Molecular genetics of the gonococcus

描述（由申请人提供）：革兰氏阴性细菌淋病奈瑟菌（淋球菌，Gc）是性传播疾病淋病的唯一病原体。作为一种人类特有的生物体，Gc不暴露于紫外线，也没有SOS系统。该提案先前已获得资助，以研究Gc RecA蛋白介导该病原体中重组和修复的机制，并确定Gc RecA与E. coliRecA酶。在过去的资助期间，我们已经表明，一个被低估的E。大肠杆菌蛋白RecX能与大肠杆菌结合。coli RecA，并通过产生RecA-DNA细丝的非活性形式来抑制SOS响应期间的链交换和辅助蛋白酶活性。我们还表明，Gc recX突变体减少了菌毛蛋白抗原变异、DNA转化和DNA修复的RecA依赖性过程，这表明，与大肠杆菌相比，coli RecX中，Gc RecX对Gc RecA的活性有正向调节作用。在下一个授予期间，我们将通过分析Gc RecX对体外Gc RecA活性的影响来确定Gc RecX刺激RecA依赖性过程的机制，以确定在没有SOS反应的情况下如何控制病原体中的重组和修复。 淋病症状的标志是主要由多形核白细胞（PMNs）组成的脓性渗出物。我们推测淋球菌最常见的损伤是中性粒细胞的氧化爆发。我们已经使用了泛奈瑟氏球菌微阵列来编目对过氧化氢处理的转录反应，并观察到79个基因在暴露于H2 O2后显著上调-包括预测的抗氧化剂，转录因子，铁调节基因，假设的ORF，伴侣蛋白等。在此期间，我们将进行额外的微阵列实验，以建立过氧化物诱导的基因表达的时间顺序。通过灭活预测的转录因子，我们可以使用微阵列来定义氧化损伤的全局调节回路。通过突变其他选定的过氧化物反应基因，我们将确定哪些上调基因编码的重要因素，保护Gc氧化应激。最后，我们将测试Gc突变体在人PMNs存在下对Gc存活的影响，以及对PMNs杀伤机制的影响。这项工作将有助于确定Gc是如何演变成破坏先天免疫反应并允许在感染人群中继续传播的。

DESCRIPTION (provided by the applicant): The Gram-negative bacterium Neisseria gonorrhoeae (the gonococcus, Gc) is the only causative agent of the sexually-transmitted disease gonorrhea. As a human-specific organism, Gc are not exposed to UV light and do not have an SOS system. This proposal was previously funded to examine the mechanisms used by the Gc RecA protein to mediate recombination and repair in this pathogen and to determine how Gc RecA differs from the E. coli RecA enzyme. In the past funding period, we have shown that an under-appreciated E. coli protein, RecX, binds E. coli RecA and inhibits its strand exchange and co-protease activities during the SOS response by creating an inactive form of the RecA-DNA filament. We have also shown that a Gc recX mutant is reduced for the RecA-dependent processes of pilin antigenic variation, DNA transformation, and DNA repair suggesting that, in contrast to E. coli RecX, Gc RecX has a positive role in regulating Gc RecA activity. During this next granting period, we will determine the mechanism by which the Gc RecX stimulates RecA-dependent processes by analyzing the effect of Gc RecX on Gc RecA activity in vitro to define how recombination and repair is controlled in a pathogen without an SOS response. The hallmark of symptomatic gonorrhea is the purulent exudate comprised mainly of polymorphonuclear cells (PMNs). We presume that the most common insult encountered by gonococci is the oxidative burst of PMNs. We have used a pan-Neisseria microarray to catalog the transcriptional response to hydrogen peroxide treatment, and have observed 79 genes that are significantly up-regulated after exposure to H2O2--including predicted antioxidants, transcription factors, iron-regulated genes, hypothetical ORFs, chaperones, and others. In this granting period, we will perform additional microarray experiments to establish the temporal order of peroxide-induced gene expression. By inactivating predicted transcription factors, we can then use microarrays to define the global regulatory circuits for oxidative damage. By mutating other selected peroxide-responsive genes, we will determine which of the up-regulated genes encode factors important for protecting Gc from oxidative stress. Finally, we will test Gc mutants for an effect on Gc survival in the presence of human PMNs, and for an effect on the killing mechanism of PMNs. This work will help establish how Gc has evolved to subvert the innate immune response and allow continued transmission within infected populations.