Effects of Mutations in Purine Salvage Gene apt on Growth and Whole Cell Autolysis in Laboratory and Clinical Isolates of Staphylococcus aureus

Our laboratory previously identified mutations in a purine salvage pathway gene (apt, encoding adenine phosphoribosyltransferase) that occurred during vancomycin selection of a Staphylococcus aureus mutant expressing a vancomycin-intermediate phenotype. Vancomycin-intermediate S. aureus (VISA) strains express altered whole cell autolysis which is thought to contribute to the overall VISA mechanism. In an effort to understand the association of the apt mutation with the VISA mechanism, vancomycin susceptibility levels, whole cell autolysis and growth rates of 2-fluroadenine (2-FA)-selected apt S. aureus mutants and their respective parent strains were analyzed. Initially, 2-FA-reduced susceptibility (2-FA^RS) apt mutants were selected from S. aureus strains SH1000, COL, MM61 and MM66. These parent-mutant strain sets were then analyzed via whole cell autolysis in 0.05 M Tris-Cl (pH 7.2) containing 0.05% Triton X-100, growth curves in Luria-Bertani broth, and vancomycin gradients. With the exception of one apt mutant (COL-2-FA^RS), most apt mutants demonstrated increased susceptibility to vancomycin. In addition, 2-FA^RS mutants demonstrated either increased (SH1000-2-FA^RS and MM61-2-FA^RS) or decreased (COL-2-FA^RS, MM66-2FA^RS) whole cell autolytic activity, while no mutant demonstrated an altered growth rate when compared to respective parent strain. The absence of growth rate alterations demonstrates that the 2-FA selection did not affect fitness in the 2-FA^RS mutants. However, all 2-FA^RS mutants investigated did demonstrate altered vancomycin susceptibility levels and whole cell autolysis rates compared to their respective parent strains. Since all VISA demonstrate altered autolysis, we hypothesize that apt mutations contribute to the VISA mechanism by altering whole cell autolysis rates.  Supported by PHS Grant R25 GM048998-13