Harnessing iron acquisition to hinder enterobacterial pathogenesis

项目总结 这一更新申请的主要目标是研究基于铁载体的免疫和 抑制大肠杆菌和非伤寒沙门氏菌生长的抗生素给药策略 (新界别)。这些革兰氏阴性兼性厌氧菌是不同患者感染的主要原因 人口。大肠杆菌包括共生生物、病原体和致病菌(通常是 无害但在某些环境中是致病的)，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，一种被称为粘附性侵袭性大肠杆菌(AIEC)的病原体是 通常从克罗恩病患者身上分离出来，克罗恩病是炎症性肠病的一种形式。NTS，包括 鼠伤寒沙门氏菌(STM)是引起炎症性腹泻的主要原因。主站点 大肠埃希氏菌和NTS的定植是胃肠道，在结肠炎和肺炎期间这些微生物在那里繁衍生息 传播到其他身体部位。最近的研究，包括我们实验室的工作，证明铁(Fe) 可获得性是大肠杆菌和沙门氏菌在肠道中定植的关键因素，促使 在这项拨款申请中提出的研究。我们的中心假设是以铁载体及其 摄取机制可以在体外和体内限制肠道病原体的生长。大肠埃希菌和沙门氏菌都能使 肠道中儿茶酚类铁载体Enterobactin(Ent)和Salmochelin(DGE，二葡萄糖基化Enterobactin) 清除宿主体内的Fe3。我们认为，阻断Ent和DGE介导的细菌获取Fe3 病原体或靶向Ent&DGE运输系统输送抗生素将提供一种抑制生长的手段 STM，AIEC和可能的其他肠道病原体在发炎的肠道中。为了支持这一概念，我们开发了 一种基于铁载体的免疫，它抑制小鼠肠道中STM和AIEC的生长，我们合成了 并评估了基于Ent&DGE支架的含铁载体-抗生素结合物(SACS)，该支架针对大肠杆菌和 扫描隧道显微镜。在目标1中，我们将生产捕获Ent和DGE的单抗，选择显示 体外对STM和AIEC的最大生长抑制活性，并研究这些抗体是否 抑制活体结肠炎期间STM和AIEC的粘膜扩张及其对肠道微生物群的影响。 在目标2中，我们将评估三种基于Ent和DGE的囊泡的抗菌活性。体外研究将在很大程度上 关注表征不同宿主环境的关键环境变量如何影响抗菌剂 SAC的活性，而体内研究将评估SAC对肠道的影响 微生物群组成以及在结肠炎期间抑制STM和AIEC的粘膜扩张。这部作品 可能导致铁载体结合抗体和铁载体-抗生素结合物的未来发展，如 限制肠道内肠道病原体和病原体的定植和致病的治疗方法。

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.