ABSTRACT: Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 µg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 µg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus.
