Genetics of Salmonella Resistance to the Inflammatory Response in the Gut

描述（由申请方提供）：非伤寒沙门氏菌，包括鼠伤寒血清型（STm），是食源性细菌病原体，在美国每年导致约140万例伤寒病例，在全球范围内导致数亿例病例。在肠中，STm诱导强烈的嗜中性炎症反应和肠上皮细胞产生抗微生物化合物。面对这种炎症反应，肠道中STm的数量急剧增加，而宿主的肠道微生物菌群急剧减少。本提案的目的是确定允许STm抵抗被肠上皮产生的抗菌剂杀死的细菌因素。 小牛是肠道STm感染的天然模型，其在临床体征、宿主反应和肠道病理学方面与人类疾病最相似。我们已经开发了一个正向遗传系统，使在小牛的回肠环结扎中筛选突变体成为可能，这是德克萨斯A&M开发的一个模型。我们已经在STm中产生了超过1000个靶向缺失菌株的集合，包括所有“沙门氏菌特异性”基因中的突变体，并且我们已经开发了基于微阵列的方法来将整个突变体集合作为单个池进行筛选。我们已经证实了该系统用于动物感染的正向遗传筛查。 在AIM-1中，我们将筛选小牛结扎回肠袢中的突变体库，以鉴定对肠上皮细胞衍生的抗菌剂敏感的突变体。 在AIM-2中，我们将在小牛结扎回肠环的竞争性感染中验证和补充这些突变体。 在AIM-3中，我们将确定这些基因中哪些对纯化的抗菌剂钙卫蛋白和肠2-防御素的抗性是重要的，这些抗菌剂已知在炎症期间由肠上皮产生。 该项目是全面确定参与肠道炎症反应的细菌基因分子机制的第一步。公共卫生相关性：沙门氏菌是食源性疾病的主要原因，每年导致约140万例腹泻病，并且是美国主要在免疫功能低下人群中与病毒，寄生虫或细菌相关的食源性疾病死亡的最常见原因。非伤寒沙门氏菌在肠道中面对宿主炎症反应时生存所使用的基因和机制尚不清楚。这些机制使沙门氏菌在感染期间在肠道中建立一个小生境，并且它们从受感染的人和牲畜的粪便物质中从这个小生境中脱落，继续传播周期。更好地了解感染这一重要阶段所涉及的基因和机制对于打破这种生物体的传播周期至关重要。这项工作将对公众健康产生直接影响。

DESCRIPTION (provided by applicant): Non-typhoidal Salmonella, including serotype Typhimurium (STm) are food-borne bacterial pathogens that cause ~1.4 million cases of diarrheal disease annually in the United States and hundreds of millions of cases worldwide. In the intestine, STm induces a strong neutrophilic inflammatory response and the production of antimicobial compounds by intestinal epithelial cells. In the face of this inflammatory response the numbers of STm in the intestine increase sharply, while the intestinal microflora of the host are dramatically reduced. The objective of this proposal is to identify the bacterial factors that allow STm to resist being killed by antimicrobials produced by the intestinal epithelium. The calf is the natural model of diarrheal STm infection that is most similar to human disease in clinical signs, host responses, and intestinal pathology. We have developed a forward genetic system to make screening for mutants feasible in ligated ileal loops in calves, a model developed at Texas A&M. We have generated a collection of over 1000 targeted deletion strains in STm, including mutants in all "Salmonella-specific" genes and we have developed microarray-based methods to screen this entire collection of mutants as a single pool. We have already confirmed the use of this system for forward genetic screening in animal infection. In AIM-1 we will screen the mutant pool in calf ligated ileal loops to identify mutants sensitive to intestinal epithelial cell-derived antimicrobials. In AIM-2 we will verify and complement these mutants in competitive infections in ligated ileal loops in calves. In AIM-3 we will determine which of these genes are important for resistance to the purified antimicrobials calprotectin and enteric 2-defensin, antimicrobials known to be produced by the intestinal epithelium during inflammation. This project is a first step toward a comprehensive determination of the molecular mechanism of the bacterial genes involved in the response to inflammation in the gut. PUBLIC HEALTH RELEVANCE: Salmonella is a leading cause of food borne illness, causing ~1.4 million cases of diarrheal disease per year and is the single most common cause of death from food-borne illnesses associated with viruses, parasites or bacteria in the US primarily in immunocompromised persons. The genes and mechanisms used by non-typhoidal Salmonellae to survive in the face of a host inflammatory response in the intestine are not well understood. These mechanisms allow Salmonellae to establish a niche in the intestine during infection, and they are shed from this niche in fecal material from infected persons and livestock continuing the cycle of transmission. Development of a better understanding of the genes and mechanisms involved in this important stage of infection is critical to breaking the cycle of transmission of this organism. This work will have a direct impact on public health.