Harnessing iron acquisition to hinder enterobacterial pathogenesis

项目总结 该应用程序的主要目标是研究基于铁载体的免疫和抗生素 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群体感染的主要原因。E. 大肠埃希氏菌包括共生生物、病原体和致病菌(通常无害但 在某些环境中致病)并引起感染，包括尿路感染(UTI)、菌血症、 脑膜炎和败血症。此外，通常分离出一种称为黏附性侵袭性大肠杆菌(AIEC)的致病变种 来自克罗恩病的患者，这是一种炎症性肠病。包括肠道沙门氏菌在内的NTS 鼠伤寒沙门氏菌(STM)是炎症性腹泻的主要原因。大肠埃希氏菌和新城疫的原发部位 定植是指胃肠道，这些细菌在结肠炎期间在那里繁衍生息，并传播给其他 身体部位。最近的研究，包括我们实验室的工作，表明铁(Fe)的有效性是一个关键 大肠杆菌和NTS在肠道中定植的进展因素，激励了在本研究中提出的研究 批准申请。我们的中心假设是，靶向铁载体及其吸收机制可能会限制 肠道病原菌在体外和体内的生长。大肠杆菌和NTS都部署儿茶酚类铁载体 肠杆菌素(Ent)和沙莫胆碱(DGE，二葡萄糖基化肠杆菌素)从宿主体内清除Fe3+。 我们建议阻断Ent和DGE介导的细菌对Fe3+的摄取或靶向Ent和DGE 输送抗生素的运输系统将提供一种抑制炎症患者STM和AIEC生长的方法 直觉。为了支持这一概念，我们开发了一种基于铁载体的免疫，该免疫基于Ent，可以抑制STM 和AIEC在小鼠肠道中的生长，我们合成并评价了铁载体-抗生素结合物(SACS)。 基于针对大肠杆菌和STM的Ent&DGE支架。在目标1中，我们将在铁的获取中使用突变体 STM和AIEC中检测CTB-Ent免疫是否能特异性抑制病原体生长的基因 以及在病原菌产生Ent和DGE时与肠粘膜的结合；确定CTB-Ent的效果 对粘膜相关微生物区系与管腔微生物区系进行免疫；并确定中和抗 ENT和DGE Ig通过限制病原体与粘膜的联系来调节保护作用。在目标2中，我们将评估 三种Ent&DGE基囊的抗菌活性。体外研究将主要集中在关键环境如何 表征不同寄主环境的变量会影响SACS的抗菌活性，而研究 在活体内将评估SAC给药对肠道微生物组组成以及对 抑制结肠炎时STM和AIEC的粘膜扩张。这项工作可能会导致未来的发展 铁载体结合抗体和囊泡作为治疗药物来限制肠道病原体和 发炎的肠道里有致病物质。

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.