ticks. TBRF is characterized by recurring episodes of high fever that coincide with the occurrence of large numbers of spirochetes in the blood. The Borrelia can reach incredible densities, as high as 108 spirochetes per milliliter, in the blood. The TBRF spirochetes must effectively evade complement in order to thrive in the bloodstream. Several TBRF spirochete species have been shown to bind the negative complement regulators factor H (FH) and C4-binding protein (C4BP). Borrelia hermsii, a TBRF spirochete endemic to North America, binds FH, FHR-1 and plasminogen via its FhbA protein. While FhbA has been hypothesized to be a critical virulence factor of B. hermsii, direct demonstration of the role of FhbA in the disease process has been precluded by the lack of a reliable genetic manipulation system for relapsing fever Borrelia. In this study, we developed and utilized a novel kanamycin resistance-green fluorescence protein construct for the site-specific allelic exchange mutagenesis of fhbA from B. hermsii strain YOR. Analysis of the B. hermsii (Bh) YORfhbA strain revealed that deletion of fhbA did not result in plasmid loss or alter the in vitro growth rate. Analyses of BhYORfhbA demonstrated that it
Abstracts / Molecular Immunology 56 (2013) 240–316
Conclusion: We have generated tools to study the role of CfHR proteins in the complement system and on the survival of N. meningitidis in human serum and found indications of direct interactions between fHbp and CfHR proteins. Our data show for the first time, to our knowledge, possible binding of CfHR proteins to meningococcal fHbp. http://dx.doi.org/10.1016/j.molimm.2013.05.205 P IEPM 9 Sequence variations and expression of the Candida albicans immune evasion proteins Gpm1 and Pra1 in clinical isolates contribute to virulence S. Luo 1,2,∗ , U.-C. Hipler 3 , C. Münzberg 2 , C. Skerka 2 , A. Verschoor 1 , P.F. Zipfel 2,4 1 Institut für Medizinische Mikrobiologie, Immunologie und Hygiene, Technische Universität München, München, Germany 2 Leibniz-Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute, Department of Infection Biology, Jena, Germany 3 Friedrich-Schiller-University Hospital, Clinic of Dermatology and Allergology, Jena, Germany 4 Friedrich-Schiller-University, Jena, Germany
Candida albicans is an important human fungal pathogen, that utilizes multiple evasion strategies to control, modulate or inhibit host immune responses. As clinical C. albicans strains vary in pathogenicity, we analyzed sequence polymorphisms and variations in expression of two central fungal immune evasion proteins: 1) phosphoglycerate mutase (Gpm1), and 2) pH-regulated antigen 1 (Pra1). In 13 clinical isolates, derived from different patient tissues, four synonymous nucleotide exchanges were identified in the 747-bp-long GPM1 gene. For the 900-bp-long PRA1 gene, 16 nucleotide exchanges were identified, representing both synonymous, as well as non-synonymous exchanges. For the PRA1 gene, a homozygous nucleotide exchange at position 73 was identified in all 13 clinical isolates tested and each expresses the Val 25 variant. Surface expression of Gpm1 and Pra1 varied 8.2 and 3.3 fold respectively, and influenced fungal immune fitness. The expression level of Gpm1 and Pra1 at C. albicans strains correlates positively with complement regulator protein binding, survival in complement active, Factor H depleted human serum, adhesion to human endothelial cells, and negatively with C3b/iC3b surface deposition. Thus, coding variations and in particular the levels of expression of two complement evasion proteins Gpm1 and Pra1 influence immune fitness and contribute to fungal virulence. http://dx.doi.org/10.1016/j.molimm.2013.05.206 P IEPM 10 Pra1 from Candida albicans has a metalloprotease function to cleave the central complement component C3 C. Pilz 1,∗ , S. Luo 1 , A. Hartmann 1 , C. Skerka 1 , P.F. Zipfel 1,2 1 2
Hans Knöll Institut Jena, Infection Biology, Jena, Germany Friedrich Schiller University, Jena, Germany
Complement as a first-line defense of human innate immunity is an immediately acting defense system that recognizes, opsonizes and eliminates infectious microbes. The opportunistic human pathogenic fungus Candida albicans regulates the host complement and expresses several immune evasion proteins. The pH regulated antigen 1 (Pra1) of C. albicans is one important fungal complement evasion protein. Secreted Pra1 binds to C3, blocks C3 activation and conversion by the C3 convertase. Attached to
the surface of C. albicans, Pra1 acquires the complement regulator Factor H and prevents C3b opsonization. Sequence analysis of Pra1 revealed a metalloprotease-like motif (H178 RXXH182 ). However, the metalloprotease proteins M35 like Deuterolysin (Aspergillus oryzae), Mep20 (A. fumigatus) and Penicillolysin (Penicillium citrinum) have a HEXXH motif at their catalytic site, which is also relevant for zinc binding. The Pra1 sequence has arginine (R) instead of the glutamic acid residue (E). The two histidine residues in the metalloprotease motif of the M35 family and in Candida Pra1 are conserved. Based on the presence of this putative metalloprotease domain we first analyzed whether Candida Pra1 displays proteolytical activity and assayed cleavage of complement protein C3. Pra1 when incubated with C3, cleaved C3 and two fragments were generated, a C3a-like (C3a-L) and a C3b-like (C3b-L) fragment. C3a-L has a higher mobility as typical C3a. N-terminal sequencing analysis of C3b-L localized the Pra1 cleavage site in the ␣-chain of C3 six amino acids prior to the cleavage site of the endogenous C3 convertase. C3b-L generated by Candida Pra1 is further processed and degraded by the host protease Factor I in the presence of the cofactor Factor H. To confirm that Candida Pra1 is a metalloprotease, the proteolytic activity of Pra1 was blocked by the metalloprotease inhibitor EDTA. The C3a-L fragment is further processed by Pra1, but not C3a, generated by the conventional C3 convertase. The cleavage of C3 by Pra1 inactivates the central complement component C3, blocks C3 central effector functions of the inflammatory and antimicrobial C3a-L activation fragment. Thus Candida Pra1 is a protease of the metalloprotease group that by cleaving host complement C3 contributes the fungal immune evasion. http://dx.doi.org/10.1016/j.molimm.2013.05.247 P IEPM 12 Genetic manipulation of the relapsing fever spirochete Borrelia hermsii permits study of the role of factor H binding and a novel complement evasion mechanism D.P. Miller ∗ , L. Fine, R.T. Marconi Virginia Commonwealth University, Microbiology and Immunology, Richmond, United States Tick-borne relapsing fever (TBRF) is the most common cause of febrile disease, surpassing even malaria, in certain regions of Africa. TBRF is also endemic to regions of North America, Central and South America, the Mediterranean, and Central Asia. The disease is caused by several different Borrelia species that are transmitted to humans through bites of infected Ornithodoros ticks. TBRF is characterized by recurring episodes of high fever that coincide with the occurrence of large numbers of spirochetes in the blood. The Borrelia can reach incredible densities, as high as 108 spirochetes per milliliter, in the blood. The TBRF spirochetes must effectively evade complement in order to thrive in the bloodstream. Several TBRF spirochete species have been shown to bind the negative complement regulators factor H (FH) and C4-binding protein (C4BP). Borrelia hermsii, a TBRF spirochete endemic to North America, binds FH, FHR-1 and plasminogen via its FhbA protein. While FhbA has been hypothesized to be a critical virulence factor of B. hermsii, direct demonstration of the role of FhbA in the disease process has been precluded by the lack of a reliable genetic manipulation system for relapsing fever Borrelia. In this study, we developed and utilized a novel kanamycin resistance-green fluorescence protein construct for the site-specific allelic exchange mutagenesis of fhbA from B. hermsii strain YOR. Analysis of the B. hermsii (Bh) YORfhbA strain revealed that deletion of fhbA did not result in plasmid loss or alter the in vitro growth rate. Analyses of BhYORfhbA demonstrated that it