ABSTRACTThe WalKR two-component system is essential for the viability ofStaphylococcus aureus, playing a central role in controlling cell wall metabolism. We produced a constitutively active form of WalR inS. aureusthrough a phosphomimetic amino acid replacement (WalRc, D55E). The strain displayed significantly increased biofilm formation and alpha-hemolytic activity. Transcriptome analysis was used to determine the full extent of the WalKR regulon, revealing positive regulation of major virulence genes involved in host matrix interactions (efb,emp,fnbA, andfnbB), cytolysis (hlgACB,hla, andhlb), and innate immune defense evasion (scn,chp, andsbi), through activation of the SaeSR two-component system. The impact on pathogenesis of varying cell envelope dynamics was studied using a murine infection model, showing that strains producing constitutively active WalRcare strongly diminished in their virulence due to early triggering of the host inflammatory response associated with higher levels of released peptidoglycan fragments. Indeed, neutrophil recruitment and proinflammatory cytokine production were significantly increased when the constitutively activewalRcallele was expressed, leading to enhanced bacterial clearance. Taken together, our results indicate that WalKR play an important role in virulence and eliciting the host inflammatory response by controlling autolytic activity.

c As a consequence of excessive antibiotic therapies in hospitalized patients, Clostridium difficile, a Gram-positive anaerobic spore-forming intestinal pathogen, is the leading cause of hospital-acquired diarrhea and colitis. Drug treatments for these diseases are often complicated by antibiotic-resistant strains and a high frequency of treatment failures and relapse; therefore, novel nonantibiotic approaches may prove to be more effective. In this study, we recombinantly expressed a prophage lysin identified from a C. difficile strain, CD630, which we named PlyCD. PlyCD was found to have lytic activity against specific C. difficile strains. However, the recombinantly expressed catalytic domain of this protein, PlyCD 1-174 , displayed significantly greater lytic activity (>4-log kill) and a broader lytic spectrum against C. difficile strains while still retaining a high degree of specificity toward C. difficile versus commensal clostridia and other bacterial species. Our data also indicated that noneffective doses of vancomycin and PlyCD 1-174 when combined in vitro could be significantly more bactericidal against C. difficile. In an ex vivo treatment model of mouse colon infection, we found that PlyCD 1-174 functioned in the presence of intestinal contents, significantly decreasing colonizing C. difficile compared to controls. Together, these data suggest that PlyCD 1-174 has potential as a novel therapeutic for clinical application against C. difficile infection, either alone or in combination with other preexisting treatments to improve their efficacy.

The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl–glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.