mgrA regulates staphylococcal virulence important for induction and progression of septic arthritis and sepsis

Septic arthritis and sepsis are common and feared complications of staphylococcal infections, and the increasing antibiotic resistance among staphylococci urge the extended research for virulence factors involved in these diseases. Staphylcoccus aureus produces a number of virulence factors controlled by several global regulatory genes including agr and sarA. MgrA is a recently identified global regulator, belonging to the SarA subfamily, which upregulates expression of several virulence factors including capsule and sortase. In addition, MgrA has been shown to regulate antibiotic resistance and decrease bacterial autolysis. In this study we have assessed the role of mgrA gene expression on induction and progression of septic arthritis and sepsis. Mice inoculated with the mgrA mutant displayed significantly less severe arthritis and showed a significantly better weight development, than wild-type inoculated mice. Importantly, all 10 mice inoculated with the mgrA mutant survived as compared to 70% mortality in the wild-type inoculated mice (p=0.003). In addition, the mgrA mutant showed significantly less bacterial persistence in kidneys as compared to the wild-type strain. We conclude that mgrA regulates virulence factors important for establishment and progression of septic arthritis and sepsis.     

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.     

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.     

Conditional mutation of an essential putative glycoprotease eliminates autolysis in Staphylococcus aureus

Our previous studies demonstrated that a putative Staphylococcus aureus glycoprotease (Gcp) is essential for bacterial survival, indicating that Gcp may be a novel target for developing antibacterial agents. However, the biological function of Gcp is unclear. In order to elucidate the reason that Gcp is required for growth, we examined the role of Gcp in bacterial autolysis, which is an important biological process for bacterial growth. Using both a spacp-regulated gcp expression strain and a TetR-regulated gcp antisense expression strain, we found that the down-regulation of gcp expression can effectively inhibit Triton X-100-induced lysis, eliminate penicillin- and vancomycin-caused cell lysis, and dramatically increase tolerance to hydrolases. Moreover, we determined whether resistance to lysis is due to a defect in murein hydrolase activity by using a zymogram analysis. The results showed that the cell lysate of a down-regulated gcp expression mutant displayed several bands of decreased murein hydrolytic activity. Furthermore, we explored the potential mechanism of Gcp's involvement in autolysis and demonstrated that Gcp may function independently from several key autolysins (Atl, LytM, and LytN) and regulators (ArlRS, Mgr/Rat, and CidA). Taken together, the above results indicate that the essential Gcp is involved in the modification of substrates of murein hydrolases as well as in the regulation of expression and/or activity of some murein hydrolases, which, in turn, may play important roles in bacterial viability.     

Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus.

Previously in our laboratory, a PCR-based strategy was used to isolate potential sensor gene fragments from the Staphyloccus aureus genome. One DNA fragment was isolated that shared strong sequence similarity to genes encoding bacterial sensor proteins, indicating that it originated from within a potential staphylococcal sensor protein gene. In this study, the DNA surrounding the PCR product origin was cloned and sequenced. This analysis revealed the presence of two genes, termed lytS and lytR, whose deduced amino acid sequences were similar to those of members of the two-component regulatory system family of proteins. S. aureus cells containing an insertional disruption of lytS exhibited a marked propensity to form aggregates in liquid culture, suggesting that alterations in cell surface components exist in this strain. Transmission electron microscopic examination of these cells revealed that the cell surface was rough and diffuse and that a large proportion of the cell population had lysed. The lytS mutant also exhibited increased autolysis and an altered level of murein hydrolase activity produced compared with the parental strain, NCTC 8325-4. These data suggest that the lytS and lytR gene products control the rate of autolysis in S. aureus by affecting the intrinsic murein hydrolase activity associated with the cell.     

Opposing Roles of the Staphylococcus aureus Virulence Regulators, Agr and Sar, in Triton X-100- and Penicillin-Induced Autolysis

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the Staphylococcus aureus virulence factor regulatory genes, agr and sar, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest that agr and sar exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.