Nutritional immunity and microbial competition during Clostridioides difficile infection

摘要 艰难梭状芽胞杆菌(以前称为艰难梭菌)是一种革兰氏阳性芽胞形成病原体， 院内感染和抗生素相关肠道感染的主要原因。艰难梭菌感染的易感性 然而，(CDI)通常伴随着抗生素治疗和随后驻留的肠道微生物区系的破坏 健康的年轻人感染人数的增加表明，还有其他因素导致CDI。至 艰难梭菌必须与宿主和肠道微生物区系的成员竞争 关键的营养物质。获得营养金属会对CDI的结果产生深远的影响，因为需要金属 辅因子约占所有蛋白质的30%。这一事实被宿主金属结合蛋白利用 在一种称为营养免疫的过程中隔离营养金属以限制微生物的生长。CDI的一个标志 是分泌强烈的毒素，对胃肠上皮造成严重损害并引发 产生促炎细胞因子和趋化因子。这些事件启动了免疫介导的 向感染部位募集炎性因子。最丰富的炎性蛋白之一 积聚在CDI部位的是钙保护素。钙保护素是中性粒细胞中含量最丰富的蛋白质，是一种 营养免疫成分，通过营养金属封存直接抑制微生物生长。 钙保护素也是一种强大的免疫调节蛋白，也是一种常见的临床炎症生物标志物，其 丰度与CDI的严重程度相关。目前尚不清楚钙保护素的大量渗透如何影响金属。 艰难梭菌在胃肠道中的利用度以及艰难梭菌与肠道成员之间的竞争 微生物区系。此外，艰难梭菌如何适应依赖钙保护素的金属限制和抵抗营养 CDI期间的豁免权仍不清楚。我们提出了一个模型，在这个模型中，营养金属起到了关键作用。 对CDI的结果。毒素引发的炎症促使免疫细胞重新聚集到肠道，从而导致 导致大量CP的积累。CP能螯合有效的营养金属，并能产生强烈的促进作用。 炎症活动。这种大规模的炎症反应和营养物质的重新分配改变了艰难梭菌的基因 表达，并影响艰难梭菌与微生物群成员之间的相互作用。最后，我们 假设艰难梭菌编码多个基因产物，这些基因产物与CP和微生物群竞争 营养金属和这种竞争对CDI的结果有深远的影响。本文件中描述的实验 该提案将测试这一模型，并确定营养免疫在艰难梭菌发病机制中的作用。 确定金属结合和免疫细胞募集在钙保护素的保护特性中的作用，以及 确定艰难梭菌在炎症过程中与微生物竞争营养金属所需的基因。 总的来说，这项提案的发现将为有效治疗或预防的发展提供信息。 CDI的治疗策略。

SUMMARY Clostridioides difficile (formerly named Clostridium difficile) is a Gram-positive, spore-forming pathogen, and the leading cause of nosocomial and antibiotic-associated intestinal infections. Susceptibility to C. difficile infection (CDI) often follows antibiotic treatment and subsequent disruption of the resident intestinal microbiota, however the rise of infections in healthy young adults suggests that there are additional factors that contribute to CDI. To colonize the gastrointestinal tract, C. difficile must compete with both the host and members of the gut microbiota for critical nutrients. Access to nutrient metals can profoundly impact the outcome of CDI as metals are required cofactors for approximately 30% of all proteins. This fact is exploited by host metal binding proteins which sequester nutrient metals to restrict microbial growth in a process termed nutritional immunity. A hallmark of CDI is the secretion of potent toxins that cause severe damage to the gastrointestinal epithelium and trigger the production of pro-inflammatory cytokines and chemokines. These events initiate the immune-mediated recruitment of inflammatory factors to the site of infection. One of the most abundant inflammatory proteins that accumulates at the site of CDI is calprotectin. Calprotectin is the most abundant protein in neutrophils and is a component of nutritional immunity that directly inhibits microbial growth through nutrient metal sequestration. Calprotectin is also a potent immunomodulatory protein, and a common clinical inflammatory biomarker whose abundance correlates with CDI severity. It is unknown how the massive infiltration of calprotectin affects metal availability in the gastrointestinal tract and shapes competition between C. difficile and members of the gut microbiota. In addition, how C. difficile adapts to calprotectin-dependent metal limitation and resists nutritional immunity during CDI remains unclear. We propose a model whereby nutrient metals make a critical contribution to the outcome of CDI. Toxin driven inflammation drives the recruitment of immune cells into the gut which leads to the accumulation of large amounts of CP. CP chelates available nutrient metals and exerts potent pro- inflammatory activities. This massive inflammatory response and redistribution of nutrients alters C. difficile gene expression and affects the interaction between C. difficile and members of the microbiota. Finally, we hypothesize that C. difficile encodes multiple gene products that compete with both CP and the microbiota for nutrient metals and this competition has a profound effect on the outcome of CDI. Experiments described in this proposal will test this model and define the contribution of nutritional immunity to the pathogenesis of C. difficile, determine the role of metal binding and immune cell recruitment in the protective properties of calprotectin, and identify C. difficile genes required to compete with the microbiome for nutrient metals during inflammation. Collectively, the findings from this proposal will inform the development of effective therapeutic or prevention strategies for the treatment of CDI.