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.     

Contribution of the Staphylococcus aureus Atl AM and GL Murein Hydrolase Activities in Cell Division, Autolysis, and Biofilm Formation

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.     

Identification and molecular characterization of an N-acetylmuramyl-L-alanine amidase Sle1 involved in cell separation of Staphylococcus aureus

We purified a peptidoglycan hydrolase involved in cell separation from a Staphylococcus aureus atl null mutant and identified its gene. Characterization of the gene product shows a 32 kDa N-acetylmuramyl-L-alanine amidase that we designated Sle1. Analysis of peptidoglycan digests showed Sle1 preferentially cleaved N-acetylmuramyl-L-Ala bonds in dimeric cross-bridges that interlink the two murein strands in the peptidoglycan. An insertion mutation of sle1 impaired cell separation and induced S. aureus to form clusters suggesting Sle1 is involved in cell separation of S. aureus. The Sle1 mutant revealed a significant decrease in pathogenesis using an acute infection mouse model. Atl is the major autolysin of S. aureus, which has been implicated in cell separation of S. aureus. Generation of an atl/sle1 double mutant revealed that the mutant cell separation was heavily impaired suggesting that S. aureus uses two peptidoglycan hydrolases, Atl and Sle1, for cell separation. Unlike Atl, Sle1 is not directly involved in autolysis of S. aureus.     

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.     

Identification and characterization of a peptidoglycan hydrolase, MurA, of Listeria monocytogenes, a muramidase needed for cell separation

A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.     

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 of LytSR-regulated genes from Staphylococcus aureus.

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.     

LytF, a novel competence-regulated murein hydrolase in the genus Streptococcus

Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.     

A new two-component regulatory system involved in adhesion, autolysis, and extracellular proteolytic activity of Staphylococcus aureus

A transposition mutant of Staphylococcus aureus was selected from the parent strain MT23142, a derivative of strain 8325. The site of transposition was near the 5' terminus of the gene arlS. ArlS exhibits strong similarities with histidine protein kinases. Sequence analysis suggested that arlS forms an operon with upstream gene arlR. The predicted product of arlR is a member of the OmpR-PhoB family of response regulators. The arlS mutant formed a biofilm on a polystyrene surface unlike the parent strain and the complemented mutant. Biofilm formation was associated with increased primary adherence to polystyrene, whereas cellular adhesion was only slightly decreased. In addition, the arlS mutant exhibited increased autolysis and altered peptidoglycan hydrolase activity compared to the parental strain and to the complemented mutant. As it has been shown for coagulase-negative staphylococci that some autolysins are able to bind polymer surfaces, these data suggest that the two-component regulatory system ArlS-ArlR may control attachment to polymer surfaces by affecting secreted peptidoglycan hydrolase activity. Finally, the arlS mutant showed a dramatic decrease of extracellular proteolytic activity, including serine protease activity, in comparison to the wild-type strain and the complemented mutant, and cells grown in the presence of phenylmethylsulfonyl fluoride (a serine protease inhibitor) showed an increased autolysin activity. Since the locus arlR-arlS strikingly modifies extracellular proteolytic activity, this locus might also be involved in the virulence of S. aureus.     

Identification of the Streptococcus mutans LytST two-component regulon reveals its contribution to oxidative stress tolerance

ABSTRACT: BACKGROUND: The S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans. RESULTS: Real-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant. CONCLUSIONS: Overall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.     

Competence-specific autolysis in Streptococcus sanguis

Streptococcus sanguis strain Wicky activated to competence for genetic transformation is known to undergo a rapid decrease in optical density upon transfer to an alkaline buffer containing reducing agents. We studied the mechanism of this autolysis-like process and made the following observations. The process was specific because preincubation of the competence inducing factor with a specific inactivating protein prevented both cellular lysis and acquisition of competence for genetic transformation. The optical density decrease of competent bacteria involved the release of a large fraction of intracellular protein, RNA and lipid. However, no hydrolysis of phospholipid and no degradation of cell wall polymers including peptidoglycan could be detected. No peptidoglycan hydrolase activity capable of degrading radiolabelled S. sanguis cell walls was detected in unfractionated S. sanguis extracts. It is suggested that autolysis of competent S. sanguis involves the activity of a novel type of murein hydrolase that introduces only a limited number of bond breaks into the peptidoglycan.     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.