Abstract
The development of chronic and recurrent Staphylococcus aureus infections is associated with the emergence of slow-growing mutants known as small-colony variants (SCVs), which are highly tolerant of antibiotics and can survive inside host cells. However, the host and bacterial factors which underpin SCV emergence during infection are poorly understood. Here, we demonstrate that exposure of S. aureus to sublethal concentrations of H2O2 leads to a specific, dose-dependent increase in the population frequency of gentamicin-resistant SCVs. Time course analyses revealed that H2O2 exposure caused bacteriostasis in wild-type cells during which time SCVs appeared spontaneously within the S. aureus population. This occurred via a mutagenic DNA repair pathway that included DNA double-strand break repair proteins RexAB, recombinase A, and polymerase V. In addition to triggering SCV emergence by increasing the mutation rate, H2O2 also selected for the SCV phenotype, leading to increased phenotypic stability and further enhancing the size of the SCV subpopulation by reducing the rate of SCV reversion to the wild type. Subsequent analyses revealed that SCVs were significantly more resistant to the toxic effects of H2O2 than wild-type bacteria. With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2. Taken together, these data reveal a mechanism by which S. aureus adapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production.
Highlights
MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdoma; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdomb; Department of Microbiology, Hammersmith Campus, Imperial College Healthcare NHS Trust, London, United Kingdomc; Department of Medicine, Imperial College London, London, United Kingdomd
To determine the effect of oxidative stress on the size of the gentamicin-resistant small-colony variants (SCVs) subpopulation, S. aureus was cultured in the presence of increasing concentrations of H2O2, paraquat, or diamide, which have previously been shown to trigger distinct changes in the staphylococcal proteome [53]
The data presented in this report reveal that these mutations increase in frequency in response to one of the major reactive oxygen species (ROS) produced by neutrophils, H2O2, via the SOS response
Summary
With the exception of heme auxotrophs, gentamicin-resistant SCVs displayed greater catalase activity than wild-type bacteria, which contributed to their resistance to H2O2 Taken together, these data reveal a mechanism by which S. aureus adapts to oxidative stress via the production of a subpopulation of H2O2-resistant SCVs with enhanced catalase production. SCVs exhibit other phenotypic characteristics which may promote survival in host tissues, including elevated rates of host cell invasion and intracellular survival, enhanced capsule production, and robust biofilm formation [5, 12, 13, 30,31,32] Several of these phenotypes are ascribed to a combination of decreased Agr activity and enhanced SigB activity, which results in strong expression of surface proteins and an absence of cytolysin production [16, 33,34,35]
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