Gonococcal Mechanisms to Evade Host Defenses

描述（由申请人提供）： 淋病奈瑟菌是一种人类病原体，引起性传播感染，称为淋病。全世界淋病的年发病率（> 1亿例）是一个主要的公共卫生问题，由于出现对多种抗生素具有耐药性的菌株而加剧。某些临床分离株表达的抗生素耐药性问题最近变得更加令人担忧，有报道称，在美国，菌株对用于淋病经验性治疗的第三代头孢菌素（头孢曲松和头孢克肟）的敏感性降低或耐药性降低。症状性淋病主要是由于宿主的炎症反应，这可能导致男性和女性的严重并发症。女性患者的侵袭性淋病可能特别急性，导致严重的妇科后果。该荣誉奖的更新致力于了解淋球菌外膜重要修饰的遗传学和分子控制，该修饰有助于淋球菌抵抗先天宿主防御的某些介质（补体和阳离子抗菌肽[CAMPs]）的能力。这种修饰包括通过称为LptA的酶将磷酸乙醇胺（PEA）连接到脂寡糖的脂质A上。在过去的资助期间，我们发现编码LptA的基因（lptA）中的无效突变使淋球菌对补体和CAMP高度敏感，抑制了淋球菌诱导的促炎反应，并在雌性小鼠下生殖道的实验感染期间降低了细菌适应性。此外，我们发现了一个多层次的，综合的和复杂的调节系统，调节lptA的表达，因此，PEA装饰的脂质A，淋球菌对CAMP的敏感性，淋球菌和炎症的体内适应性。该系统是本次续期申请的重点。整个调控系统由用于lptA表达的操纵子内的多个启动子、lptA编码序列内的相位可变多核苷酸重复序列（其可导致高频率的LptA截短）和称为MisR的DNA结合蛋白的基因激活能力组成。在申请资助期间，将确定这些监管程序，并确定其对CAMP耐药性的影响。具体目标1中体现的研究将定义和表征用于lptA表达的启动子，它们的差异使用和对亚致死水平抗菌剂的反应。将鉴定调节这些启动子的使用的转录因子，以便理解lptA表达如何在转录水平上被控制。此外，将在实验室维持的和最近的临床菌株中定义LptA的时相可变产生。具体目标2中体现的研究致力于了解MisR调节lptA表达的机制，以及该控制系统如何影响淋球菌对CAMPs的抗性和独特脂质A结构的产生。拟议工作的成功完成将显着推进我们的知识，淋球菌如何可以逃避宿主防御和调节促炎反应，通过使用基因控制系统。重要的是，这些结果还应该为未来开发可用于治疗淋病的新型抗菌药物和治疗策略提供重要的见解。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae is a human pathogen that causes the sexually transmitted infection termed gonorrhea. The yearly incidence of gonorrhea worldwide (>100 million cases) is a major public health problem, which is compounded by the emergence of strains resistant to multiple antibiotics. The problem of antibiotic resistance expressed by certain clinical isolates has been made more worrisome recently with reports of strains expressing decreased susceptibility or resistance to the third generation cephalosporins (ceftriaxone and cefixime) that have been used for empiric therapy of gonorrhea in the United States. Symptomatic gonorrhea is largely due to the host's inflammatory response, which can result in severe complications for men and women. Invasive gonorrhea in the female patient can be particularly acute resulting in severe gynecologic consequences. This Merit Award renewal is dedicated to understanding the genetics and molecular control of an important modification of the gonococcal outer membrane that contributes to the ability of gonococci to resist certain mediators (complement and cationic antimicrobial peptides [CAMPs]) of innate host defense. This modification consists of the attachment of phosphoethanolamine (PEA) to the lipid A of lipooligosaccharide by an enzyme termed LptA. During the past funding period we showed that null mutations in the gene (lptA) encoding LptA rendered gonococci hyper-susceptible to complement and CAMPs, depressed the pro- inflammatory response induced by gonococci and decreased bacterial fitness during an experimental infection of the lower genital tract of female mice. Further, we uncovered a multi-layered, integrated and complex regulatory system that modulates lptA expression and, as a consequence, PEA decoration of lipid A, gonococcal susceptibility to CAMPs, in vivo fitness of gonococci and inflammation. This system is the focus of this renewal application. The overall regulatory system consists of multiple promoters within an operon for lptA expression, a phase variable polynucleotide repeat sequence within the lptA coding sequence that can cause truncation of LptA at high frequencies and the gene-activating capacity of a DNA-binding protein termed MisR. During the requested funding period, these regulatory processes will be defined and their impact on CAMP resistance will be determined. The studies embodied in Specific Aim 1 will define and characterize the promoters used for lptA expression, their differential use and response to sub-lethal levels of antimicrobials. Transcriptional factors that modulate use of these promoters will be identified so as to understand how lptA expression is controlled at the transcriptional level. Additionally, the phase variable production of LptA will be defined in laboratory-maintained and recent clinical strains. The studies embodied in Specific Aim 2 are dedicated to understanding the mechanisms by which MisR regulates lptA expression and how this control system influences gonococcal resistance to CAMPs and production of unique lipid A structures. The successful completion of the proposed work will significantly advance our knowledge regarding how gonococci can evade host defenses and modulate pro-inflammatory responses through the use of gene control systems. Importantly, the results should also provide important insights for the future development of novel antimicrobial agents and therapeutic strategies that can be used in the treatment of gonorrhea.