REGULATION OF MULTIDRUG RESISTANCE IN S AUREUS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 去年，在INBRE计划中，我们的实验室迅速进入了对金黄色葡萄球菌的夫西地酸反应的分析。类固醇抗生素中的呋西地酸，通过阻止核糖体释放延伸因子G(EF-G)来抑制蛋白质合成。我们现在已经确定了金黄色葡萄球菌中的梭西地酸-刺激隆，以及金黄色葡萄球菌在获得导致夫西地酸耐药的染色体突变后发生的生物学变化。我们利用了尖端技术：金黄色葡萄球菌泛基因组微阵列(版本IV)和阵列数据分析协议(NIAID的病原体功能基因组资源中心)，以及比较基因组测序(CGS)。 呋西地酸诱导后，416个基因上调，378个基因下调。一个上调的基因(Est，12.2倍)编码一种羧酸酯酶，这是一类已知在类固醇新陈代谢中发挥作用的酶。最高上调的两个基因编码葡萄球菌分泌抗原(SSAA)(22.2倍)和推测的EMRB-QacA多药外排泵(17.2倍)。双组分调控系统YycGF的反应调节因子YycF已被证明与SSAA启动子结合，并且yycF被呋西地酸下调(-2.1倍)。SSAA和EMRB/qacA转录降低(分别为-3.11倍和-1.76倍)的自发yycG(组氨酸激酶)点突变显示对呋西地酸的耐受性降低(e-1.7倍)。呋西地酸诱导还上调了延伸因子-G基因(FUSA，2.1倍)和25个核糖体蛋白基因(2-6倍)。下调最多的基因是推测的出口酸性磷酸酶(-11.8倍)。呋西地酸诱导还下调了21个蛋白质降解基因(-2.1~11.1倍)、10个tRNA氨酰化基因(-2.2~4.1倍)和15个嘌呤生物合成基因(-2.3~7.4倍)。我们认为，呋西地酸上调EST可能导致呋西地酸的部分降解和失活以及核糖体蛋白基因的上调，蛋白质降解、tRNA氨酰化和嘌呤生物合成的下调可能是对呋西地酸毒性的补偿。我们还展示了SSAA、ErmB/QacA和YycGF在金黄色葡萄球菌对呋西地酸的反应中的作用。 利用金黄色葡萄球菌SH1000菌株产生1、2步呋喃西地酸抗性(FusR)突变体。不出所料，两个突变体都发生了FusA的突变。第一步的突变体还显示了一个假定的噬菌体蛋白的突变，而第二步的突变体则在AGRA和一个Arac样的转录调控因子上存在额外的突变。与SH1000相比，两个突变体都表现出全面的转录变化和生长速度下降。虽然两个FusR突变体之间有一些共同的转录变化，但在各个突变体的转录本中也存在着广泛的差异。与SH1000相比，这两个突变株对环丙沙星、松油消毒剂乙锭、酒精和三氯生的敏感性增加。这些易感性的增加归因于：MGRA和MARR同系物上调以及相关的NorB和BLT样多药外排泵基因下调；葡萄糖素生物合成基因(CRTM和crtN)下调；编码酒精脱氢酶(Adh1)的基因；以及编码enoyl-acyl载体蛋白还原酶(Fabi)的基因(-2.4至-2.9倍)。我们的结论是，FusR突变会导致广泛的转录组改变，这些改变可能会增加适应成本，而FusR突变显示出对多种抗菌素的敏感性降低。

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.