scholarly journals Clumping Factor A Interaction with Complement Factor I Increases C3b Cleavage on the Bacterial Surface of Staphylococcus aureus and Decreases Complement-Mediated Phagocytosis

2010 ◽  
Vol 78 (4) ◽  
pp. 1717-1727 ◽  
Author(s):  
Pamela S. Hair ◽  
Charlene G. Echague ◽  
Amber M. Sholl ◽  
Justin A. Watkins ◽  
Joan A. Geoghegan ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Protein ◽  
Factor H ◽  
Complement Factor ◽  
Wild Type ◽  
Bacterial Surface ◽  
Complement Control Protein ◽  
Factor I ◽  
Protein Factor ◽  
Fibrinogen Binding

ABSTRACT The human complement system is important in the immunological control of Staphylococcus aureus infection. We showed previously that S. aureus surface protein clumping factor A (ClfA), when expressed in recombinant form, bound complement control protein factor I and increased factor I cleavage of C3b to iC3b. In the present study, we show that, compared to the results for the wild type, when isogenic ClfA-deficient S. aureus mutants were incubated in serum, they bound less factor I, generated less iC3b on the bacterial surface, and bound fewer C3 fragments. It has been shown previously that two amino acids in ClfA (P336 and Y338) are essential for fibrinogen binding. However, S. aureus expressing ClfA(P336A Y338S) was less virulent than ClfA-deficient strains in animal models. This suggested that ClfA contributed to S. aureus virulence by a mechanism different than fibrinogen binding. In the present study, we showed that S. aureus expressing ClfA(P336A Y338S) was more susceptible to complement-mediated phagocytosis than a ClfA-null mutant or the wild type. Unlike ClfA, ClfA(P336A Y338S) did not enhance factor I cleavage of C3b to iC3b and inhibited the cofactor function of factor H. Fibrinogen enhanced factor I binding to ClfA and the S. aureus surface. Twenty clinical S. aureus strains all expressed ClfA and bound factor I. High levels of factor I binding by clinical strains correlated with poor phagocytosis. In summary, our results suggest that the interaction of ClfA with factor I contributes to S. aureus virulence by a complement-mediated mechanism.

scholarly journals Impaired Immunogenicity of a Meningococcal Factor H-Binding Protein Vaccine Engineered To Eliminate Factor H Binding

2010 ◽  
Vol 17 (7) ◽  
pp. 1074-1078 ◽  
Author(s):  
Peter T. Beernink ◽  
Jutamas Shaughnessy ◽  
Sanjay Ram ◽  
Dan M. Granoff
Keyword(s):  
Binding Protein ◽  
Factor H ◽  
Vaccine Antigen ◽  
Mouse Strains ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complement Factor ◽  
Protein Vaccine ◽  
Wild Type ◽  
Bacterial Surface ◽  
Cast Doubt

ABSTRACT Meningococcal factor H-binding protein (fHbp) is a promising antigen that is part of two vaccines in clinical development. The protein specifically binds human complement factor H (fH), which downregulates complement activation on the bacterial surface and enables the organism to evade host defenses. In humans, the vaccine antigen forms a complex with fH, which may affect anti-fHbp antibody repertoire and decrease serum bactericidal activity by covering important fHbp epitopes. In a recent study, fHbp residues in contact with fH were identified from a crystal structure. Two fHbp glutamate residues that mediated ion-pair interactions with fH were replaced with alanine, and the resulting E218A/E239A mutant no longer bound the fH fragment. In the present study, we generated the E218A/E239A mutant recombinant protein and confirmed the lack of fH binding. By enzyme-linked immunosorbent assay (ELISA), the mutant fHbp showed similar respective concentration-dependent inhibition of binding of four bactericidal anti-fHbp monoclonal antibodies (MAbs) to fHbp, compared with inhibition by the soluble wild-type protein. In two mouse strains, the mutant fHbp elicited up to 4-fold-lower IgG anti-fHbp antibody titers and up to 20-fold-lower serum bactericidal titers than those elicited by the wild-type fHbp vaccine. Thus, although introduction of the two alanine substitutions to eliminate fH binding did not appear to destabilize the molecule globally, the mutations resulted in decreased immunogenicity in mouse models in which neither the mutant nor the wild-type control vaccine bound fH. These results cast doubt on the vaccine potential in humans of this mutant fHbp.

scholarly journals Cleavage of Complement C3b to iC3b on the Surface of Staphylococcus aureus Is Mediated by Serum Complement Factor I

2004 ◽  
Vol 72 (5) ◽  
pp. 2858-2863 ◽  
Author(s):  
K. M. Cunnion ◽  
P. S. Hair ◽  
E. S. Buescher
Keyword(s):  
Staphylococcus Aureus ◽  
Human Serum ◽  
Serum Proteins ◽  
Regulatory Protein ◽  
Factor H ◽  
Complement Factor ◽  
Serum Factor ◽  
Complement Protein ◽  
Factor I ◽  
Serotype 5

ABSTRACT Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.

scholarly journals Impaired Immunogenicity of Meningococcal Neisserial Surface Protein A in Human Complement Factor H Transgenic Mice

2015 ◽  
Vol 84 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Eduardo Lujan ◽  
Rolando Pajon ◽  
Dan M. Granoff
Keyword(s):  
Transgenic Mice ◽  
Outer Membrane ◽  
Protein A ◽  
Surface Protein ◽  
Factor H ◽  
Antibody Responses ◽  
Complement Factor ◽  
Wild Type ◽  
Protective Antibody ◽  
Bactericidal Antibody

Neisserial surface protein A (NspA) is a highly conserved outer membrane protein previously investigated as a meningococcal vaccine candidate. Despite eliciting serum bactericidal activity in mice, a recombinant NspA vaccine failed to elicit serum bactericidal antibodies in a phase 1 clinical trial in humans. The discordant results may be explained by the recent discovery that NspA is a human-specific ligand of the complement inhibitor factor H (FH). Therefore, in humans but not mice, NspA would be expected to form a complex with FH, which could impair human anti-NspA protective antibody responses. To investigate this question, we immunized human FH transgenic BALB/c mice with three doses of recombinant NspA expressed inEscherichia colimicrovesicles, with each dose being separated by 3 weeks. Three of 12 (25%) transgenic mice and 13 of 14 wild-type mice responded with bactericidal titers of ≥1:10 in postimmunization sera (P= 0.0008, Fisher's exact test). In contrast, human FH transgenic and wild-type mice immunized with a control meningococcal native outer membrane vesicle vaccine had similar serum bactericidal antibody responses directed at PorA, which is not known to bind human FH, and a mutant factor H binding protein (FHbp) antigen with a >50-fold lower level of FH binding than wild-type FHbp antigen binding.Thus, human FH can impair anti-NspA serum bactericidal antibody responses, which may explain the poor immunogenicity of the NspA vaccine previously tested in humans. A mutant NspA vaccine engineered to have decreased binding to human FH may increase protective antibody responses in humans.

Identification of Staphylococcus aureus surface protein SdrE as a complement factor H-binding molecule

2011 ◽  
Vol 48 (14) ◽  
pp. 1695 ◽  
Author(s):  
K.M. Cunnion ◽  
J.A. Sharp ◽  
P.S. Hair ◽  
C.G. Echague ◽  
M.D. Ward ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Protein ◽  
Factor H ◽  
Complement Factor H ◽  
Complement Factor

scholarly journals A short sequence within subdomain N1 of region A of the Staphylococcus aureus MSCRAMM clumping factor A is required for export and surface display

Microbiology ◽  
2014 ◽  
Vol 160 (4) ◽  
pp. 659-670 ◽  
Author(s):  
Niamh McCormack ◽  
Timothy J. Foster ◽  
Joan A. Geoghegan
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cytoplasmic Membrane ◽  
Signal Sequence ◽  
Surface Display ◽  
Surface Protein ◽  
Covalent Attachment ◽  
Bacterial Surface ◽  
Fibrinogen Binding ◽  
Fibronectin Binding

Clumping factor A (ClfA) is the archetypal fibrinogen-binding surface protein of Staphylococcus aureus and a member of the microbial surface component recognizing adhesive matrix molecules (MSCRAMM) family. An N-terminal signal sequence directs export of the MSCRAMM by the Sec pathway and the C-terminal cell wall-anchoring domain allows covalent attachment of ClfA to peptidoglycan by sortase. Region A of ClfA comprises three independently folded subdomains N1, N2 and N3. Subdomains N2N3 comprise IgG-like folds and promote fibrinogen binding. Nothing is known about the structure or function of subdomain N1. Here we demonstrate an unexpected role for N1 in the export and surface localization of ClfA. Attempted expression of a ClfA variant lacking subdomain N1 resulted in impaired growth of S. aureus and accumulation of ClfA protein in the cytoplasm and cytoplasmic membrane. The presence of residues 211–228 of N1 was required to allow display of ClfA on the bacterial surface. The importance of this region was confirmed when a ClfA variant lacking residues 211–220 was also mislocalized to the cytoplasm and cytoplasmic membrane. However, these residues were not required for export of ClfA lacking the Ser-Asp repeats that link region A to the wall-anchoring domain. Similarly, subdomain N1 of a related MSCRAMM fibronectin-binding protein B was required for export and surface display of the full-length protein, but not a derivative lacking fibronectin-binding repeats. In summary, we demonstrate that residues in the N1 subdomain are required for export and cell wall localization of S. aureus MSCRAMM proteins.

scholarly journals The N2N3 domains of ClfA, FnbpA, and FnbpB in Staphylococcus aureus bind to human complement factor H, and their antibodies enhance the bactericidal capability of human blood

2020 ◽  
Author(s):  
Xinrui Mao ◽  
Junghyun Kim ◽  
QingFeng Zhang ◽  
TingTing Jiang ◽  
Dong Ho Ahn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Surface ◽  
Human Blood ◽  
Bacterial Cell ◽  
Regulatory Protein ◽  
Factor H ◽  
Complement Factor ◽  
Bacterial Cell Surface ◽  
Protein Factor

Abstract In the complement system, the opsonin C3b binds to the bacterial cell surface and mediates the opsonophagocytosis. However, the cell wall protein SdrE of Staphylococcus aureus inhibits the C3b activity by recruiting the complement regulatory protein factor H (fH). SdrE binds to fH via its N-terminal N2N3 domain, which are also found in six other staphylococcal cell wall proteins. In this study, we report that not only the N2N3 domain of SdrE but also those of ClfA, FnbpA, and FnbpB can bind to fH. When immobilized on a microplate, the N2N3 domains recruited fH and enhanced the factor I (fI)-mediated cleavage of C3b. When mixed with fH and S. aureus cells, the N2N3 domains inhibited the fH binding to S. aureus cells and reduced the fI-mediated C3b cleavage on the bacterial cell surface. The F(ab)′2 fragments of the rabbit N2N3 antibodies also inhibited the fH-binding to the S. aureus cell surface. When added to human blood, the N2N3 antibodies or the N2N3 domain proteins significantly increased the bactericidal activity. Based on these results, we conclude that, in S. aureus, not only SdrE but also ClfA, FnbpA, and FnbpB can contribute to the inhibition of C3b-mediated opsonophagocytosis.

scholarly journals Staphylococcus aureus SdrE captures complement factor H's C-terminus via a novel ‘close, dock, lock and latch' mechanism for complement evasion

2017 ◽  
Vol 474 (10) ◽  
pp. 1619-1631 ◽  
Author(s):  
Yingjie Zhang ◽  
Minhao Wu ◽  
Tianrong Hang ◽  
Chengliang Wang ◽  
Ye Yang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Evasion ◽  
Alternative Pathway ◽  
Regulatory Protein ◽  
Surface Protein ◽  
Factor H ◽  
Host Cells ◽  
Main Body ◽  
Complement Factor ◽  
C Terminus

Complement factor H (CFH) is a soluble complement regulatory protein essential for the down-regulation of the alternative pathway on interaction with specific markers on the host cell surface. It recognizes the complement component 3b (C3b) and 3d (C3d) fragments in addition to self cell markers (i.e. glycosaminoglycans, sialic acid) to distinguish host cells that deserve protection from pathogens that should be eliminated. The Staphylococcus aureus surface protein serine–aspartate repeat protein E (SdrE) was previously reported to bind human CFH as an immune-evasion tactic. However, the molecular mechanism underlying SdrE–CFH-mediated immune evasion remains unknown. In the present study, we identified a novel region at CFH's C-terminus (CFH1206–1226), which binds SdrE N2 and N3 domains (SdrEN2N3) with high affinity, and determined the crystal structures of apo-SdrEN2N3 and the SdrEN2N3–CFH1206–1226 complex. Comparison of the structure of the CFH–SdrE complex with other CFH structures reveals that CFH's C-terminal tail flips from the main body to insert into the ligand-binding groove of SdrE. In addition, SdrEN2N3 adopts a ‘close’ state in the absence of CFH, which undergoes a large conformational change on CFH binding, suggesting a novel ‘close, dock, lock and latch' (CDLL) mechanism for SdrE to recognize its ligand. Our findings imply that SdrE functions as a ‘clamp' to capture CFH's C-terminal tail via a unique CDLL mechanism and sequesters CFH on the surface of S. aureus for complement evasion.

scholarly journals New functional and structural insights from updated mutational databases for complement factor H, Factor I, membrane cofactor protein and C3

2014 ◽  
Vol 34 (5) ◽  
Author(s):  
Elizabeth Rodriguez ◽  
Pavithra M. Rallapalli ◽  
Amy J. Osborne ◽  
Stephen J. Perkins
Keyword(s):  
Alternative Pathway ◽  
Factor H ◽  
Complement Factor H ◽  
Complement C3 ◽  
Complement Factor ◽  
Membrane Cofactor Protein ◽  
Complement Alternative Pathway ◽  
Factor I ◽  
Mutational Hotspots ◽  
Structural Insights

A new compilation of 324 mutations in four major proteins from the complement alternative pathway reveals mutational hotspots in factor H and complement C3, and less so in factor I and membrane cofactor protein. Their associations with function are discussed.

IMMU-12. TUMOR CELL IDO ENHANCES IMMUNE SUPPRESSION AND DECREASES SURVIVAL INDEPENDENT OF TRYPTOPHAN METABOLISM IN GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi94-vi94
Author(s):  
Lijie Zhai ◽  
April Bell ◽  
Erik Ladomersky ◽  
Kristen Lauing ◽  
Lakshmi Bollu ◽  
...  
Keyword(s):  
Overall Survival ◽  
T Cells ◽  
Regulatory T Cells ◽  
Cell Lines ◽  
Factor H ◽  
Lower Grade ◽  
Complement Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Human Gbm

Abstract OBJECTIVE Indoleamine 2,3-dioxygenase 1 (IDO; IDO1) is an immune checkpoint that’s characterized as a potent immunosuppressive mediator through its ability to metabolize tryptophan and wild-type IDH patient-resected glioblastoma (GBM) expresses IDO in ≥ 95% of cases. Recent findings from our group led us to investigate the alternative hypothesis that IDO possesses immunosuppressive effects that are independent of its associated metabolic activity. METHODS Murine GBM cell lines that overexpress either wild-type or enzyme-null IDO were created for in vivo characterization of IDO enzyme-independent immunosuppressive function. Microarray was conducted to identify human IDO expression-correlated genes, which were further investigated in human GBM cell lines, patient GBM tissues and plasma, as well as the TCGA database. Ex vivo cell co-culture assays and syngeneic mouse orthotopic GBM models were employed to study immunosuppressive mechanisms. RESULTS Here, we demonstrate that non-enzymic IDO activity decreases survival in experimental animals and increases the expression of immunosuppressive complement factor H (CFH) in human GBM. CFH mRNA levels positively correlate with those of IDO and many other immunosuppressive genes in patient resected GBM and can be applied as a prognostic marker in both lower grade gliomas and GBM. Similar to IDO, the increased expression of CFH in patient-resected glioma was positively correlated with an increased signature for regulatory T cells (Tregs) and myeloid-derived suppressive cells (MDSCs). High expression of CFH in tumor cells increases intratumoral Tregs levels and decreases overall survival in mice with GBM, while inducing tumor associated macrophage cell differentiation. CONCLUSIONS Here, we demonstrated that glioblastoma (GBM) cell IDO promotes the accumulation of intratumoral FoxP3+ regulatory T cells (Tregs) and tumor progression while decreasing overall survival - independent of IDO enzyme activity. Our study reveals a targetable non-metabolic IDO-dependent mechanism for future therapeutic intervention in patients with GBM.

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