Molecular genetics of the gonococcus

描述（由申请人提供）：革兰氏阴性菌淋病奈瑟菌（淋球菌，Gc）是淋病的唯一病原体。作为一种人类特有的生物，Gc不会暴露在紫外线下，也没有SOS系统。该提案先前获得资助，旨在研究Gc RecA蛋白介导该病原体重组和修复的机制，并确定Gc RecA与大肠杆菌RecA酶的区别。在过去的资助期内，我们已经证明了一种被低估的大肠杆菌蛋白RecX与大肠杆菌RecA结合，并通过产生一种无活性形式的RecA- dna丝，在SOS反应期间抑制其链交换和共蛋白酶活性。我们还发现，Gc recX突变体在依赖RecA的匹林抗原变异、DNA转化和DNA修复过程中减少，这表明，与大肠杆菌recX相比，Gc recX在调节Gc RecA活性方面具有积极作用。在接下来的研究中，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依赖过程的机制，以确定在没有SOS反应的情况下病原体中如何控制重组和修复。

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.