Integration of nutrient availability, TCA cycle activity, and colonization factor expression in Campylobacter jejuni

项目摘要 空肠弯曲菌是世界上细菌性胃肠炎的主要致病菌。人类 感染通常通过摄入受污染的食物，特别是家禽，导致 细菌在结肠中繁殖并引起严重的炎症和腹泻。虽然感染和疾病是 经常会发生自我限制、持续的定植和慢性疾病。尽管C. 空肠对人类健康的影响，很少有人知道在宿主-病原体界面发生的相互作用， 感染，包括如何C.空肠感觉并适应宿主肠道环境以促进感染。这 主要是由于这种生物体与其他胃肠道病原体的进化分歧， 限制了从这些生物体中发现的相关性，并需要对弯曲杆菌进行专门研究 属为此，我们的小组先前在C中确定了一个独特的调节因子。空肠，我们称之为HeuR， 促进天然鸟类宿主的最大定殖，随后发现对 调节几个遗传决定因素，包括那些参与从宿主血红素中获得铁的决定因素， 蛋氨酸的生物合成。此外，HeuR及其下游目标是有效入侵或 持续存在于人类结肠细胞中，这表明这些机制需要更好地理解，因为它们是 显然与动物感染有关。 我们初步确定，这种新的调节剂结合几个TCA中间体， TCA循环活性控制定植决定因子的表达。首先，我们将确定所有的直接目标， HeuR和检查TCA中间体是否影响HeuR结合那些DNA靶标的能力， 基因表达。此外，我们将确定这种新型调节剂的配体结合基序，以及它如何促进 HeuR活性。第二，因为我们已经确定C。空肠TCA中间丰度受 铁限制，我们将使用质量同位素分析，以确定点沿着C。空肠TCA循环， 受铁限制的影响，并确定改变TCA循环活性是否影响HeuR依赖性 定殖决定簇表达。此外，我们将直接确定每种TCA酶 质量同位素分析表明，受铁限制。最后，HeuR的直接目标之一， 确定可能受TCA中间体依赖性结合影响的是C的血红素利用系统。 空肠。这个系统在C语言中的特性描述得很差。空肠，我们将研究这个系统是否有助于铁 在动物感染过程中获得，并将努力充分表征血红素利用的过程， 在未来的研究中可以利用它来抑制C.空肠。

Project Summary Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis in the world. Human infection often occurs through the ingestion of contaminated food, especially poultry, which leads to the bacterium colonizing the colon and causing severe inflammation and diarrhea. While infection and disease is often self-limiting, persistent colonization and chronic diseases do occur. Despite the significant impacts of C. jejuni on human health, very little is known about the interactions that occur at the host-pathogen interface during infection, including how C. jejuni senses and adapts to the host intestinal environment to promote infection. This is primarily due to the evolutionary divergence of this organism from other gastrointestinal pathogens, which limits the relevance of findings from those organisms and necessitates specific study of the Campylobacter genus. To that end, our group previously identified a unique regulator in C. jejuni, which we call HeuR, that promotes maximum colonization of a natural avian host and was subsequently found to positively or negatively regulate several genetic determinants, including those involved in the acquisition of iron from host heme and the biosynthesis of methionine. In addition, HeuR and its downstream targets are required for efficient invasion or persistence in human colonocytes, which suggests these mechanisms need to be better understood as they are clearly involved in infection of animals. We preliminarily determined that this novel regulator binds several TCA intermediates and may sense TCA cycle activity to control expression of colonization determinants. First, we will define all direct targets of HeuR and examine whether TCA intermediates impact the ability of HeuR to bind those DNA targets and impact gene expression. Additionally, we will identify the ligand binding motif of this novel regulator and how it facilitates HeuR activity. Second, because we have determined that C. jejuni TCA intermediate abundance is affected by iron-restriction, we will use mass isotopomer analysis to identify the points along the C. jejuni TCA cycle that are affected by iron-limitation and determine whether altering TCA cycle activity affects HeuR-dependent colonization determinant expression. In addition, we will directly determine the levels at which each TCA enzyme indicated by mass isotopomer analysis is affected by iron-limitation. Lastly, one of the direct targets of HeuR we identified that may be impacted by TCA intermediate-dependent binding is the heme utilization system of C. jejuni. This system is poorly characterized in C. jejuni and we will examine whether this system facilitates iron acquisition during animal infection and will work to fully characterize the process of heme utilization so that it can be leveraged in future studies to inhibit the infection potential of C. jejuni.