Gonococcal Nuclease Mediated Escape from Neutrophil Extracellular Traps

描述（由申请人提供）：淋病奈瑟菌（Gc）引起性传播感染淋病，淋病是美国第二常见的可报告细菌感染。由于Gc对多种抗生素具有耐药性，淋病仍然是一个突出的公共卫生问题，2011年发现了完全耐药的分离株。急性淋病的临床特征是中性粒细胞聚集到感染部位。虽然中性粒细胞具有多种抗菌武器库，但Gc在中性粒细胞存在下存活。我的实验室正在研究Gc抵抗中性粒细胞清除的细胞和分子机制。中性粒细胞清除细胞外细菌的一种方法是释放中性粒细胞细胞外陷阱（NETs）。net由染色质和相关的阳离子抗菌蛋白组成。我们的研究小组发现Gc分泌一种与金黄色葡萄球菌热核酸酶同源的核酸酶，称为Nuc。我们发现，与亲本Gc或nuc补体Gc相比，当原代人中性粒细胞感染nuc突变Gc时，NET完整性增加。中性粒细胞暴露后，nuc突变菌的恢复较少，添加外源dna酶可以增强nuc突变菌的恢复。根据这些观察结果，我们假设Gc Nuc是一种新的毒力因子，使Gc能够逃离net。在这个提议中，我们将研究Gc Nuc如何提高细菌从net中的回收率。在Specific Aim 1中，我们将测试重组Nuc、表达Nuc的Gc与Nuc突变体Gc及其上清液降解人中性粒细胞释放的NET DNA的能力。我们还将检查急性淋病患者的渗出液中是否存在NETs，以及它们是否易受Nuc活性的影响。具体目标2将研究Nuc的表达如何允许Gc逃避net。我们将确定Nuc表达如何影响Gc与体外和人体形成的NETs共定位。我们将使用读出单个细菌活力的染料来确定NETs是否对Gc具有杀菌作用，以及Nuc表达如何影响Gc细胞外活力。本实验将揭示Nuc在Gc发病机制中的作用机制。通过揭示细胞外Gc如何利用Nuc抵抗先天免疫系统的清除，这些研究可能突出了Nuc作为治疗干预多药耐药淋病的新靶点。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Gc) causes the sexually transmitted infection gonorrhea, the second-most prevalent reportable bacterial infection in the United States. Gonorrhea remains a prominent public health problem due to Gc resistance to multiple antibiotics, and completely drug-resistant isolates were identified in 2011. The clinical hallmark of acute gonorrhea is the recruitment of neutrophils to the site of infection. Although neutrophils possess a diverse antimicrobial arsenal, Gc survives in the presence of neutrophils. My laboratory is investigating the cellular and molecular mechanisms used by Gc to resist neutrophil clearance. One approach used by neutrophils to clear extracellular bacteria is the release of neutrophil extracellular traps (NETs). NETs consist of chromatin with associated cationic antimicrobial proteins. Our research team has found that Gc secretes a nuclease with homology to Staphylococcus aureus thermonuclease, called Nuc. We detected increased NET integrity when primary human neutrophils were infected with nuc mutant Gc, compared with parent or nuc complement Gc. Fewer nuc mutant bacteria were recovered after exposure to neutrophils, and nuc mutant recovery was enhanced by adding exogenous DNase. From these observations, we hypothesize that Gc Nuc is a novel virulence factor that enables Gc to escape NETs. In this proposal, we will investigate how Gc Nuc enhances bacterial recovery from NETs. In Specific Aim 1, we will test the ability of recombinant Nuc, as well as Nuc-expressing vs. nuc mutant Gc and their supernatants, to degrade NET DNA released by primary human neutrophils. We will also examine if NETs are present in exudates from individuals with acute gonorrhea and if they are susceptible to Nuc activity. Specific Aim 2 will examine how expression of Nuc allows Gc to escape NETs. We will determine how Nuc expression affects Gc colocalization with NETs formed ex vivo and in humans. We will use dyes that read out the viability of individual bacteria to determine if NETs are bactericidal for Gc, and how Nuc expression affects Gc extracellular viability. The experiments outlined in this proposal will revea the mechanism by which Nuc contributes to Gc pathogenesis. By revealing how extracellular Gc uses Nuc to resist clearance by the innate immune system, these studies may highlight Nuc as a new target for therapeutic intervention against multidrug-resistant gonorrhea.