walK and clpP mutations confer reduced vancomycin susceptibility in Staphylococcus aureus

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Vancomycin-intermediate Staphylococcus aureus (VISA) develops resistance through spontaneous mutations.
• Previous studies identified mutations in vraSR and graRS as key to the VISA phenotype.
• Understanding the genetic basis of VISA is crucial for combating antibiotic resistance.

Purpose of the Study:

• To identify novel genetic mutations responsible for vancomycin resistance in a laboratory-derived VISA strain.
• To confirm the contribution of identified mutations to vancomycin resistance through gene replacement.
• To investigate the prevalence of these mutations in clinical VISA isolates.

Main Methods:

• Gene sequencing to identify mutations in the laboratory-derived VISA strain LR5P1-V3.
• Gene replacement experiments to introduce walK and clpP mutations into a susceptible strain.
• Phenotypic analysis including cell wall thickness, growth rate, and autolytic activity.
• Transcriptional profiling of mutant strains.
• Mutation prevalence study on 39 clinical VISA strains.

Main Results:

• Identified deletion in walK and truncating mutation in clpP as responsible for vancomycin resistance in LR5P1-V3.
• Gene replacement confirmed that walK or clpP mutations individually increased vancomycin MIC, with both mutations synergistically raising it to 4 mg/liter.
• Mutant strains exhibited VISA-like characteristics: thickened cell wall, slow growth, and reduced autolysis.
• Transcriptional profiling revealed widespread gene expression changes in response to walK and clpP mutations.
• Mutation prevalence study showed walRK (61.5%) and clpP (7.7%) mutations are present in clinical VISA strains, with walRK being most common.

Conclusions:

• Mutations in walK and clpP represent a novel combination of genetic events contributing to vancomycin resistance in Staphylococcus aureus.
• The walK and clpP mutations confer VISA phenotype, including altered cell wall and reduced autolysis.
• WalK and ClpP mutations are prevalent in clinical VISA isolates, highlighting their significance in antibiotic resistance.