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. 2009 Jan 15;199(2):209-18.
doi: 10.1086/595740.

Role of mgrA and sarA in methicillin-resistant Staphylococcus aureus autolysis and resistance to cell wall-active antibiotics

María Pilar Trotonda  1 , Yan Q Xiong, Guido Memmi, Arnold S Bayer, Ambrose L Cheung
Affiliations

Role of mgrA and sarA in methicillin-resistant Staphylococcus aureus autolysis and resistance to cell wall-active antibiotics

María Pilar Trotonda et al. J Infect Dis. .

Abstract

Background: We have previously shown the importance of mgrA and sarA in controlling autolysis of Staphylococcus aureus, with MgrA and SarA both being negative regulators of murein hydrolases.

Methods: In this study, we analyzed the effects of mgrA and sarA on antibiotic-mediated lysis in vitro and on the responses to cell wall-active antibiotic therapy in an experimental endocarditis model by use of 2 representative MRSA strains: the laboratory strain COL and the community-acquired clinical strain MW2.

Results: We found that mgrA and sarA independently down-regulated sarV (a marker for autolysis), although the alteration in sarV expression did not correlate directly with the autolysis profiles of single mgrA and sarA mutants. Importantly, the mgrA/sarA double mutants of both strains were more autolytic than the single mutants in vitro. We demonstrated that, despite equivalent intrinsic virulences of the parent strains and their isogenic mgrA/sarA double mutants in the endocarditis model, oxacillin and vancomycin treatment of the mgrA/sarA double mutants yielded significant reductions in vegetation bacterial densities in vivo, compared with treatment of their respective parent strains.

Conclusions: These results suggest that down-regulation of mgrA/sarA in combination with use of cell wall-active antibiotics may represent a novel approach to treat MRSA infections.

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Figures

Figure 1
Figure 1
Dose response of oxacillin mediated lysis in COL (A) and MW2 (B). Growth curve of the parental strains, their respective isogenic mgrA, sarA and mgrA/sarA mutants at different concentrations of oxacillin. Wild-type S. aureus strains (◆;), ΔmgrA (■), ΔsarA (▲) and ΔmgrA/ΔsarA mutants (X).
Figure 2
Figure 2
Triton X-100- (A) and oxacillin- (B) (1/10 MICs) induced autolysis over time at 30°C with the COL strain set. Wild-type COL strain (◆), ΔmgrA (■), ΔsarA (▲) and ΔmgrA/ΔsarA mutants (X). The Y-axis represents percent lysis.
Figure 3
Figure 3
Zymographic analyses of extracellular murein hydrolases in S. aureus strains COL (A and B) and MW2 (C and D) and their isogenic mutants. Lane 1, wild-type; lane 2, ΔmgrA; lane 3, ΔsarA; lane 4, ΔmgrA/ΔsarA; lane 5, ΔsarV; lane 6, wild type hyper-expressing sarV. Processed Atl is indicated by unfilled block arrow. The glucosaminidase (51 kDa) and amidase bands (62 kDa) are indicated by arrow heads and filled blocked arrows, respectively. The short and long narrow arrows highlight unidentified murein hydrolase bands.
Figure 4
Figure 4
sarV expression in S. aureus COL and MW2 and their respective mgrA, sarA and mgrA/sarA double mutants. (A) sarV promoter activation was represented as mean fluorescence/OD650 ratio from triplicate readings at different times during growth. Wild-type strains (◆), ΔmgrA (■), ΔsarA (▲), ΔmgrA/ΔsarA (X) and sarV mutant (o). The standard errors of the means are indicted for each time points. The final time point at 7 hrs is used for statistical analysis (*). Using the Student t test, differences are noted between COL vs. double mutant (p <0.02), COL sarA mutant vs. double mutant (p< 0.046), MW2 vs. double mutant (p<0.01) and MW2 sarA mutant vs. double mutant (p < 0.01). (B) SarV protein expression by immunoblots. A 14 kDa band was recognized by polyclonal antibodies against SarV in S. aureus COL and MW2 and their respective mgrA, sarA and mgrA-sarA double mutants and the sarV hyperexpressor strains. Lane 1, wild-type; lane 2, ΔmgrA; lane 3, ΔsarA; lane 4, ΔmgrA/ΔsarA; lane 5, ΔsarV; lane 6, sarV hyperexpressor.
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