Identification of CodY-Regulated Factors that Control Sporulation in Clostridioides difficile

项目摘要/摘要 艰难梭状芽胞杆菌是一种厌氧芽胞形成病原体，可导致约500,000人感染，13,000人 美国每年的死亡人数和医疗费用约为10亿美元。艰难梭菌感染导致 严重腹泻和结肠炎。感染是通过摄入艰难梭菌孢子传播的，艰难梭菌孢子 可以在氧气、消毒剂、化学物质和辐射中存活。摄取后，孢子萌发成营养体。 肠道内的细胞。这些营养细胞定植于胃肠道，产生毒素并形成 更多的孢子。孢子的形成是由胃肠道中病原体的营养匮乏引起的。 艰难梭菌通过特定的转录调控因子(如Cody)感知营养的可利用性。科迪是一个 一种全球转录调节因子，存在于低G+C革兰氏阳性细菌中，并对细胞内产生反应 支链氨基酸和GTP的浓度。尽管已知艰难梭菌Cody突变体 超孢子化，Cody抑制产孢量的方法目前还不清楚。基于 Cody对产孢量的抑制及其作为转录调节因子的作用，我推测Cody 通过控制一种产孢因子的表达来调节产孢量(S)。在初步研究中，我 对Cody和野生型菌株进行转录转录分析，结合Cody结合图谱 位点和转录敲除。通过这些实验，我确定了Cody直接调节的因子 影响产孢量。前三位候选基因分别是CDR20291_1334、CDR20291_1851-1853和 CDR20291-2485-2486。为了检验假设，我建议完成以下具体目标：目标1，我将评估 CDR20291_1334、CDR20291_1851-1853和CDR20291_2485-2486在产孢量中的作用 无效突变体，并随后分析它们对产孢量和转录图谱的贡献。还有，我会的 用生长曲线、荧光显微镜和扫描电子显微镜研究艰难梭菌中这些基因的基本功能 免疫共沉淀实验。在目标2中，我将比较Cody结合与野生型上的这些靶基因 用凝胶迁移率改变分析检测含有突变Cody结合位点的等位基因和变异 通过互补研究评估Cody依赖调控对产孢率的影响 这些基因含有野生型和突变的Cody盒。这些目标的完成将确定科迪- 控制孢子形成并通过Cody揭示其在孢子形成调节中的作用的调控基因。

PROJECT SUMMARY/ABSTRACT Clostridioides difficile is an anaerobic spore-forming pathogen that causes around 500,000 infections, 13,000 deaths, and healthcare costs of around 1 billion dollars annually in the United States. C. difficile infections cause severe diarrhea and inflammation of the colon. Infection is transmitted by ingestion of C. difficile spores, which can survive oxygen, disinfectants, chemicals, and radiation. After ingestion, spores germinate into vegetative cells within the intestine. These vegetative cells colonize the gastrointestinal tract, generating toxins and forming more spores. Spore formation is triggered by nutritional deprivation of the pathogen in the gastrointestinal tract. Nutrient availability is are sensed in C. difficile by specific transcriptional regulators, such as CodY. CodY is a global transcription regulator that is present in low G + C Gram-positive bacteria and responds to intracellular concentrations of branched-chain amino acids and GTP. Although it is known that C. difficile codY mutants hyper-sporulate, the means by which CodY represses sporulation is not currently understood. Based on the sporulation repression by CodY and its role as a transcriptional regulator, I hypothesize that CodY regulates sporulation by controlling the expression of a sporulation factor(s). In preliminary studies, I performed transcriptomic analyses of codY and wild-type strains, in combination with mapping of CodY-binding sites and transcriptional knockdown. Through these experiments I identified direct CodY-regulated factors that impact sporulation. The top three candidate genes are CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486. To test the hypothesis, I propose to complete these Specific Aims: Aim 1, I will assess the roles of CDR20291_1334, CDR20291_1851-1853, and CDR20291_2485-2486 in sporulation by generating null mutants and subsequently analyzing their contribution to sporulation and transcriptional profiles. Also, I will characterize the basic functions of these genes in C. difficile using growth curves, fluorescent microscopy, and co-immunoprecipitation experiments. In Aim 2, I will compare CodY binding to these target genes on wild-type alleles and variants containing mutated CodY binding sites by electrophoretic mobility shift assay, followed by assessment of the impact of CodY-dependent regulation on sporulation frequency via complementation studies with these genes containing wild-type and mutated CodY boxes. The completion of these aims will identify CodY- regulated genes that control sporulation and uncover their role in sporulation regulation through CodY.