REGULATION OF MULTIDRUG RESISTANCE IN S AUREUS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。在水杨酸浓度诱导多种抗菌素耐药性（2 mM）的金黄色葡萄球菌生长导致生长抑制。由于生长缓慢的细菌表现出更高的抗菌素耐药性，水杨酸的生长抑制可能有助于水杨酸诱导的抗菌素耐药性机制。此外，与合作者Wayne Van Vorheiss （NMSU）一起，我们证明在暴露于2 mM水杨酸盐期间，SH1000培养物的氧气消耗减少了31.2% (13.0 vs 9.0 ul) (p 0.01)， 50分钟后二氧化碳产量减少了35.4% (p 0.01) （11.3 vs 17.4 ul）。这种降低的代谢活性与转录分析结果相关。特别是编码甘油醛-3-磷酸脱氢酶的gapA2的表达被水杨酸下调。GapA活性也降低了约2倍，而在未经处理的细胞裂解物中，GapA抑制剂碘乙酸酯抑制GapA活性，2mm水杨酸对活性没有影响。这表明水杨酸盐在转录水平上抑制GapA活性。水杨酸存在下的生长也下调了pgi的转录活性，pgi编码葡萄糖-6-磷酸异构酶。Pgi催化葡萄糖-6-磷酸转化为果糖-6-磷酸，其活性也是糖酵解发生所必需的。与上述基因相反，在金黄色葡萄球菌葡萄糖酸操纵子（gntRKP）中发现的基因gntK和gntP在水杨酸存在下生长后显着上调。在含有水杨酸盐的培养基中添加过量的葡萄糖酸盐可以改善水杨酸盐的生长抑制作用。由于没有证据表明金黄色葡萄球菌中存在enterner - doudoroff通路，因此葡萄糖酸盐可能在金黄色葡萄球菌中通过戊糖磷酸循环（PPC）最初被氧化。我们假设，如果水杨酸处理细胞的代谢依赖于葡萄糖代谢途径，那么葡萄糖的添加和相关的分解代谢物抑制将有助于水杨酸的毒性。正如预期的那样，葡萄糖的加入加剧了水杨酸盐的生长抑制作用。另一种在水杨酸存在下生长受到抑制的基因是pckA，它编码磷酸烯醇丙酮酸羧激酶。PckA利用GTP将草酰乙酸转化为磷酸烯醇丙酮酸，这是糖异生所必需的。总的来说，我们推测水杨酸可以抑制分解代谢产物的抑制，抑制糖酵解途径和糖异生，从而抑制整体葡萄糖代谢，并诱导戊糖磷酸循环引发的葡萄糖酸氧化，该循环提供能量和代谢物，允许水杨酸暴露期间持续生长。需要更多的实验来确定这种中枢代谢的改变如何促进水杨酸诱导的多种抗菌素耐药机制。拓扑异构酶IV是金黄色葡萄球菌中氟喹诺酮类药物的主要靶点，金黄色葡萄球菌独立的单步氟喹诺酮耐药突变体具有编码拓扑异构酶IV亚基parE和parC的基因突变。福西地酸与延伸因子G （EF-G）、GTP/GDP和核糖体的复合物结合，抑制肽合成易位步骤后EF-G-GDP的释放，从而抑制感菌的生长。在金黄色葡萄球菌中，编码EF-G的染色体上定位的基因fusA的突变通过降低药物对蛋白质合成机制的亲和力而导致对夫西地酸的抗性。金黄色葡萄球菌在水杨酸存在下的生长导致对抗生素环丙沙星和夫西地酸的敏感性降低。在水杨酸存在下生长后，我们通过苏格兰分析捕获了基因parE和fusA，并通过实时PCR分析证实了基因的水杨酸诱导性。在阵列分析中，水杨酸盐也上调了fusA（1.16倍）。由于水杨酸暴露会提高parE和fusA的转录，可能是抗菌靶点的增加分别增加了对环丙沙星和福西地酸的内在耐药性。水杨酸存在下的生长也会下调mgrA， mgrA编码抗菌外排泵（NorA、NorB、NorC和Tet38）基因的负调节因子。我们推测，在水杨酸诱导的早期，这些外排泵的一个或多个转录单位可能会短暂上调，导致泵的产生增加，但随后又恢复到诱导前的活性水平。由于金黄色葡萄球菌在水杨酸存在下的生长降低了对夫西地酸和普通房屋清洁剂的敏感性，并降低了mgrA的表达，我们假设mgrA突变体应该表现出对夫西地酸和普通房屋清洁剂的敏感性降低。mgrA：猫突变体对普通房屋清洁剂的易感性降低（），但对夫西地酸的易感性增加。此外，在所有药物梯度中添加水杨酸盐导致所有mgrA::cat突变体的敏感性降低。需要更多的研究来确定由mgrA控制的外排泵是否也有助于葡萄球菌细胞对普通房屋清洁剂的反应。总的来说，由于mgrA失活不影响GapA活性，也不抑制水杨酸诱导的多种抗菌素耐药性，因此水杨酸反应似乎以不依赖于mgrA的方式起作用。有趣的是，水杨酸诱导并没有调节mgra调控的毒力基因（如agr、hla、sarS和spa）的表达。然而，由MgrA负调控的主要双功能自溶素在水杨酸的作用下被上调。除了mgrA，我们注意到编码sarA负调节因子的基因sarR也被水杨酸盐的生长下调。先前我们已经证明sarA是表达内在多重抗微生物药物耐药性所必需的，并且sarA调节是由水杨酸诱导的金黄色葡萄球菌。然而，sarA的突变和转录分析表明，水杨酸对多种抗菌素耐药性的影响与sarA无关。

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. Growth of S. aureus with salicylate at concentrations that induce multiple antimicrobial resistance (2 mM) leads to growth inhibition. Since slow-growing bacteria express increased antimicrobial resistance, growth inhibition by salicylate may contribute to the salicylate-inducible antimicrobial resistance mechanism. In addition, with collaborator Wayne Van Vorheiss (NMSU) we demonstrated that O2 consumption by SH1000 cultures was reduced (p 0.01) during exposure to 2 mM salicylate by 31.2% (13.0 vs 9.0 ul) and CO2 production was reduced (p 0.01) by 35.4% after 50 min (11.3 vs 17.4 ul). This reduced metabolic activity correlate with the transcriptional analyses findings. In particular, the expression of gapA2 which encodes glyceraldehyde-3-phosphate dehydrogenase is downregulated by salicylate. GapA activity is also decreased ~ 2-fold and while the GapA inhibitor iodoacetate inhibits GapA activity in untreated cell lysates, 2 mM salicylate had no effect on activity. This demonstrates that salicylate inhibits GapA activity at the level of transcription. Growth in the presence of salicylate also downregulated the transcriptional activity of pgi, which encodes glucose-6-phosphate isomerase. Pgi catalyzes the interconversion of glucose-6-phosphate to fructose-6-phosphate and its activity is also required for glycolysis to occur. In contrast to the genes above, the genes found within the S. aureus gluconate operon (gntRKP) gntK and gntP were significantly upregulated following growth in the presence of salicylate.The addition of gluconate in excess to growth media containing salicylate ameliorated the growth inhibitory effect(s) of salicylate. Since there is no evidence of an Entner-Doudoroff pathway in S. aureus, gluconate is probably initially oxidized via the pentose phosphate cycle (PPC) in S. aureus. We hypothesized that if the metabolism of the salicylate-treated cell relied on a gluconate metabolic pathway, than the addition of glucose and the associated catabolite repression would contribute to salicylate toxicity. As expected, the addition of glucose exacerbated the growth inhibitory effects of salicylate. Another gene repressed by growth in the presence of salicylate is pckA, which encodes phosphoenolpyruvate carboxykinase. PckA converts phophoenolpyruvate from oxaloacetate using GTP and is required for gluconeogenesis Collectively, we speculate that salicylate derepresses catabolite repression, inhibiting the glycolytic pathway and gluconeogenesis stiffling overall glucose metabolism, and induces gluconate oxidation initiated with the pentose phosphate cycle that provides energy and metabolites allowing for continued growth during salicylate exposure. More experimentation is required to determine how this alteration in central metabolism contributes to the salicylate-inducible multiple antimicrobial resistance mechanism. Topoisomerase IV is the primary target of fluoroquinolones in S. aureus, and independent single-step fluoroquinolone-resistant mutants of S. aureus possess mutations in the genes encoding both topoisomerase IV subunits, parE and parC. Fusidic acid binds to the complex of elongation factor G (EF-G), GTP/GDP and the ribosome, inhibiting the release of EF-G-GDP after the translocation step of peptide synthesis, thereby inhibiting growth of susceptible bacteria. Mutations in fusA, the chromosomally located gene encoding EF-G in S. aureus leads to fusidic acid resistance by reducing the affinity of the drug for the protein synthesis machinery. Growth of S. aureus in the presence of salicylate leads to a reduction in susceptibility to the antibiotics ciprofloxacin and fusidic acid. We have captured the genes parE and fusA via SCOTS analysis following growth in the presence of salicylate and confirmed the salicylate inducibility of genes via real-time PCR analysis. fusA also appeared upregulated by salicyate on the array analysis (1.16-fold). Since salicylate exposure elevates parE and fusA transcription, it is possible that an increased production of antimicrobial target increases intrinsic resistance to ciprofloxacin and fusidic acid respectively. Growth in the presence of salicylate also downregulates mgrA which encodes a negative regulator of genes encoding the antimicrobial efflux pumps, NorA, NorB, NorC, and Tet38. We speculate that early on during salicylate induction one or more of these efflux pump transcriptional units may be upregulated transiently leading to increased production of a pump(s), but then returns to its pre-induced activity level. Since growth of S. aureus in the presence of salicylate reduces susceptibility to fusidic acid and common house cleaners, and reduces mgrA expression, we hypothesized that mgrA mutants should demonstrate reduced susceptibility to fusidic acid and a common house cleaner. mgrA::cat mutants demonstrated reduced susceptibility to a common house cleaner (), yet increased susceptibility to fusidic acid. Furthermore, the addition of salicylate to all drug gradients led to reduced susceptibility in all mgrA::cat mutants. More research is required to determine if the efflux pumps controlled by mgrA are also contributing to the response of the staphylococcal cell to common house cleaners. Overall, since mgrA inactivation does not affect GapA activity, nor inhibit salicylate-inducible multiple antimicrobial resistance, it appears that salicylate response functions in a mgrA-independent fashion. Interestingly, salicylate-induction did not modulate the expression of MgrA-regulated virulence genes (e.g. agr, hla, sarS, and spa). However, the major bifunctional autolysin atl negatively regulated by MgrA, was upregulated by growth with salicylate . Besides mgrA, we note that the gene encoding the negative regulator of sarA, sarR was also downregulated by growth with salicylate. Previously we have demonstrated that sarA is required for the expression of intrinsic multiple antimicrobial resistance and sarA regulation was induced by salicylate in S. aureus. However, mutational and transcriptional analysis of sarA, demonstrate that the salicylate effect on multiple antimicrobial resistance is sarA-independent.