Gonococcal Nuclease Mediated Escape from Neutrophil Extracellular Traps

描述(申请人提供)：淋病奈瑟氏菌(GC)引起的性传播感染淋病，第二流行的报告细菌感染在美国。由于GC对多种抗生素的耐药性，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菌株。急性淋病的临床特征是中性粒细胞聚集到感染部位。虽然中性粒细胞拥有多种抗菌剂，但GC在中性粒细胞存在的情况下仍能存活。我的实验室正在研究GC用来抵抗中性粒细胞清除的细胞和分子机制。中性粒细胞清除胞外细菌的一种方法是释放中性粒细胞胞外陷阱(Net)。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。我们检测到当原代人类中性粒细胞感染nuc突变体GC时，与亲本或nuc补体GC相比，其网络完整性增加。中性粒细胞作用后，nuc突变菌数减少，外源DNA酶的加入可提高nuc突变菌的回收率。根据这些观察，我们假设GC Nuc是一种新的毒力因子，使GC能够逃脱Net。在这项提案中，我们将调查GC Nuc如何促进Net中的细菌回收。在具体目标1中，我们将测试重组Nuc以及表达Nuc的突变株GC及其上清液降解原代人中性粒细胞释放的净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Net，以及它们是否容易受到Nuc活动的影响。特定目标2将研究Nuc的表达如何使GC逃脱Net。我们将确定Nuc的表达如何影响GC与体外和人类形成的网络的共存。我们将使用读取单个细菌活性的染料来确定Net是否对GC具有杀菌作用，以及Nuc的表达如何影响GC的细胞外活性。这项建议中概述的实验将揭示Nuc促进GC发病的机制。通过揭示细胞外GC如何利用Nuc来抵抗天然免疫系统的清除，这些研究可能会突出Nuc作为治疗耐多药淋病的新靶点。

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.