Mutated response regulator graR is responsible for phenotypic conversion of Staphylococcus aureus from heterogeneous vancomycin-intermediate resistance to vancomycin-intermediate resistance

Abstract

Multistep genetic alteration is required for methicillin-resistant Staphylococcus aureus (MRSA) to achieve the level of vancomycin resistance of vancomycin-intermediate S. aureus (VISA). In the progression of vancomycin resistance, strains with heterogeneous vancomycin resistance, designated hetero-VISA, are observed. In studying the whole-genome sequencing of the representative hetero-VISA strain Mu3 and comparing it with that of closely related MRSA strains Mu50 (VISA) and N315 (vancomycin-susceptible S. aureus [VSSA]), we identified a mutation in the response regulator of the graSR two-component regulatory system. Introduction of mutated graR, designated graR*, but not intact graR, designated graRn, could convert the hetero-VISA phenotype of Mu3 into a VISA phenotype which was comparable to that of Mu50. The same procedure did not appreciably increase the vancomycin resistance of VSSA strain N315, indicating that graR* expression was effective only in the physiological milieu of hetero-VISA cell to achieve a VISA phenotype. Interestingly, the overexpression of graR* increased the daptomycin MICs in both Mu3 and N315 and decreased the oxacillin MIC in N315.

FIG. 1.

Cell wall thickness of Mu3, Mu50, and graR-related Mu3 derivative strains Six representative transmission electron microscopy photos showing various cell wall thicknesses of each strain are presented. Mu3(pgraR*) showed a very thick cell wall (37.88 ± 11.31 nm), which was comparable to that of Mu50 (35.02 ± 4.01 nm) and was much thicker than that of Mu3(pYT3) (26.11 ± 3.66 nm). Mu3(pgraRn) also showed increased cell wall thickness (32.50 ± 5.09 nm), but it was less significant than that of Mu3(pgraR*).

FIG. 2.

Autolysis assay of Mu3, Mu50, and graR-related Mu3 derivative strains Symbols: open circles, Mu3; closed circles, Mu3(pgraR*); open triangles, Mu3ΔgraSR; closed triangles, Mu3(pgraRn); open squares, Mu50. Note that the introduction of pgraR* significantly decreased the autolysis activity of Mu3 to a level equal to that of Mu50. The autolysis of Mu3(pgraRn) was also reduced but not as significantly as with Mu3(pgraR*). The deletion of graSR significantly enhanced the autolytic activity of Mu3.

FIG. 3.

Growth rates of Mu3, Mu50, and graR-related Mu3-derivative strains Symbols, strains, and doubling times (in minutes) are as follows: open circles, Mu3 (29.88); closed circles, Mu3(pgraR*) (41.52); open triangles, Mu3ΔgraSR (26.66); closed triangles, Mu3(pgraRn) (30.12); open squares, Mu50 (34.27); closed squares, Mu3(pYT3) (31.52). Note that the introduction of graR* (and graR) decreased the growth rate of Mu3, while the deletion of graSR increased it.

FIG. 4.

Microarray image analysis for Mu3 graR transformants. Microarray image analysis was done using ImaGene 4.0 software (BioDiscovery, Inc. Los Angeles, CA) (A) (Left) Representative image of a microarray slide showing all florescence spots with different intensities; (right) scatter plot diagram of normalized spot intensities in the analysis of Mu3(pgraR*)/Mu3(pgraRn). Seven spots were identified where the signal intensities were significantly higher in strain Mu3(pgraR*) than in Mu3(pgraRn). (B) (Left) Magnified view of the above-described seven spots in three representative sets of microarray analysis; (right) the spots mentioned above correspond to the genes vraFG, vraDE, isaB, fmtC(mprF), and SAS091.