Gonococcal Mechanisms to Evade Host Defenses

描述(由申请人提供)： 淋病奈瑟氏菌是一种人类病原体，可引起被称为淋病的性传播感染。全世界每年的淋病发病率(1亿例)是一个主要的公共卫生问题，对多种抗生素产生抗药性的菌株的出现更是雪上加霜。近年来，一些临床分离株对第三代头孢菌素(头孢曲松和头孢克辛)的敏感性降低或耐药性下降的报道使某些临床分离株的耐药性问题更加令人担忧。有症状的淋病在很大程度上是由于宿主的炎症反应，这可能会导致男性和女性的严重并发症。侵袭性淋病在女性患者中可能特别严重，导致严重的妇科后果。此次获奖更新致力于了解淋球菌外膜的一种重要修饰的遗传学和分子控制，这种修饰有助于淋球菌抵抗某些天然宿主防御介质(补体和阳离子抗菌肽[cAMP])的能力。这种修饰包括通过一种名为LPTA的酶将磷乙醇胺(PEA)附着到脂寡糖的脂A上。在过去的资助期间，我们发现编码LPTA的基因(LPTA)的零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，并降低了雌性小鼠下生殖道实验感染期间的细菌适应性。此外，我们发现了一个多层的、集成的和复杂的调控系统，它调节LPTA的表达，结果是，PEA修饰类脂A，淋球菌对cAMP的易感性，淋球菌在体内的适合性和炎症。该系统是本次续费申请的重点。整个调控系统包括LPTA表达操纵子中的多个启动子，LPTA编码序列中可导致LPTA高频截断的相位可变的多核苷酸重复序列，以及称为MISR的DNA结合蛋白的基因激活能力。在所要求的供资期间，将确定这些监管程序，并确定其对营地抵抗的影响。具体目标1中包含的研究将定义和表征用于LPTA表达的启动子、它们的不同使用以及对亚致死水平的抗菌剂的反应。调节这些启动子使用的转录因子将被识别，以便了解LPTA表达是如何在转录水平上被控制的。此外，LPTA的阶段变量生产将在实验室维护的和最近的临床菌株中定义。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，以及这一控制系统如何影响淋球菌对cAMP的耐药性和独特的类脂A结构的产生。这项拟议工作的成功完成将极大地提高我们对淋球菌如何通过使用基因控制系统逃避宿主防御和调节促炎反应的知识。重要的是，这一结果也应该为未来开发可用于治疗淋病的新型抗菌剂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.