Sequence diversity in the A domain of Staphylococcus aureus fibronectin-binding protein A.

BACKGROUND: Fibronectin-binding protein A (FnBPA) mediates adhesion of Staphylococcus aureus to fibronectin, fibrinogen and elastin. We previously reported that S. aureus strain P1 encodes an FnBPA protein where the fibrinogen/elastin-binding domain (A domain) is substantially divergent in amino acid sequence from the archetypal FnBPA of S. aureus NCTC8325, and that these variations created differences in antigenicity. In this study strains from multilocus sequence types (MLST) that spanned the genetic diversity of S.aureus were examined to determine the extent of FnBPA A domain variation within the S. aureus population and its effect on ligand binding and immuno-crossreactivity. RESULTS: Seven different isotype forms (I - VII) of the FnBPA A domain were identified which were between 66 to 76% identical in amino acid sequence in any pair-wise alignment. The fnbA allelic variants in strains of different multilocus sequence type were identified by DNA hybridization using probes specific for sequences encoding the highly divergent N3 sub-domain of different isotypes. Several isotypes were not restricted to specific clones or clonal complexes but were more widely distributed. It is highly likely that certain fnbA genes have been transferred horizontally. Residues lining the putative ligand-binding trench were conserved, which is consistent with the ability of each A domain isotype to bind immobilized fibrinogen and elastin by the dock-latch-lock mechanism. Variant amino acid residues were mapped on a three-dimensional model of the FnBPA A domain and were predicted to be surface-exposed. Polyclonal antibodies raised against the recombinant isotype I A domain bound that protein with a 4 - 7 fold higher apparent affinity compared to the A domains of isotypes II - VII, while some monoclonal antibodies generated against the isotype I A domain showed reduced or no binding to the other isotypes. CONCLUSION: The FnBPA A domain occurs in at least 7 different isotypes which differ antigenically and exhibit limited immuno-crossreactivity, yet retain their ligand-binding functions. Antigenic variation of the FnBPA A domain may aid S. aureus to evade the host's immune responses. These findings have implications for the development of vaccines or immunotherapeutics that target FnBPA.

A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB.

Device-associated infections involving biofilm remain a persistent clinical problem. We recently reported that four methicillin-resistant Staphylococcus aureus (MRSA) strains formed biofilm independently of the icaADBC-encoded exopolysaccharide. Here, we report that MRSA biofilm development was promoted under mildly acidic growth conditions triggered by the addition of glucose to the growth medium. Loss of sortase, which anchors LPXTG-containing proteins to peptidoglycan, reduced the MRSA biofilm phenotype. Furthermore introduction of mutations in fnbA and fnbB, which encode the LPXTG-anchored multifunctional fibrinogen and fibronectin-binding proteins, FnBPA and FnBPB, reduced biofilm formation by several MRSA strains. However, these mutations had no effect on biofilm formation by methicillin-sensitive S. aureus strains. FnBP-promoted biofilm occurred at the level of intercellular accumulation and not primary attachment. Mutation of fnbA or fnbB alone did not substantially affect biofilm, and expression of either gene alone from a complementing plasmid in fnbA fnbB mutants restored biofilm formation. FnBP-promoted biofilm was dependent on the integrity of SarA but not through effects on fnbA or fnbB transcription. Using plasmid constructs lacking regions of FnBPA to complement an fnbAB mutant revealed that the A domain alone and not the domain required for fibronectin binding could promote biofilm. Additionally, an A-domain N304A substitution that abolished fibrinogen binding did not affect biofilm. These data identify a novel S. aureus biofilm phenotype promoted by FnBPA and FnBPB which is apparently independent of the known ligand-binding activities of these multifunctional surface proteins.

Molecular basis for Staphylococcus aureus-mediated platelet aggregate formation under arterial shear in vitro.

OBJECTIVE: Staphylococcus aureus is the most frequent causative organism of infective endocarditis (IE) and is characterized by thrombus formation on a cardiac valve that can embolize to a distant site. Previously, we showed that S. aureus clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA) can stimulate rapid platelet aggregation. METHODS AND RESULTS: In this study we investigate their relative roles in mediating aggregate formation under physiological shear conditions. Platelets failed to interact with immobilized wild-type S. aureus (Newman) at shear rates <500 s(-1) but rapidly formed an aggregate at shear rates >800 s(-1). Inactivation of the ClfA gene eliminated aggregate formation at any shear rate. Using surrogate hosts that do not interact with platelets bacteria overexpressing ClfA supported rapid aggregate formation under high shear with a similar profile to Newman whereas bacteria overexpressing FnBPA did not. Fibrinogen binding to ClfA was found to be essential for aggregate formation although fibrinogen-coated surfaces only allowed single-platelets to adhere under all shear conditions. Blockade of the platelet immunoglobulin receptor Fc gammaRIIa inhibited aggregate formation. CONCLUSIONS: Thus, fibrinogen and IgG binding to ClfA is essential for aggregate formation under arterial shear conditions and may explain why S. aureus is the major cause of IE.

Capsular polysaccharide masks clumping factor A-mediated adherence of Staphylococcus aureus to fibrinogen and platelets.

BACKGROUND: Clumping factor A (ClfA) is a Staphylococcus aureus cell wall-associated adhesin that mediates staphylococcal binding to fibrinogen and platelets. Our goals were to determine whether expression of capsular polysaccharide (CP) affected ClfA-mediated adherence of S. aureus and to assess whether the length of the ClfA repeat region influenced this interaction. METHODS: ClfA constructs with repeat regions of different lengths were introduced into isogenic S. aureus strains that expressed CP5, CP8, or no CP. S. aureus binding to fibrinogen was assessed in rabbit plasma and on fibrinogen-coated microtiter plates. Adherence of S. aureus strains to platelets was evaluated by flow cytometry and confocal microscopy. RESULTS: As the length of the ClfA repeat region increased, binding of acapsular S. aureus to fibrinogen-coated microtiter plates was enhanced. By contrast, encapsulated S. aureus expressing the full-length ClfA were poorly adherent. The acapsular S. aureus mutant strain showed a 2-fold increase in platelet binding, compared with the isogenic encapsulated strains. By contrast, platelet aggregation was unaffected by CP production. CONCLUSION: CP expression inhibits S. aureus ClfA-mediated binding to fibrinogen and platelets, and a full-length repeat region cannot overcome this inhibition. These findings have important implications for vaccine development, given that CP may mask surface adhesins.

Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B.

Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.

Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus.

The fibronectin binding protein, FnBPA, is a multifunctional microbial surface component recognizing adhesive matrix molecule (MSCRAMM) that promotes bacterial adherence to immobilized fibrinogen and elastin via the N-terminal A domain. The binding site for fibrinogen and elastin was localized to subdomains N2N3. A three-dimensional structural model of FnBPA was created based on the known crystal structure of the domains N2N3 of clumping factor A (ClfA). The role of individual residues in the putative ligand binding trench was examined by testing the affinity of mutants for fibrinogen and elastin. Two residues (N304 and F306) were crucial for binding both ligands and are in the equivalent positions to residues known to be important for fibrinogen binding by ClfA. A peptide comprising the C-terminus of the gamma-chain of fibrinogen and a monoclonal anti-rAFnBPA antibody were potent inhibitors of the FnBPA-elastin interaction. This suggests that FnBPA binds to fibrinogen and elastin in a similar manner. Amino acid sequence divergence of 26.5% occurred between the A domains of FnBPA from strains 8325-4 and P1. Most variant residues were predicted to be located on the surface of domains N2N3 while few occurred in the putative ligand binding trench and the latching peptide explaining limited immunocross reactivity while ligand binding activity is conserved.

Clumping factor A of Staphylococcus aureus inhibits phagocytosis by human polymorphonuclear leucocytes.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.

The role of Staphylococcus aureus adhesins in the pathogenesis of ventricular assist device-related infections.

Ventricular assist devices (VADs) are an important form of therapy for end-stage congestive heart failure. However, infection of the VAD, which is often caused by Staphylococcus aureus, poses a major threat to survival. Using a novel in vitro binding assay with VAD membranes and a heterologous lactococcal system of expression, we identify 3 S. aureus proteins--clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as the main factors involved in adherence to VAD polyurethane membranes. Adherence is greatly diminished by long implantation times, reflecting a change in topological features of the VAD membrane, and is primarily mediated by the FnBPA domains in the staphylococcal proteins. We also compare the adherence of S. aureus mutant strains and show that other staphylococcal components appear to be involved in adherence to VAD membranes. Finally, we demonstrate that ClfA, FnBPA, and FnBPB mediate bacterial infection of implanted murine intra-aortic polyurethane patches.

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor.

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin-binding proteins FnBPA and FnBPB are major platelet-activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen-binding A domain or the fibronectin-binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA-promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV-3) specific for the FcgammaRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcgammaRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV-3 suggesting that the IgG-FcgammaRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A.

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA-promoted platelet activation. Expression of ClfA from a nisin-inducible promoter demonstrated that a minimum level of surface-expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcgammaRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface-bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcgammaRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen-independent pathway required IgG and complement deposition to trigger platelet aggregation.