Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus

Abstract

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcus aureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

The two-component regulator WalKR provides an essential link between cell wall homeostasis with DNA replication inStaphylococcus aureus

“…However, contrary to our expectation that activation of WalKR, and therefore down regulation of ltaS, would increase Congo red susceptibility, we observed an increase in susceptibility to Congo red of WalKR down mutants. Although this did not match our prediction, similar results have been recently reported, with laboratory evolution of VISA strains showing increased Congo red susceptibility in strains with and without WalKR mutations 70,71 . We propose that Congo red susceptibility arises due to genotype-naïve pleotropic effects of the VISA phenotype on cell wall structure, limiting its utility as a direct ltaS probe in this instance.…”

The evolution of diverse antimicrobial responses in vancomycin-intermediateStaphylococcus aureusand its therapeutic implications

“…The blue-shaded cells identify mutations that have been previously observed in laboratory and clinical isolates. [14][15][16][17][18][19][20][21][22][23][24][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] We list the affected genes along the bottom in order of the total number of mutations.…”