walKandclpPMutations Confer Reduced Vancomycin Susceptibility inStaphylococcus aureus

Abstract

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) is generated from vancomycin-susceptibleStaphylococcus aureusby multiple spontaneous mutations. We previously reported that sequential acquisition of mutations in the two-component regulatory systemsvraSRandgraRSwas responsible for the VISA phenotype of strain Mu50. Here we report on the identification of a novel set of regulator mutations, a deletion mutation in two-component regulatory systemwalRK(synonyms,vicRKandyycFG), and a truncating mutation in a proteolytic regulatory gene,clpP, responsible for the raised vancomycin resistance in a laboratory-derived VISA strain, LR5P1-V3. The contributory effect of the two mutations to vancomycin resistance was confirmed by introducing thewalKandclpPmutations into the vancomycin-susceptible parent strain N315LR5P1 by a gene replacement procedure. The vancomycin MIC of N315LR5P1 was raised from 1 to 2 mg/liter by the introduction of thewalKorclpPmutation, but it was raised to 4 mg/liter by the introduction of both thewalKandclpPmutations. The vancomycin MIC value of the double mutant was equivalent to that of strain LR5P1-V3. Like VISA clinical strains, LR5P1-V3 and the double mutant strain LR5P1walK*clpP* exhibited a thickened cell wall, slow growth, and decreased autolytic activity. Transcriptional profiles of the mutants with gene replacements demonstrated that introduction of both thewalKandclpPmutations could alter expression of dozens or hundreds of genes, including those involved in cell envelope and cellular processes, intermediary metabolism, and information pathway. A mutation prevalence study performed on 39 worldwide clinical VISA strains showed that 61.5, 7.7, 10.3, and 20.5% of VISA strains harbored mutations inwalRK,clpP,graRS, andvraSR, respectively. The mutation ofwalRKwas most frequently carried by VISA strains. Together, these results suggested that the mutations ofwalKandclpPidentified in LR5P1-V3 constitute a new combination of genetic events causing vancomycin resistance inStaphylococcus aureus.