Subproject 2: Identification of Pathways that Can be Targeted for the Development of Novel Therapies for MRSA

摘要 耐甲氧西林金黄色葡萄球菌(MRSA)是引起严重社区和医院相关疾病的主要原因 感染。除了获得抗生素耐药性外，金黄色葡萄球菌还会转化为持久的耐药形式 在这种情况下，传统的治疗方法无能为力。耐药的持久细胞是顽固性疾病的原因 慢性感染。为了解决对新的抗MRSA疗法的迫切需求，我们开发了一种线虫- 耐甲氧西林金黄色葡萄球菌筛选平台，以确定具有抗耐药金黄色葡萄球菌活性的化合物。化验鉴定出化合物 既有体内药效又有较低的寄主毒性。我们结合了一个完整的动物体内筛查，大约有82,000个合成的 体外筛选具有潜在抗MRSA活性的化合物 杀死顽固的姐妹亚群。我们重点研究了四种化合物，CD437，nTZDpa，bithionol， 和PQ401，这四种药物都能根除活跃生长的MRSA和静止期MRSA持久细胞 通过破坏它们的膜脂双层。这些化合物表现出快速的杀伤率，与其他 抗生素，极低的抗药性选择概率，以及细菌对哺乳动物的高选择性 膜。使用二硫代酚和nTZDpa以及这些化合物的类似物，我们发现了抗持久活性 这些化合物的活性与它们增加膜流动性的能力呈正相关。所有四种化合物 之前被研究为潜在的治疗药物，二硫代酚是一种前FDA批准的驱虫药。 尽管膜破坏化合物有可能杀死顽固不化的细菌，但人们普遍认为它们的 由于缺乏对哺乳动物膜上细菌的选择性，治疗应用受到限制。 然而，我们的结果表明，胆固醇在哺乳动物中的存在，而不是在细菌膜中的存在 为识别/设计无毒的抗膜干扰化合物提供了一个重要的机会。 因此，我们提出了以下两个目标，以确定膜破坏化合物的特征 与它们杀死顽固性细菌的能力有关，但不会破坏哺乳动物的细胞膜。在目标1中，我们将利用 生化、生理、生物物理和冷冻-EM技术，以1)进一步研究 化合物杀死持久者的能力及其增加膜流动性的能力，以及2)识别 膜破坏化合物的作用机理和靶点。我们还建议测试一下 协同作用下金黄色葡萄球菌皮下感染模型中的膜干扰化合物 并确定这些化合物是否也针对粪肠球菌细胞膜。在目标2中，我们 将利用保尔森实验室(核心B)开发的高通量微流控技术 作为“母机”：1)分离生长中稀有的耐甲氧西林金黄色葡萄球菌细胞；2)与 Walker实验室在母机器中使用TN-seq来鉴定MRSA转座子插入突变体 对膜破坏化合物的敏感性。拟议项目的成功完成将增强 我们有能力识别和/或设计有效和安全的抗生素来治疗难以治疗的革兰氏阳性感染。

SUMMARY Methicillin-resistant S. aureus (MRSA) are leading causes of serious community and hospital-associated infection. In addition to acquiring antibiotic resistance, S. aureus converts to a persistent antibiotic-tolerant form in which traditional treatments are powerless. Antibiotic-tolerant persister cells are responsible for recalcitrant chronic infections. To address the urgent need for new anti-MRSA therapeutics, we developed a C. elegans- MRSA screening platform to identify compounds with activity against MRSA. The assay identifies compounds with both in vivo efficacy and low host toxicity. We combined a whole animal in vivo screen of ~82,000 synthetic compounds for ones that exhibit potential anti-MRSA activity with in vitro screens for compounds with the ability to kill the stubborn persister subpopulation. We have focused on four compounds, CD437, nTZDpa, bithionol, and PQ401, all four of which eradicate both actively growing MRSA and stationary phase MRSA persister cells by disrupting their membrane lipid bilayers. These compounds exhibit fast killing rates, synergism with other antibiotics, extremely low probabilities of resistance selection, and high selectivity for bacterial over mammalian membranes. Using bithionol and nTZDpa and analogs of these compounds, we found that anti-persister activity of these compounds positively correlates with their ability to increase membrane fluidity. All four compounds have been previously studied as potential therapeutics and bithionol is a former FDA-approved anthelmintic. Despite the potential of membrane-disrupting compounds to kill persisters, it is generally thought that their therapeutic applications are limited due to a lack of selectivity for bacterial over mammalian membranes. However, our results show that the presence of cholesterol in mammalian but not in bacterial membranes provides an important opportunity to identify/design non-toxic anti-persister membrane-disrupting compounds. We therefore propose the following two aims to identify features of membrane-disrupting compounds that correlate with their ability to kill persisters but not disrupt mammalian membranes. In Aim 1, we will utilize biochemical, physiological, biophysical, and cryo-EM techniques to 1) further investigate the correlation between the ability of compounds to kill persisters and their ability to increase membrane fluidity, and 2) to identify the mechanisms of action and targets of membrane-disrupting compounds. We also propose to test the efficacy of membrane-disrupting compounds in a murine S. aureus subcutaneous abscess infection model in collaboration with the Hooper lab and determine whether the compounds also target E. faecalis cell membranes. In Aim 2, we will utilize high throughput microfluidics technology developed in the Paulsson laboratory (Core B) and referred to as “mother machines” to: 1) isolate rare MRSA persister cells in growing cultures, and 2) in collaboration with the Walker lab, use Tn-seq in mother machines to identify MRSA transposon insertion mutants with enhanced susceptibility to membrane disrupting compounds. Successful completion of the proposed project will enhance our ability to identify and/or design efficacious and safe antibiotics for hard to treat Gram positive infections.