Identification of daptomycin resistance mechanisms in Clostridioides difficile

项目摘要 在美国，艰难梭菌每年造成近50万例感染，导致近200万人死亡。 3万人死亡CDC宣布C。difficile是对公共卫生的“紧急”威胁，是最高的威胁类别。 迫切需要新的治疗方法。我们许多最有用的抗生素都是针对细胞包膜的。不过小 关于C.很难这些细胞被 应激反应系统可能在抗生素耐药性中起重要作用。通过了解C.艰难 响应细胞包膜压力，我们可能会发现更好的治疗选择C。艰难感染。苏罗托霉素， 靶向肽聚糖合成的达托霉素衍生物显示出作为C. 很难然而，相互矛盾的3期临床试验已经停止了surotomycin作为治疗 C.艰难感染。我们使用多种遗传学方法来确定涉及达托霉素的关键因子 阻力通过分离自发的达托霉素抗性突变体并进行Tn-seq，我们鉴定了两种 不同的双组分调节系统，DraRS和DapRS，它们参与达托霉素耐药性。我们 将追求三个具体目标，剖析这些调节剂在控制达托霉素耐药性中的作用， 细胞被膜应激反应在目标1中，我们将定义DraRS调控系统如何有助于抗生素 阻力我们将测试功能获得型和功能丧失型draRS突变体两者对对耐药性的影响。 许多细胞被膜应力和细胞被膜生物发生。我们还将定义DraR调节子， 确定单个基因对抗生素抗性和细胞包膜生物发生和胁迫的贡献 反应在目标2中，我们将剖析DraR的活性是如何响应细胞包膜应激而被控制的。我们 初步数据表明，DraRS被抗生素激活，破坏了脂质-II循环。我们将使用CRISPRi来测试 该模型通过遗传重演抗生素的作用并确定对DraR激活的作用。 我们还将分离不能对细胞包膜应激作出反应的DraS突变体。在目标3中，我们将定义 DapRS和DapRS调节基因hexSDF在细胞包膜生物发生中的作用。我们的数据显示 是生产一种独特的糖脂所必需的，糖脂占C.艰难 膜的我们将定义DapR调节子以及单个调节子成员如何对达托霉素起作用 电阻、细胞电荷和脂质含量以及细胞包膜应激反应。 这些目标将促进我们对C的理解。很难定义它如何抵抗细胞壁的作用 抗生素如达托霉素和万古霉素。

Project Summary Clostridioides (Clostridium) difficile causes nearly 500,000 infections a year in the United States, leading to nearly 30,000 deaths. The CDC has declared C. difficile an “urgent” threat to public health, the highest threat category. New treatments are sorely needed. Many of our most useful antibiotics target the cell envelope. However, little is known about cell envelope biogenesis or cell envelope stress response in C. difficile. These cell envelope stress response systems may play an important role in antibiotic resistance. By understanding how C. difficile responds to cell envelope stress we may uncover better treatment options for C. difficile infections. Surotomycin, a daptomycin derivative, which targets peptidoglycan synthesis showed promise as a potential treatment for C. difficile. However, conflicting phase 3 clinical trials have halted development of surotomycin as a treatment for C. difficile infections. We used multiple genetic approaches to identify key factors involved in daptomycin resistance. By isolating spontaneous daptomycin resistant mutants and performing Tn-seq we identified two different two component regulatory systems, DraRS and DapRS, that are involved in daptomycin resistance. We will pursue three specific aims to dissect the role of these regulators in controlling daptomycin resistance and cell envelope stress response. In Aim 1 we will define how the DraRS regulatory system contributes to antibiotic resistance. We will test both gain-of-function and loss-of-function draRS mutants for their effect on resistance to a number of cell envelope stresses and on cell envelope biogenesis. We will also define the DraR regulon and determine the contribution of individual genes to antibiotic resistance and cell envelope biogenesis and stress response. In Aim 2 we will dissect how activity of DraR is controlled in response to cell envelope stress. Our preliminary data suggest DraRS is activated by antibiotics disrupt the lipid-II cycle. We will use CRISPRi to test this model by genetically recapitulating the effects of antibiotics and determining the effect on DraR activation. We will also isolate mutants of DraS that are unable to respond to cell envelope stress. In Aim 3 we will define the role of DapRS and the DapRS-regulated genes hexSDF in cell envelope biogenesis. Our data suggest they are required for production of a unique glycolipid which makes up 16% of the polar lipids in the C. difficile membrane. We will define the DapR regulon and how individual regulon members contribute to daptomycin resistance, cell charge, and lipid content and cell envelope stress response. Together these aims will advance our understanding of C. difficile by defining how it resists cell-wall acting antibiotics like daptomycin and vancomycin.