REGULATION OF MULTIDRUG RESISTANCE IN S AUREUS

金黄色葡萄球菌多重耐药性的调控

• 批准号: 7720451
• 负责人: JOHN E GUSTAFSON
• 金额: $ 4.18万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720451
• 关键词: Acid Phosphatase; Acyl Carrier Protein; Alcohol dehydrogenase; Alcohols; Anabolism; Antibiotics; Antigens; Bacteriophages; Binding; Biological; Carboxylic Ester Hydrolases; Ciprofloxacin; Class; Computer Retrieval of Information on Scientific Projects Database; Data Analyses; Disinfectants; Down-Regulation; Enzymes; Ethidium; Funding; Fusidic Acid; Gene Expression Profile; Gene Proteins; Genes; Genetic Transcription; Genomics; Grant; Growth; Homologous Gene; Individual; Institution; Laboratories; Lead; Multi-Drug Resistance; Mutation; Oils; Oxidoreductase; Peptide Elongation Factor G; Play; Predisposition; Protein Biosynthesis; Proteins; Protocols documentation; Purines; Rate; Regulation; Research; Research Personnel; Resistance; Resources; Ribosomal Proteins; Ribosomes; Role; Source; Staphylococcus aureus; Steroids; System; Technology; Toxic effect; Transfer RNA Aminoacylation; Triclosan; United States National Institutes of Health; Up-Regulation; antimicrobial; base; carboxylesterase; comparative; cost; efflux pump; fitness; functional genomics; mutant; pathogen; prevent; programs; promoter; protein degradation; protein-histidine kinase; purine; response; staphyloxanthin; steroid metabolism

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 去年，在 INBRE 计划中，我们的实验室迅速开始分析金黄色葡萄球菌的夫西地酸反应。 类固醇抗生素中的夫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。我们现在已经描述了金黄色葡萄球菌中的夫西地酸刺激物以及在获得导致夫西地酸抗性的染色体突变后金黄色葡萄球菌中发生的生物学改变。我们利用尖端技术做到了这一点：金黄色葡萄球菌泛基因组微阵列（第四版）和阵列数据分析方案（NIAID 的病原体功能基因组资源中心）以及比较基因组测序 (CGS)。 夫西地酸诱导导致 416 个基因上调和 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代谢中发挥作用的酶。两个上调程度最高的基因编码葡萄球菌分泌抗原 (SsaA)（22.2 倍）和推定的 EmrB-QacA 多药外排泵（17.2 倍）。双组分调节系统 YycGF 的响应调节剂 YycF 已被证明可结合 SsaA 启动子，并且 yycF 被夫西地酸下调（-2.1 倍）。 ssaA 和 emrB/qacA 转录减少（分别为 -3.11 倍和 -1.76 倍）的自发 yycG（组氨酸激酶）点突变体表现出夫西地酸抗性降低（e -1.7 倍）。夫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（增加 2-6 倍）。下调幅度最大的基因是假定的输出酸性磷酸酶（-11.8 倍）。夫西地酸诱导还下调了 21 个蛋白质降解基因（-2.1 至 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）。我们的结论是，夫西地酸est的上调可能导致夫西地酸的部分降解和失活以及核糖体蛋白基因的上调，而蛋白质降解、tRNA氨酰化和嘌呤生物合成的下调可能会补偿夫西地酸的毒性。我们还证明了 SsaA、ErmB/QacA 和 YycGF 在金黄色葡萄球菌对夫西地酸的反应中的作用。 利用金黄色葡萄球菌菌株 SH1000 产生第一步和第二步夫西地酸抗性 (FusR) 突变体。正如预期的那样，两个突变体中都发生了fusA突变。第一步突变体还表现出假定的噬菌体蛋白的突变，而第二步突变体则在 agrA 和类似 araC 的转录调节因子中含有额外的突变。与 SH1000 相比，两种突变体都表现出全面的转录改变和生长速度降低。虽然两个 FusR 突变体之间存在一些转录改变，但各个突变体转录组中也存在明显的广泛差异。与 SH1000 相比，两种突变体都表现出对环丙沙星、乙锭、松油消毒剂、酒精和三氯生的敏感性增加。这些增加的敏感性归因于： mgrA 和 marR 同源物的上调以及相关的norB 和 blt 样多药外排泵基因的下调；葡萄球菌黄素生物合成基因（crtM 和 crtN）的下调；编码乙醇脱氢酶（adh1）的基因；和编码烯酰基-酰基载体蛋白还原酶（fabI）的基因（-2.4至-2.9倍）。我们得出的结论是，FusR 突变导致广泛的转录组改变，这些改变可能会增加适应成本，并且 FusR 突变体表现出对多种抗菌药物的敏感性降低。