: 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. ? ? ?
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