Gonococcal Infection and Gene Expression in Female Mice

描述(申请人提供)：淋病奈瑟菌逃避女性生殖道先天防御的能力被认为是多因素和复杂的。抗氧化剂可以保护淋球菌不被吞噬细胞产生的活性氧类杀死。唾液酸转移酶(LST)和其他因素促进补体介导的防御逃避。这些因子保护淋球菌免受天然宿主防御系统逃避的证据主要基于体外检测。在一等奖的支持下，我们建立了首个可复制的淋球菌生殖道感染的小动物模型。这一模型为我们提供了一种有价值和独特的研究工具来测试淋球菌与宿主天然防御的相互作用。为了满足体内研究假想的有助于逃避PMN和补体介导的杀伤的因素的需要，我们在这里将I。在实验小鼠生殖道感染期间，测量已知的淋球菌的抗氧化防御系统(过氧化氢酶、细胞色素C过氧化物酶、锰摄取、蛋氨酸亚砜还原酶)在防止人类中性粒细胞死亡和存活方面的相对贡献。我们将在假设直接防御氧化应激的基因(Kat、CCP、mntC、MSRA)中构建单突变和双突变，并测试它们在人和鼠中性粒细胞吞噬细胞杀伤中存活的能力，以及感染正常小鼠和NADPH氧化酶缺陷小鼠的能力；2.明确淋球菌唾液酸基转移酶在赋予小鼠对嗜中性粒细胞吞噬细胞杀伤的抵抗力和提高淋球菌在小鼠下生殖道中的存活率中的作用我们将确定LST缺陷的淋球菌是由于摄取增加还是由于诱导更强的呼吸爆发而对PMN杀伤更敏感。我们将利用C3和C4缺陷小鼠和NADPH缺陷小鼠来测试PMN杀伤试验做出的预测，III。)确定观察到的厌氧培养的淋球菌对雌二醇处理的小鼠的感染性增加以及对正常人类血清杀菌活性增强的基础。我们将测试DNA微阵列技术确定的可能增加体内存活率的基因中的突变，以确定它们是否与厌氧诱导的感染性增加有关。我们将评估厌氧诱导的亚硝酸还原酶(ANIA)在体内赋予优势和增加血清抵抗力方面的作用，方法是测试ANIA突变体在小鼠中的传染性，并利用ANIA特异性抗血清来阻断厌氧生长的淋球菌和补体之间的相互作用。

DESCRIPTION (provided by applicant): The capacity of N. gonorrhoeae to evade innate defenses in the female genital tract is hypothesized to be multifactorial and complex. Antioxidant factors may protect gonococci from killing by reactive oxygen species produced by phagocytes. Sialyltranferase (Lst) and other factors promote evasion of complement-mediated defenses. Evidence that these factors protect gonococci against evasion of innate host defenses is based primarily on in vitro assays. With the support of the first award, we developed the first reproducible small animal model of gonococcal genital tract infection. This model provides us with a valuable and unique research tool to test gonococcal interactions with host innate defenses. To satisfy the need for in vivo studies on factors hypothesized to contribute to evasion of PMN and complement-mediated killing, here we will i.) measure the relative contribution of the known antioxidant defenses of N. gonorrhoeae (catalase, cytochrome C peroxidase, manganese uptake, methionine sulfoxide reductase) in protection from killing by human PMNs and in survival during experimental murine genital tract infection. We will construct single and double mutants in genes hypothesized to directly defend against oxidative stress (kat, ccp, mntC, msrA), and test their capacity to survive opsonophagocytic killing by human and murine PMNs, and to infect normal mice and NADPH oxidase-deficient mice; ii.) define the role of gonococcal sialyltransferase in conferring resistance to opsonophagocytic killing by murine PMNs and in enhancing survival of N. gonorrhoeae in the murine lower genital tract We will determine if Lst-deficient gonococci are more senstive to PMN killing due to increased uptake or the induction of a stronger respiratory burst. We will utilize C3 and C4-deficient mice and NADPH-deficient mice to test predictions made from PMN killing assays, iii.) Determine the basis for the observed increased infectivity of anaerobically grown N. gonorrhoeae for estradiol-treated mice and for increased resistance to the bactericidal activity of normal human serum. We will test mutants in genes that may confer increased survival in vivo as identified by DNA microarray technology to see if they are responsible for anaerobically-induced increased infectivity. We will assess the role of anaerobically induced nitrite reductase (AniA) in conferring an advantage in vivo and increased resistance to serum by testing the infectivity of an aniA mutant in mice, and by utilizing AniA-specific antiserum to block interactions between anaerobically grown gonococci and complement.