Harnessing iron acquisition to hinder enterobacterial pathogenesis

项目摘要 该更新申请的主要目的是研究基于铁载体的免疫， 旨在抑制大肠杆菌和非伤寒沙门氏菌生长的抗生素递送策略 （NTS）。这些革兰氏阴性兼性厌氧菌是各种患者感染的主要原因 人口。E.大肠杆菌包括细菌生物体、病原体和致病菌（通常是 无害但在某些情况下是致病的）并引起包括尿路感染（UTI）在内的感染， 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）是 通常从克罗恩病患者中分离出来，克罗恩病是一种炎症性肠病。NTS，包括 肠道沙门氏菌血清型鼠伤寒沙门氏菌（STm）是炎症性腹泻的主要原因。主站点 大肠大肠杆菌和NTS定殖是胃肠道，这些生物体在结肠炎期间在胃肠道中茁壮成长， 传播到其他身体部位。最近的研究，包括我们实验室的工作，表明铁（Fe） 可用性是E.大肠杆菌和沙门氏菌在肠道中的定植， 在这项研究申请中提出。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在体外和体内的生长。E.大肠杆菌和沙门氏菌 儿茶酸铁载体肠杆菌素（Ent）和Salmochelin（DGE，二葡糖基化肠杆菌素）， Fe 3+从主机。我们认为，阻断Ent& DGE介导的细菌对Fe ~（3+）的摄取， 病原体或靶向Ent&DGE运输系统以递送抗生素将提供抑制生长的手段 STm，AIEC和其他可能的肠道病原体在发炎的肠道。为了支持这一观点，我们开发了 一种基于铁载体的免疫，抑制STm和AIEC在小鼠肠道中的生长，我们合成了 并基于靶向E.杆菌和 STm。在目标1中，我们将生产捕获Ent&DGE的单克隆抗体，选择表现出 体外对STm和AIEC的最大生长抑制活性，并研究这些抗体是否 在体内结肠炎期间抑制STm和AIEC的粘膜扩张以及它们对肠道微生物组的影响。 在目标2中，我们将评估三种基于Ent& DGE的SAC的抗微生物活性。体外研究将在很大程度上 重点关注表征不同宿主环境的关键环境变量如何影响抗菌药物 体内研究将评估SAC给药对肠道的影响， 在结肠炎期间，STm和AIEC的粘膜扩张的抑制作用。这项工作 可能导致铁载体结合抗体和铁载体-抗生素缀合物的未来发展， 治疗剂以限制发炎肠道中的肠道病原体和致病生物的定殖。

PROJECT SUMMARY The primary objective of this renewal 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 Salmonella 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 Salmonella deploy the catecholate siderophores enterobactin (Ent) and salmochelin (DGE, diglucosylated enterobactin) in the gut to scavenge Fe3+ from the host. We propose that 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, AIEC and possibly other enteric pathogens in the inflamed gut. In support of this notion, we developed a siderophore-based immunization 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 produce monoclonal antibodies that capture Ent&DGE, select the antibodies that exhibit the greatest growth inhibitory activity against STm and AIEC in vitro, and investigate whether these antibodies inhibit mucosal expansion of STm and AIEC during colitis in vivo as well as their impact on the gut microbiome. 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 siderophore-antibiotic conjugates as therapeutics to limit colonization and of enteric pathogens and pathobionts in the inflamed gut.