Experimental human infection with isogenic mutants of Neisseria gonorrhoeae

 描述(申请人提供)：淋病仍然是一个全球公共卫生问题，据世卫组织估计，2008年有超过1.06亿例淋病病例，2012年向美国疾控中心报告的病例超过334,000例，而且发病率还在上升。随着淋病奈瑟菌(GC)耐药性的增加，以及没有新的抗菌疗法在酝酿中，发生无法治愈的淋病的可能性是令人不安的。无法治疗的GC和随之而来的艾滋病毒传播增加带来的潜在不利健康后果令人震惊，并突出表明迫切需要新的治疗和疫苗目标。我们的长期目标是了解GC发病的分子机制并鉴定感染所需的细菌结构，目的是发现GC疫苗和/或治疗的潜在新靶点。在自然界中，GC只感染人类，这限制了动物模型结果在临床上可翻译的程度。因此，我们建议进行一项临床试验，目标是1)确定男性尿路感染对几种GC细胞包膜结构的需求，以及2)表征人类对缺乏这些结构的突变的免疫反应。试验的预期结果是确定人类感染所需的一个或多个GC包膜结构，这将验证这些结构是否为GC疫苗或治疗的潜在新靶点。聚集在一起的研究团队管理着北卡罗来纳大学的实验性人类淋球菌感染项目，这是目前世界上唯一的此类项目，为在受控条件下研究人类感染中的GC因素和宿主反应提供了独特的机会。男性志愿者实验性尿路感染是安全的，野生型GC会引发自然淋病的体征和症状。这项拟议的试验集中在几种表面结构上，这些结构介导了GC对人类先天免疫反应的抵抗。我们假设，与野生型GC相比，含有改变或消除这些结构的突变的GC菌株的实验性男性尿路感染将显示出突变感染性降低、炎症减少或GC突变清除增加。我们将使用非竞争性感染来衡量GC存活率和宿主免疫反应，以及GC基因表达的变化。我们提出了对三种GC包膜结构的研究，我们已经在实验室和小鼠模型中显示了对人类先天免疫细胞和人类衍生抗菌化合物的中间抗性。目的1重点研究影响GC MtrCDE外排泵的突变，该泵输出宿主抗菌化合物，在人类细胞系统和小鼠生殖器感染中对GC感染起重要作用。目的2重点研究改变脂质A结构的突变，它对体外细胞内GC存活很重要，并影响小鼠GC存活。最后，目标3侧重于GC酶的突变，这些突变导致在人类细胞系统和小鼠中调节先天免疫反应的肽聚糖亚单位的释放。我们在试验中测试的GC包膜结构还没有在人类感染中得到很好的研究，它们代表了新的潜在的治疗和/或疫苗靶点。

 DESCRIPTION (provided by applicant): Gonorrhea remains a global public health problem with over 106 million cases estimated by WHO in 2008 and over 334,000 cases reported to the US CDC in 2012, and rates are on the rise. With increasing Neisseria gonorrhoeae (GC) antibiotic resistance and no new antimicrobial therapies in the pipeline, the possibility of incurable gonorrhea is uncomfortably real. The potential adverse health consequences of untreatable GC and attendant increases in HIV transmission are alarming and highlight the urgent need for new therapeutic and vaccine targets. Our long term goals are to understand the molecular mechanisms of GC pathogenesis and identify bacterial structures required for infection with the aim of discovering potential novel targets for GC vaccines and/or treatments. In nature, GC only infects humans, which limits the extent to which results in animal models are clinically translatable. Accordingly, we propose a clinical trial with the immediate goals of 1) determining the requirement for several GC cell envelope structures in male urethral infection and 2) characterizing human immune responses to mutants lacking these structures. The expected outcome of the trial is the identification of one or more GC envelope structures required for human infection, which will validate these structures as potential novel targets for GC vaccines or treatments. The assembled research team manages the experimental human gonococcal infection program at the University of North Carolina, which is currently the only one of its kind in the world, providing a unique opportunity to study GC factors and host responses in human infection under controlled conditions. Experimental urethral infection of male volunteers is safe, and wild-type GC elicit signs and symptoms of natural gonorrhea. The proposed trial focuses on several surface structures that mediate GC resistance to human innate immune responses. We hypothesize that experimental male urethral infection with GC strains containing mutations that alter or eliminate these structures will show reduced mutant infectivity, decreased inflammation or increased GC mutant clearance compared to wild-type GC. We will use noncompetitive infections to measure GC survival and host immune responses in addition to changes in GC gene expression. We propose studies of three GC envelope structures that we have shown mediate resistance to human innate immune cells and human-derived antimicrobial compounds in the lab and in a mouse model. Aim 1 focuses on mutations affecting the GC MtrCDE efflux pump, which exports host antimicrobial compounds and is important for GC infection in human cell systems and in murine genital infection. Aim 2 focuses on mutations that alter the structure of lipid A, which is important for intracellular GC survival in vitro, and influences GC survival in mice. Finally, Aim 3 focuses on mutations in GC enzymes that cause release of peptidoglycan subunits that modulate innate immune responses in human cell systems and in mice. The GC envelope structures that we aim to test in the trial have not been well-studied in human infection, and they represent novel potential therapeutic and/or vaccine targets.