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

Importance of the Global Regulators Agr and SaeRS in the Pathogenesis of CA-MRSA USA300 Infection

CA-MRSA infection, driven by the emergence of the USA300 genetic background, has become epidemic in the United States. USA300 isolates are hypervirulent, compared with other CA- and HA-MRSA strains, in experimental models of necrotizing pneumonia and skin infection. Interestingly, USA300 isolates also have increased expression of core genomic global regulatory and virulence factor genes, including agr and saeRS. To test the hypothesis that agr and saeRS promote the observed hypervirulent phenotype of USA300, isogenic deletion mutants of each were constructed in USA300. The effects of gene deletion on expression and protein abundance of selected downstream virulence genes were assessed by semiquantitative real-time reverse-transcriptase PCR (qRT-PCR) and western blot, respectively. The effects of gene deletion were also assessed in mouse models of necrotizing pneumonia and skin infection. Deletion of saeRS, and, to a lesser extent, agr, resulted in attenuated expression of the genes encoding α-hemolysin (hla) and the Panton-Valentine leukocidin (lukSF-PV). Despite the differences in hla transcription, the toxin was undetectable in culture supernatants of either of the deletion mutants. Deletion of agr, but not saeRS, markedly increased the expression of the gene encoding protein A (spa), which correlated with increased protein abundance. Each deletion mutant demonstrated significant attenuation of virulence, compared with wild-type USA300, in mouse models of necrotizing pneumonia and skin infection. We conclude that agr and saeRS each independently contribute to the remarkable virulence of USA300, likely by means of their effects on expression of secreted toxins

Genetic Diversity of Staphylocoagulase Genes (coa): Insight into the Evolution of Variable Chromosomal Virulence Factors in Staphylococcus aureus

. Although SCs have been classified into 10 serotypes based on the differences in the antigenicity, genetic bases for their diversities and relatedness to chromosome types are poorly understood. type except for the cases of CC1 and CC8, which contained two and three different SC types, respectively. loci, resulting in the carriage of the combinations of allotypically different important virulence determinants in staphylococcal chromosome

Factors Contributing to the Biofilm-Deficient Phenotype of Staphylococcus aureus sarA Mutants

Mutation of sarA in Staphylococcus aureus results in a reduced capacity to form a biofilm, but the mechanistic basis for this remains unknown. Previous transcriptional profiling experiments identified a number of genes that are differentially expressed both in a biofilm and in a sarA mutant. This included genes involved in acid tolerance and the production of nucleolytic and proteolytic exoenzymes. Based on this we generated mutations in alsSD, nuc and sspA in the S. aureus clinical isolate UAMS-1 and its isogenic sarA mutant and assessed the impact on biofilm formation. Because expression of alsSD was increased in a biofilm but decreased in a sarA mutant, we also generated a plasmid construct that allowed expression of alsSD in a sarA mutant. Mutation of alsSD limited biofilm formation, but not to the degree observed with the corresponding sarA mutant, and restoration of alsSD expression did not restore the ability to form a biofilm. In contrast, concomitant mutation of sarA and nuc significantly enhanced biofilm formation by comparison to the sarA mutant. Although mutation of sspA had no significant impact on the ability of a sarA mutant to form a biofilm, a combination of protease inhibitors (E-64, 1-10-phenanthroline, and dichloroisocoumarin) that was shown to inhibit the production of multiple extracellular proteases without inhibiting growth was also shown to enhance the ability of a sarA mutant to form a biofilm. This effect was evident only when all three inhibitors were used concurrently. This suggests that the reduced capacity of a sarA mutant to form a biofilm involves extracellular proteases of all three classes (serine, cysteine and metalloproteases). Inclusion of protease inhibitors also enhanced biofilm formation in a sarA/nuc mutant, with the combined effect of mutating nuc and adding protease inhibitors resulting in a level of biofilm formation with the sarA mutant that approached that of the UAMS-1 parent strain. These results demonstrate that the inability of a sarA mutant to repress production of extracellular nuclease and multiple proteases have independent but cumulative effects that make a significant contribution to the biofilm-deficient phenotype of an S. aureus sarA mutant

Functional Amyloids Composed of Phenol Soluble Modulins Stabilize Staphylococcus aureus Biofilms

Staphylococcus aureus is an opportunistic pathogen that colonizes the skin and mucosal surfaces of mammals. Persistent staphylococcal infections often involve surface-associated communities called biofilms. Here we report the discovery of a novel extracellular fibril structure that promotes S. aureus biofilm integrity. Biochemical and genetic analysis has revealed that these fibers have amyloid-like properties and consist of small peptides called phenol soluble modulins (PSMs). Mutants unable to produce PSMs were susceptible to biofilm disassembly by matrix degrading enzymes and mechanical stress. Previous work has associated PSMs with biofilm disassembly, and we present data showing that soluble PSM peptides disperse biofilms while polymerized peptides do not. This work suggests the PSMs' aggregation into amyloid fibers modulates their biological activity and role in biofilms