Alpha-toxin is required for biofilm formation by Staphylococcus aureus

Staphylococcus aureus is a common pathogen associated with nosocomial infections. It can persist in clinical settings and gain increased resistance to antimicrobial agents through biofilm formation. We have found that alpha-toxin, a secreted, multimeric, hemolytic toxin encoded by the hla gene, plays an integral role in biofilm formation. The hla mutant was unable to fully colonize plastic surfaces under both static and flow conditions. Based on microscopy studies, we propose that alpha-hemolysin is required for cell-to-cell interactions during biofilm formation.     

Phosphodiesterase activity of CvfA is required for virulence in Staphylococcus aureus

We previously identified the cvfA gene (SA1129) as a novel virulence regulator in Staphylococcus aureus using the silkworm infection model. The cvfA gene, which is conserved among various pathogenic bacteria, contributes to the expression of the agr locus, a global virulence regulator that controls the expression of genes encoding various exoproteins, such as hemolysin. CvfA protein has a transmembrane domain, an RNA binding domain (KH domain), and a metal-dependent phosphohydrolase domain (HD domain). We report here the purification of recombinant CvfA protein from a membrane fraction of Escherichia coli by measuring its phosphodiesterase activity. Purified CvfA protein hydrolyzed the phosphodiester linkage of 2',3'-cyclic AMP, 2',3'-cyclic GMP, and 2',3'-cyclic phosphate at the 3'-terminal of RNA in the presence of Mn(2+). CvfA mutant proteins with amino acid substitutions in the HD domain had significantly decreased phosphodiesterase activity. Furthermore, mutated cvfA genes encoding proteins with low phosphodiesterase activity did not complement the decreased hemolysin production or the attenuated killing ability against silkworms in the cvfA deletion mutant. These results suggest that the phosphodiesterase activity of CvfA protein is required for virulence in S. aureus.     

Staphylococcus aureus IsdB is a hemoglobin receptor required for heme iron utilization

The pathogenesis of human infections caused by the gram-positive microbe Staphylococcus aureus has been previously shown to be reliant on the acquisition of iron from host hemoproteins. The iron-regulated surface determinant system (Isd) encodes a heme transport apparatus containing three cell wall-anchored proteins (IsdA, IsdB, and IsdH) that are exposed on the staphylococcal surface and hence have the potential to interact with human hemoproteins. Here we report that S. aureus can utilize the host hemoproteins hemoglobin and myoglobin, but not hemopexin, as iron sources for bacterial growth. We demonstrate that staphylococci capture hemoglobin on the bacterial surface via IsdB and that inactivation of isdB, but not isdA or isdH, significantly decreases hemoglobin binding to the staphylococcal cell wall and impairs the ability of S. aureus to utilize hemoglobin as an iron source. Stable-isotope-tracking experiments revealed removal of heme iron from hemoglobin and transport of this compound into staphylococci. Importantly, mutants lacking isdB, but not isdH, display a reduction in virulence in a murine model of abscess formation. Thus, IsdB-mediated scavenging of iron from hemoglobin represents an important virulence strategy for S. aureus replication in host tissues and for the establishment of persistent staphylococcal infections.     

Mathematical modelling of the agr operon in Staphylococcus aureus

Staphylococcus aureus is a pathogenic bacterium that utilises quorum sensing (QS), a cell-to-cell signalling mechanism, to enhance its ability to cause disease. QS allows the bacteria to monitor their surroundings and the size of their population, and S. aureus makes use of this to regulate the production of virulence factors. Here we describe a mathematical model of this QS system and perform a detailed time-dependent asymptotic analysis in order to clarify the roles of the distinct interactions that make up the QS process, demonstrating which reactions dominate the behaviour of the system at various timepoints. We couple this analysis with numerical simulations and are thus able to gain insight into how a large population of S. aureus shifts from a relatively harmless state to a highly virulent one, focussing on the need for the three distinct phases which form the feedback loop of this particular QS system.     

Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death

The ability of Staphylococcus aureus to adapt to various conditions of stress is the result of a complex regulatory response. Previously, it has been demonstrated that Clp homologues are important for a variety of stress conditions, and our laboratory has shown that a clpC homologue was highly expressed in the S. aureus strain DSM20231 during biofilm formation relative to expression in planktonic cells. Persistence and long-term survival are a hallmark of biofilm-associated staphylococcal infections, as cure frequently fails even in the presence of bactericidal antimicrobials. To determine the role of clpC in this context, we performed metabolic, gene expression, and long-term growth and survival analyses of DSM20231 as well as an isogenic clpC allelic-replacement mutant, a sigB mutant, and a clpC sigB double mutant. As expected, the clpC mutant showed increased sensitivity to oxidative and heat stresses. Unanticipated, however, was the reduced expression of the tricarboxylic acid (TCA) cycle gene citB (encoding aconitase), resulting in the loss of aconitase activity and preventing the catabolization of acetate during the stationary phase. clpC inactivation abolished post-stationary-phase recovery but also resulted in significantly enhanced stationary-phase survival compared to that of the wild-type strain. These data demonstrate the critical role of the ClpC ATPase in regulating the TCA cycle and implicate ClpC as being important for recovery from the stationary phase and also for entering the death phase. Understanding the stationary- and post-stationary-phase recovery in S. aureus may have important clinical implications, as little is known about the mechanisms of long-term persistence of chronic S. aureus infections associated with formation of biofilms.     

An oxidation-sensing mechanism is used by the global regulator MgrA in Staphylococcus aureus

Staphylococcus aureus is a human pathogen responsible for most wound and hospital-acquired infections. The protein MgrA is both an important virulence determinant during infection and a regulator of antibiotic resistance in S. aureus. The crystal structure of the MgrA homodimer, solved at 2.86 A, indicates the presence of a unique cysteine residue located at the interface of the protein dimer. We discovered that this cysteine residue can be oxidized by various reactive oxygen species, such as hydrogen peroxide and organic hydroperoxide. Cysteine oxidation leads to dissociation of MgrA from DNA and initiation of signaling pathways that turn on antibiotic resistance in S. aureus. The oxidation-sensing mechanism is typically used by bacteria to counter challenges of reactive oxygen and nitrogen species. Our study reveals that in S. aureus, MgrA adopts a similar mechanism but uses it to globally regulate different defensive pathways.     

Characterization of RAT,an autolysis regulator in Staphylococcus aureus

In trying to identify genetic loci involved in the regulation of cap5 genes in Staphylococcus aureus, we isolated a transposon mutant that exhibited a growth defect, enhanced autolysis and increased sensitivity to Triton X-100 and penicillin, attributable in part to increased murein hydrolase activity. Analysis of the chromosomal sequence flanking the transposon insertion site revealed that the gene disrupted in the mutant encodes an open reading frame of 147 amino acids. We named this gene rat, which stands for regulator of autolytic activity. Sequence analysis indicated that Rat is homologous to the MarR and, to a lesser extent, the SarA protein families. Mutations in rat resulted in decreased expression of known autolytic regulators lytSR, lrgAB and arlRS. Gel shift studies indicated that Rat binds to the lytRS and arlRS promoters, thus confirming Rat as a DNA-binding protein to these known repressors of autolytic activity. As anticipated, rat appears to be a negative regulator of autolysin genes including lytM and lytN. These data suggest that the rat gene product is an important regulator of autolytic activity in S. aureus.     

The nitrosative stress response of Staphylococcus aureus is required for resistance to innate immunity

Staphylococcus aureus is a highly virulent human pathogen with an extensive array of strategies to subvert the innate immune response. An important aspect of innate immunity is the production of the nitrogen monoxide radical (Nitric Oxide, NO.). Here we describe an adaptive response to nitrosative stress that allows S. aureus to replicate at high concentrations of NO.. Microarray analysis revealed 84 staphylococcal genes with significantly altered expression following NO. exposure. Of these, 30 are involved with iron-homeostasis, potentially under the control of the Fur regulator. Another seven induced genes are involved in hypoxic/fermentative metabolism, including the flavohaemoprotein, Hmp. The SrrAB two-component system has been shown to regulate the expression of many of the NO.-induced metabolic genes. Indeed, inactivation of hmp, srrAB and fur resulted in heightened NO. sensitivity. Hmp was responsible for c. 90% of measurable staphylococcal NO. consumption and therefore critical for efficient NO. detoxification. While SrrAB was required for maximal hmp expression, srrAB mutants still exhibited significant NO. scavenging and NO.-dependent induction of hmp. Yet S. aureus lacking SrrAB were more sensitive to nitrosative stress than hmp mutants, indicating that the contribution of SrrAB to NO. resistance extends beyond the regulation of hmp expression. Both Hmp and SrrAB were required for full virulence in a murine sepsis model, however, only the attenuation of the hmp mutant was restored by the abrogation of host NO. production. Thus, the S. aureus Hmp protein has evolved to serve as an iNOS-dependent virulence determinant.