Gonococcal Infection and Gene Expression in Female Mice

描述（由申请人提供）：淋病奈瑟菌逃避女性生殖道先天防御的能力被假设为多因素和复杂的。抗氧化因子可以保护淋球菌不被吞噬细胞产生的活性氧杀死。唾液转移酶（Lst）和其他因子促进补体介导的防御逃避。这些因素保护淋球菌免受先天宿主防御逃避的证据主要基于体外试验。在第一个奖项的支持下，我们开发了第一个可重复的淋球菌性生殖道感染小动物模型。这个模型为我们提供了一个有价值的和独特的研究工具来测试淋球菌与宿主先天防御的相互作用。为了满足体内研究的需要，假设有助于逃避PMN和补体介导的杀伤，在这里，我们将i)测量已知的淋病奈瑟菌抗氧化防御（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）在保护人类PMN的杀伤和在实验小鼠生殖道感染期间的生存中的相对贡献。我们将构建假设直接防御氧化应激的基因（kat, ccp, mntC, msrA）的单突变体和双突变体，并测试它们在人类和小鼠PMNs的促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；ii.)确定淋球菌唾液酰基转移酶在抵抗小鼠PMN的抗噬细胞杀伤和提高淋病奈瑟菌在小鼠下生殖道中的存活方面的作用。我们将确定lst缺乏的淋球菌对PMN杀伤是否更敏感，原因是吸收增加或诱导更强的呼吸爆发。我们将使用C3和c4缺陷小鼠和nadph缺陷小鼠来测试PMN杀死试验的预测结果。确定观察到的厌氧生长的淋病奈瑟菌对雌二醇处理小鼠的感染性增加和对正常人血清杀菌活性增加的抵抗力的基础。我们将测试DNA微阵列技术鉴定的可能增加体内存活率的基因突变，以确定它们是否与厌氧诱导的感染性增加有关。我们将通过在小鼠中测试厌氧诱导的亚硝酸盐还原酶（AniA）突变体的感染性，以及利用AniA特异性抗血清阻断厌氧生长的淋球菌与补体之间的相互作用，来评估AniA在赋予体内优势和增加血清抗性方面的作用。

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.