Identification of the Staphylococcus aureus MSCRAMM clumping factor B (ClfB) binding site in the αC-domain of human fibrinogen

Clumping factor B (ClfB) of Staphylococcus aureus binds to cytokeratin 10 and to fibrinogen. In this study the binding site in human fibrinogen was localized to a short region within the C terminus of the Aα-chain. ClfB only bound to the Aα-chain of fibrinogen in a ligand-affinity blot and in solid-phase assays with purified recombinant fibrinogen chains. A variant of fibrinogen with wild-type Bβ- and γ-chains but with a deletion that lacked the C-terminal residues from 252–610 of the Aα-chain did not support adherence of S. aureus Newman expressing ClfB. A series of truncated mutants of the recombinant Aα-chain were tested for their ability to support adherence of S. aureus Newman ClfB+, which allowed the binding site to be localized to a short segment of the unfolded flexible repeated sequence within the C terminus of the Aα-chain. This was confirmed by two amino acid substititions within repeat 5 of the recombinant Aα-chain which did not support adherence of Newman ClfB+. Lactococcus lactis expressing ClfB mutants with amino acid substitutions (N256 and Q235) located in the putative ligand-binding trench between domains N2 and N3 of the A-domain were defective in adherence to immobilized fibrinogen and cytokeratin 10, suggesting that both ligands bind to the same or overlapping regions

A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system

Shotgun Phage Display - Selection for Bacterial Receptins or other Exported Proteins

Shotgun phage display cloning involves construction of libraries from randomly fragmented bacterial chromosomal DNA, cloned genes, or eukaryotic cDNAs, into a phagemid vector. The library obtained consists of phages expressing polypeptides corresponding to all genes encoded by the organism, or overlapping peptides derived from the cloned gene. From such a library, polypeptides with affinity for another molecule can be isolated by affinity selection, panning. The technique can be used to identify bacterial receptins and identification of their minimal binding domain, and but also to identify epitopes recognised by antibodies. In addition, after modification of the phagemid vector, the technique has also been used to identify bacterial extracytoplasmic proteins

Crystal Structures Reveal the Multi-Ligand Binding Mechanism of Staphylococcus aureus ClfB

Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties

Evolutionary Analyses of Staphylococcus aureus Identify Genetic Relationships between Nasal Carriage and Clinical Isolates

Nasal carriage of Staphylococcus aureus has long been hypothesized to be a major vector for the transmission of virulent strains throughout the community. To address this hypothesis, we have analyzed the relatedness between a cohort of nasal carriage strains and clinical isolates to understand better the genetic conformity therein. To assess the relatedness between nasal carriage and clinical isolates of S. aureus, a genetic association study was conducted using multilocus sequence typing (MLST) and typing of the hypervariable regions of clumping factor and fibronectin binding protein genes. At all loci analyzed, genetic associations between both nasal carriage and clinical isolates were observed. Computational analyses of MLST data indicate that nasal carriage and clinical isolates belong to the same genetic clusters (clades), despite differences in sequence type assignments. Genetic analyses of the hypervariable regions from the clumping factor and fibronectin binding protein genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates within our cohort, but they also exhibit 100% sequence similarity within these regions. The findings of this report indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of morbidity and mortality

The Multifunctional LigB Adhesin Binds Homeostatic Proteins with Potential Roles in Cutaneous Infection by Pathogenic Leptospira interrogans

Leptospirosis is a potentially fatal zoonotic disease in humans and animals caused by pathogenic spirochetes, such as Leptospira interrogans. The mode of transmission is commonly limited to the exposure of mucous membrane or damaged skin to water contaminated by leptospires shed in the urine of carriers, such as rats. Infection occurs during seasonal flooding of impoverished tropical urban habitats with large rat populations, but also during recreational activity in open water, suggesting it is very efficient. LigA and LigB are surface localized proteins in pathogenic Leptospira strains with properties that could facilitate the infection of damaged skin. Their expression is rapidly induced by the increase in osmolarity encountered by leptospires upon transition from water to host. In addition, the immunoglobulin-like repeats of the Lig proteins bind proteins that mediate attachment to host tissue, such as fibronectin, fibrinogen, collagens, laminin, and elastin, some of which are important in cutaneous wound healing and repair. Hemostasis is critical in a fresh injury, where fibrinogen from damaged vasculature mediates coagulation. We show that fibrinogen binding by recombinant LigB inhibits fibrin formation, which could aid leptospiral entry into the circulation, dissemination, and further infection by impairing healing. LigB also binds fibroblast fibronectin and type III collagen, two proteins prevalent in wound repair, thus potentially enhancing leptospiral adhesion to skin openings. LigA or LigB expression by transformation of a nonpathogenic saprophyte, L. biflexa, enhances bacterial adhesion to fibrinogen. Our results suggest that by binding homeostatic proteins found in cutaneous wounds, LigB could facilitate leptospirosis transmission. Both fibronectin and fibrinogen binding have been mapped to an overlapping domain in LigB comprising repeats 9–11, with repeat 11 possibly enhancing binding by a conformational effect. Leptospirosis patient antibodies react with the LigB domain, suggesting applications in diagnosis and vaccines that are currently limited by the strain-specific leptospiral lipopolysaccharide coats

A Structural Model of the Staphylococcus aureus ClfA–Fibrinogen Interaction Opens New Avenues for the Design of Anti-Staphylococcal Therapeutics

The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) γ-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin αIIbβ3 and ClfA bind to the same segment in the Fg γ-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the “Dock, Lock and Latch” mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not αIIbβ3 may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures

Bacteriophage Lysin Mediates the Binding of Streptococcus mitis to Human Platelets through Interaction with Fibrinogen

The binding of bacteria to human platelets is a likely central mechanism in the pathogenesis of infective endocarditis. We have previously found that platelet binding by Streptococcus mitis SF100 is mediated by surface components encoded by a lysogenic bacteriophage, SM1. We now demonstrate that SM1-encoded lysin contributes to platelet binding via its direct interaction with fibrinogen. Far Western blotting of platelets revealed that fibrinogen was the major membrane-associated protein bound by lysin. Analysis of lysin binding with purified fibrinogen in vitro confirmed that these proteins could bind directly, and that this interaction was both saturable and inhibitable. Lysin bound both the Aα and Bβ chains of fibrinogen, but not the γ subunit. Binding of lysin to the Bβ chain was further localized to a region within the fibrinogen D fragment. Disruption of the SF100 lysin gene resulted in an 83±3.1% reduction (mean ± SD) in binding to immobilized fibrinogen by this mutant strain (PS1006). Preincubation of this isogenic mutant with purified lysin restored fibrinogen binding to wild type levels. When tested in a co-infection model of endocarditis, loss of lysin expression resulted in a significant reduction in virulence, as measured by achievable bacterial densities (CFU/g) within vegetations, kidneys, and spleens. These results indicate that bacteriophage-encoded lysin is a multifunctional protein, representing a new class of fibrinogen-binding proteins. Lysin appears to be cell wall-associated through its interaction with choline. Once on the bacterial surface, lysin can bind fibrinogen directly, which appears to be an important interaction for the pathogenesis of endocarditis