REGULATION OF MULTIDRUG RESISTANCE IN S AUREUS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This last year in the INBRE program our laboratory has moved swiftly into the analysis of the fusidic acid response of Staphylococcus aureus. Fusidic acid in a steroid antibiotic that inhibits protein synthesis by preventing release of elongation factor G (EF-G) from the ribosome. We have now characterized the fusidic acid-stimulon in S. aureus and the biological alterations that occur in Staphylococcus aureus following the acquisition of chromosomal mutations leading to fusidic acid resistance. We have done this utilizing cutting edge technology: S. aureus pangenome microarrays (Version IV) and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center), as well as comparative genomic sequencing (CGS). Fusidic acid induction led to the upregulation of 416 genes and downregulation of 378 genes. One upregulated gene (est, 12.2-fold) encodes a carboxylesterase, a class of enzymes known to play a role in steroid metabolism. The two most highly upregulated genes encode the staphylococcal secretory antigen (SsaA) (22.2-fold) and a putative EmrB-QacA multidrug efflux pump (17.2-fold). The response regulator YycF of the two-component regulatory system YycGF has been shown to bind the SsaA promoter, and yycF is downregulated by fusidic acid (-2.1-fold). A spontaneous yycG (histidine kinase) point mutant with reduced ssaA and emrB/qacA transcription (-3.11 and -1.76-fold respectively) demonstrated reduced fusidic acid resistance (e -1.7-fold). Fusidic acid induction also upregulated the elongation factor-G gene (fusA, 2.1-fold) and 25 ribosomal protein genes (2-6-fold increases). The most downregulated gene was a putative exported acid phosphatase (-11.8-fold). Fusidic acid induction also downregulated 21 protein degradation genes (-2.1 to 11.1-fold), 10 tRNA aminoacylation genes (-2.2-4.1-fold) and 15 purine biosynthesis genes (-2.3-7.4 fold). We conclude that fusidic acid upregulation of est might lead to the partial degradation and inactivation of fusidic acid and the upregulation in ribosome protein genes, and downregulation of protein degradation, tRNA aminoacylation and purine biosynthesis might compensate for fusidic acid toxicity. We also demonstrate a role for SsaA, ErmB/QacA and YycGF in the response of S. aureus to fusidic acid. S. aureus strain SH1000 was utilized to generate 1st- and 2nd-step fusidic acid-resistant (FusR) mutants. Mutations in fusA occurred in both mutants as expected. The 1st-step mutant also demonstrated mutations in a putative phage protein, while the 2nd-step mutant harbored additional mutations in agrA and an araC-like transcriptional regulator. Compared to SH1000, both mutants demonstrated sweeping transcriptional alterations and reduced growth rates. While some transcriptional alterations were shared between the two FusR mutants, broad profile differences were also evident in the individual mutant transcriptomes. Compared to SH1000, both mutants demonstrated increased susceptibility to ciprofloxacin, ethidium, a pine-oil based disinfectant, alcohols and triclosan. These increased susceptibilities were attributed to: upregulation of mgrA and marR-homologues and associated downregulation of the norB and blt-like multidrug efflux pump genes; downregulation of staphyloxanthin biosynthesis genes (crtM and crtN); a gene encoding an alcohol dehydrogenase (adh1); and a gene encoding an enoyl-acyl carrier protein reductase (fabI) (-2.4 to -2.9-fold). We conclude that FusR mutations lead to extensive transcriptome alterations and these alterations probably increase fitness costs and tjhat FusR mutants demonstrate reduced susceptibility to multiple antimicrobials.