Effect of Quorum Sensing on N. gonorrhoeae infection of human PMN's

描述(由申请人提供)： 淋球菌(Ng)是一种典型的隐形病原体，它使用多种机制来逃避人类的免疫防御，因此再次感染是一个问题。这挫败了疫苗开发的尝试，同时抗菌素的抗药性使有效的治疗变得更加困难和昂贵。有必要采取新的方法来确定控制这种重要病原体的方法，这种病原体在全世界造成近6500万例病例，其中大多数发生在贫困人口和欠发达国家。我们实验室的研究表明，Ng具有在宫颈上皮细胞表面形成生物膜的能力。作为这些研究的一部分，我们一直在研究淋球菌LuxS在生物膜形成中的作用，并有证据表明，淋球菌有一个功能强大的群体感应系统，它用来调节生物膜形成过程中的基因表达。此外，我们的研究表明，Ng自动诱导因子-2(AI-2)与两个组分调控基因misRS相互作用。在MISRS控制的大约80个基因中，有淋球菌热核酸酶，它是重塑生物膜DNA基质的关键，以及参与肽聚糖生物合成和细胞分裂的基因。我们还观察到SIR-2脱乙酰酶的一个同源物和一个新的乙酰转移酶也受到MISRS的调控，在这些观察的基础上，我们进行了N-赖氨酸乙酰化的蛋白质组学研究，我们发现在一个Ng LuxS突变体中，N‘-赖氨酸乙酰化显著增加。许多研究表明，细菌群剂可以影响真核细胞中的信号转导。我们以前对金黄色葡萄球菌的研究表明，在吞噬体的范围内，自身诱导物的水平足够高，足以通过AGR系统引起基因表达的改变，并促进吞噬细胞中生存所必需的毒力因子的表达。淋病奈瑟菌可以在吞噬体内存活，并在中性粒细胞中诱导抗凋亡事件。淋球菌只有一个AI-2系统，缺乏产生酰基高丝氨酸内酯的酶。我们感兴趣的是研究淋球菌产生的AI-2和N‘-赖氨酸乙酰化是否在吞噬后的发病机制中发挥作用。我们将在这个建议中测试的假设是，一个完整的Ng群体感应系统在PMN吞噬体内发挥作用，是Ng在该环境中生存所必需的。我们将通过以下具体目标来解决这一假设： 特异性目的1：确定人PMN摄入Ng的群体感应系统的激活状态；特异性目标2：确定淋球菌群体感应系统的突变是否以及如何影响Ng在PMN中的存活和携带Ng的PMN的细胞生物学。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Ng) is a prototypic stealth pathogen that uses multiple mechanisms to evade human immune defenses; consequently re-infection is a problem. This has frustrated attempts at vaccine development, while resistance to antimicrobials has made effective therapy more difficult and costly. New approaches are necessary to identify means to control this important pathogen, which causes almost 65 million cases on a worldwide basis, the majority in indigent populations and in less well-developed countries. Studies in our laboratory have demonstrated that Ng has the ability to form biofilms on the cervical epithelial cell surface. As part of these studies, we have been examining the role of gonococcal luxS in biofilm formation and have evidence that the gonococcus has a functional quorum sensing system that it uses to modify gene expression during biofilm formation. Furthermore, our studies have shown that the Ng autoinducer -2 (AI-2) interacts with the two component regulatory genes, misRS. Among the approximately 80 genes under the control of misRS are the gonococcal thermonuclease that is critical to remodeling the biofilm DNA matrix, and genes involved in peptidoglycan biosynthesis and cell division. We also observed that a homolog of a SIR-2 deacetylase and a novel acetyltransferase are also regulated by misRS, and based on these observations, we undertook proteomic studies of N-lysine acetylation and we discovered that N¿-lysine acetylation increases significantly in a Ng luxS mutant. A number of studies have shown that bacterial quorum agents can impact on signaling in eukaryotic cells. Our previous studies with Staphylococcus aureus have shown that within the confines of the phagolysome, autoinducer levels are sufficiently high to cause modifications in gene expression through the agr system and promote expression of virulence factors integral to survival in phagocytes. N. gonorrhoeae can survive within the phagolysome and induces an anti-apoptotic event in neutrophils. The gonococcus only has an AI-2 system lacking the enzymes to produce acylhomoserine lactone. Our interest is to examine whether the AI-2 produced by N. gonorrhoeae and N¿-lysine acetylation might play a role in pathogenesis after phagocytosis. The hypothesis that we will test in this proposal is that an intact Ng quorum sensing system functions within the PMN phagolysome and is necessary for survival of Ng in that environment. We will resolve this hypothesis by the following specific aims: Specific Aim 1: Determine the state of activation of the quorum sensing system of Ng ingested by human PMN and Specific Aim 2: Determine if and how mutations in the gonococcal quorum sensing system affect both the survival of Ng in PMN and the cell biology of Ng-laden PMN.