Experimental human infection with isogenic mutants of Neisseria gonorrhoeae

 描述（由申请人提供）：淋病仍然是一个全球公共卫生问题，2008年WHO估计有超过1.06亿例病例，2012年向美国CDC报告了超过334，000例病例，并且发病率正在上升。随着淋病奈瑟菌（GC）抗生素耐药性的增加，并且没有新的抗菌治疗方法在管道中，淋病不可治愈的可能性是令人不安的真实的。无法治疗的胃癌的潜在不良健康后果和随之而来的艾滋病毒传播的增加令人震惊，并突出了对新的治疗和疫苗目标的迫切需要。我们的长期目标是了解GC发病机制的分子机制，并确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仅感染人类，这限制了动物模型结果的临床可转化程度。因此，我们提出了一项临床试验，其直接目标是：1）确定男性尿道感染中对几种GC细胞包膜结构的需求; 2）表征人类对缺乏这些结构的突变体的免疫应答。该试验的预期结果是鉴定出人类感染所需的一种或多种GC包膜结构，这将验证这些结构作为GC疫苗或治疗的潜在新靶点。组装的研究团队管理北卡罗来纳州大学的实验性人类淋球菌感染项目，该项目目前是世界上唯一的此类项目，为在受控条件下研究GC因子和人类感染中的宿主反应提供了独特的机会。男性志愿者的实验性尿道感染是安全的，野生型GC引起自然淋病的体征和症状。拟议的试验集中在几个表面结构，介导GC抵抗人类先天免疫反应。我们假设，实验男性尿道感染GC菌株含有突变，改变或消除这些结构将显示减少突变感染，减少炎症或增加GC突变体清除野生型GC相比。我们将使用非竞争性感染来测量GC存活率和宿主免疫反应，以及GC基因表达的变化。我们提出了三个GC包膜结构的研究，我们已经证明介导的人类先天免疫细胞和人源性抗微生物化合物在实验室和小鼠模型中的耐药性。目的1关注影响GC MtrCDE外排泵的突变，该外排泵输出宿主抗微生物化合物，并且对人类细胞系统和鼠生殖器感染中的GC感染很重要。目的2关注改变脂质A结构的突变，这对体外细胞内GC存活很重要，并影响小鼠中GC的存活。最后，目标3关注GC酶的突变，这些突变导致肽聚糖亚基的释放，肽聚糖亚基调节人类细胞系统和小鼠的先天免疫应答。我们在试验中测试的GC包膜结构尚未在人类感染中得到充分研究，它们代表了新的潜在治疗和/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.