Role of ArlRS in autolysis in methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains

Abstract

Autolysis plays an essential role in bacterial cell division and lysis with β-lactam antibiotics. Accordingly, the expression of autolysins is tightly regulated by several endogenous regulators, including ArlRS, a two component regulatory system that has been shown to negatively regulate autolysis in methicillin-sensitive Staphylococcus aureus (MSSA) strains. In this study, we found that inactivation of arlRS does not play a role in autolysis of methicillin-resistant S. aureus (MRSA) strains, such as community-acquired (CA)-MRSA strains USA300 and MW2 or the hospital-acquired (HA)-MRSA strain COL. This contrasts with MSSA strains, including Newman, SH1000, RN6390, and 8325-4, where autolysis is affected by ArlRS. We further demonstrated that the striking difference in the roles of arlRS between MSSA and MRSA strains is not due to the methicillin resistance determinant mecA. Among known autolysins and their regulators, we found that arlRS represses lytN, while no effect was seen on atl, lytM, and lytH expression in both CA- and HA-MRSA strains. Transcriptional-fusion assays showed that the agr transcripts, RNAII and RNAIII, were significantly more downregulated in the arlRS mutant of MW2 than the MSSA strain Newman. Importantly, provision of agr RNAIII in trans to the MW2 arlRS mutant via a multicopy plasmid induced autolysis in this MRSA strain. Also, the autolytic phenotype in the arlRS mutant of MSSA strain Newman could be rescued by a mutation in either atl or lytM. Together, these data showed that ArlRS impacts autolysis differently in MSSA and MRSA strains.

Fig 1

Increased autolysis of the arlRS mutant of strain Newman in TSB supplemented with 0.02% Triton X-100. (A and B) Growth curves of MW2, Newman, and their isogenic arlRS mutants with and without 0.02% Triton X-100. Overnight cultures were diluted to an OD₆₅₀ of 0.1 in TSB, supplemented with 0.02% Triton X-100, and grown at 37°C with shaking. The asterisk indicates statistical significance of the growth of the arlRS mutant of Newman versus the parent strain at all time points by a paired Student t test (P < 0.001). WT, wild type. (C) Map of the arlRS TCRS locus. Shown are the open reading frame designations of the MW2 and Newman genomes, secondary gene names, proposed functions, translated protein sizes (amino acids [aa]), and the region deleted in the present study. (D) Deletion of arlRS does not significantly affect murein hydrolase activity in Newman (New) and MW2. Shown are zymogram analyses of cell extracts from S. aureus MW2, Newman, and their isogenic arlRS mutants. Equivalent amounts of cell extracts were separated on an 8% SDS-polyacrylamide gel containing heat-killed S. aureus RN4220 cells (left) or Micrococcus lysodeikticus (right). The resolved gels were washed with water, incubated with lysis buffer (50 mM Tris, 0.1% Triton X-100, 10 mM MgCl₂, and CaCl₂) for 18 to 24 h, and stained with 0.5% methylene blue. Areas of murein hydrolase activity are indicated by clear zones (the same results were obtained with heat-killed cells of Newman and MW2 arlRS mutants).

Fig 2

Effects of complementation on autolysis of arlRS mutants of Newman and MW2. (A) Complementation of arlRS on the chromosome or in trans with pEPSA5::arlRS restored wild-type levels of growth in the presence of Triton X-100. Shown are growth curves of Newman, its isogenic arlRS mutant, and complemented strains with and without 0.02% Triton X-100. The asterisk indicates statistical significance of the growth of the arlRS mutant versus the parent strain Newman at all time points by a paired Student t test (P < 0.001). (B) Effect of mecA on autolysis of arlRS mutants of Newman and MW2. Deletion of mecA in the arlRS mutant of MW2 did not affect growth under autolysis-inducing conditions, while cross-complementation of the arlRS mutant of Newman had no effect on restoration of the defect in Triton X-100-induced autolysis. Shown are growth curves of the Newman wild type and the arlRS mutant carrying pEPSA5::mecA, MW2 mecA, and mecA-arlRS mutants in TSB-0.02% Triton X-100. The asterisk indicates statistical significance of the growth of the arlRS mutant versus its isogenic parent strain Newman at all time points by a paired Student t test (P < 0.001).

Fig 3

Effects of the arlRS mutation on the expression of autolysin genes and known lytic regulators. Shown is Northern blot analysis of lytN, lrgAB, and lytRS (A) and of mgrA, RNAII, and RNAIII (B) expression. The blots were hybridized with a DNA probe specific for each gene radiolabeled with [α-³²P]dCTP. Below are shown the ethidium bromide-stained rRNAs, indicating equivalent amounts of RNA in each sample. The RNAs for each strain were extracted at OD₆₅₀s of 0.7, 1.1, and 1.7, corresponding to exponential, late-exponential, and early-stationary phases, respectively. The experiment was repeated at least three times with similar results. A representative experiment is shown.

Fig 4

Regulation of ArlRS on RNAIII. Shown is the expression of GFP_uvr driven by the agr P3 promoter in overnight cultures. Promoter activity was plotted as mean fluorescence/OD₆₅₀ from 3 clones in triplicate. The experiments were repeated three times, with one set shown. The asterisk indicates statistical significance of the indicated strain compared to MW2 by a paired Student t test (P < 0.001). comp, complementation.

Fig 5

Overexpression of RNAIII affects growth of the arlRS mutant of MW2 under autolysis-inducing conditions. (Left) Growth curves of MW2 and its isogenic arlRS mutant carrying pRN6735 with 0.02% Triton X-100. (Right) Expression of RNAIII of the arlRS mutant of MW2 with and without induction with a sub-MIC concentration of oxacillin. The asterisk indicates statistical significance at OD₆₅₀ for growth of the arlRS mutant of MW2 overexpressing agr at all time points by a paired Student t test (P < 0.001).

Fig 6

Deletion of atl or lytM affects murein hydrolase activity and restores growth in a Newman arlRS mutant strain under autolysis-inducing conditions. (A) Zymogram analysis of cell extracts from S. aureus Newman and its isogenic arlRS, lytN, lytM, atl, and double mutants. Equivalent amounts of cell extracts were separated on an 8% SDS-polyacrylamide gel containing heat-killed S. aureus RN4220 cells. After electrophoresis, the gels were washed with water and then buffer and finally stained with 0.5% methylene blue as described in Materials and Methods. Areas of murein hydrolase activity are indicated by clear zones. The arrows indicate bands of increased activity in lytM and arlRS-lytM mutants. (B) Growth curves of Newman and its isogenic arlRS, lytN, lytM, atl, and double mutants with 0.02% Triton X-100. Overnight cultures were diluted to an OD₆₅₀ of 0.1 in TSB, supplemented with 0.02% Triton X-100, and grown at 37°C with shaking. The asterisk indicates statistical significance of growth of arlRS and arlRS-lytN mutants versus the parent strain Newman at all time points by a paired Student t test (P < 0.001).