Harnessing iron acquisition to hinder enterobacterial pathogenesis

项目摘要 本申请的主要目的是研究铁载体免疫和抗生素 旨在抑制大肠杆菌和非伤寒沙门氏菌（NTS）生长的递送策略。这些 革兰氏阴性兼性厌氧菌是不同患者人群感染的主要原因。E. 大肠杆菌包括寄生生物、病原体和致病菌（通常无害但 在某些情况下是致病的）并引起包括尿路感染（UTI），菌血症， 脑膜炎和败血症此外，一种称为粘附侵袭性E.大肠杆菌（AIEC）通常是分离的 克隆氏病患者，一种炎症性肠病。NTS，包括肠道沙门氏菌 鼠伤寒血清型（STm）是炎性腹泻的主要原因。E. coli和NTS 定植是胃肠道，这些生物体在结肠炎期间在那里茁壮成长，并传播到其他组织。 身体部位。最近的研究，包括我们实验室的工作，表明铁（Fe）的可用性是关键 E.大肠杆菌和NTS在肠道中的定植，激发了本研究中提出的研究。 补助金申请我们的中心假设是，靶向铁载体及其摄取机制可以限制 肠道病原体体外和体内生长。E.大肠杆菌和NTS部署儿茶酚铁载体 肠杆菌素（Ent）和沙门氏菌螯铁素（DGE，二葡糖基化肠杆菌素）对来自宿主的Fe 3+的耐受性。 我们建议阻断细菌病原体对Ent& DGE介导的Fe ~（3+）的摄取或靶向Ent&DGE 递送抗生素的转运系统将提供一种抑制STm和AIEC在发炎患者中生长的手段。 直觉为了支持这一观点，我们开发了一种基于铁载体的免疫接种， 和AIEC在小鼠肠道中的生长，我们合成并评估了铁载体-抗生素缀合物（SAC） 基于靶向E. coli和STm。在目标1中，我们将在Fe获取中使用突变体 STm和AIEC中的基因以测试CTB-Ent免疫是否导致病原体生长的特异性抑制 以及病原菌产生Ent时与肠道黏膜的结合情况决定CTB-Ent的作用 免疫对粘膜相关微生物群与腔微生物群的影响;并确定是否中和抗- Ent和DGE IG通过限制病原体与粘膜的结合来介导保护作用。在目标2中，我们将评估 图4示出了三种基于Ent& DGE的SAC的抗微生物活性。体外研究将主要集中在如何关键的环境 表征不同宿主环境的变量影响SAC的抗菌活性，而研究 体内试验将评估SAC给药对肠道微生物组组成以及 抑制结肠炎期间STm和AIEC的粘膜扩张。这项工作可能会导致未来的发展， 铁载体结合抗体和SAC作为限制肠道病原体定殖的治疗剂， 在发炎的肠道里发现致病菌

PROJECT SUMMARY The primary objective of this application is to investigate siderophore-based immunization and antibiotic delivery strategies designed to inhibit the growth of Escherichia coli and non-typhoidal Salmonella (NTS). These Gram-negative facultative anaerobic bacteria are major causes of infections in diverse patient populations. E. coli includes commensal organisms, pathogens, and pathobionts (organisms that are usually harmless but are pathogenic in some settings) and cause infections that include urinary tract infections (UTI), bacteremia, meningitis, and sepsis. Moreover, a pathovar known as adherent-invasive E. coli (AIEC) is commonly isolated from patients with Crohn’s disease, a form of inflammatory bowel disease. NTS, including Salmonella enterica serovar Typhimurium (STm), are major causes of inflammatory diarrhea. The primary site of E. coli and NTS colonization is the gastrointestinal tract, where these organisms thrive during colitis and disseminate to other body sites. Recent studies, including work from our laboratories, demonstrate that iron (Fe) availability is a key factor for the progression of E. coli and NTS colonization in the gut, motivating the research proposed in this grant application. Our central hypothesis is that targeting siderophores and their uptake machineries can limit enteric pathogen growth in vitro and in vivo. Both E. coli and NTS deploy the catecholate siderophores enterobactin (Ent) and salmochelin (DGE, diglucosylated enterobactin) in the gut to scavenge Fe3+ from the host. We propose that blocking Ent&DGE-mediated Fe3+ acquisition by bacterial pathogens or targeting Ent&DGE transport systems to deliver antibiotics will provide a means to inhibit the growth of STm and AIEC in the inflamed gut. In support of this notion, we developed a siderophore-based immunization based on Ent that inhibits STm and AIEC growth in the murine gut, and we synthesized and evaluated siderophore-antibiotic conjugates (SACs) based on the Ent&DGE scaffold that target E. coli and STm. In Aim 1, we will use mutants in Fe acquisition genes in STm and AIEC to test whether CTB-Ent immunization results in specific inhibition of pathogen growth and association with the gut mucosa when the pathogen produces Ent&DGE; determine the effect of CTB-Ent immunization on the mucosal-associated versus luminal microbiota; and ascertain whether neutralizing anti- Ent&DGE Ig mediate protection by limiting pathogen association with the mucosa. In Aim 2, we will evaluate the antimicrobial activity of three Ent&DGE-based SACs. Studies in vitro will largely focus on how key environmental variables that characterize diverse host environments affect the antimicrobial activity of SACs, whereas studies in vivo will evaluate the consequences of SAC administration on the gut microbiome composition as well as on inhibiting mucosal expansion of STm and AIEC during colitis. This work may lead to future development of siderophore-binding antibodies and SACs as therapeutics to limit colonization of enteric pathogens and pathobionts in the inflamed gut.