Gonococcal Nuclease Mediated Escape from Neutrophil Extracellular Traps

淋球菌核酸酶介导中性粒细胞胞外陷阱的逃逸

基本信息

批准号：
8680531
负责人：
Alison K Criss
金额：
$ 23.7万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-05 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8680531
关键词：
Acute Affect Antibiotics Antibodies Bacteria Bacterial Infections Cells Chromatin Clinical Complement DNA Deoxyribonucleases Drug resistance Dyes Exposure to Exudate Fluorescent Dyes Future Gonorrhea Gram-Positive Bacteria Histones Homologous Gene Human Immune system Individual Infection Infection prevention Inflammation Laboratories Leukocyte Elastase Measures Mediating Microbial Biofilms Micrococcal Nuclease Molecular Morbidity - disease rate Multi-Drug Resistance Neisseria gonorrhoeae Neutrophil Infiltration Parents Pathogenesis Phagocytosis Phorbol Esters Physiological Production Property Proteins Public Health Reactive Oxygen Species Reading Recombinants Recovery Reporting Research Resistance Sexually Transmitted Diseases Site Staphylococcus aureus Structure Surface Testing Therapeutic Therapeutic Intervention United States Vaccines Vertebral column Virulence Factors antimicrobial bactericide base chemokine combat efflux pump extracellular fighting in vivo killings microorganism mutant neutrophil novel novel therapeutic intervention nuclease public health relevance research study scaffold therapy development

项目摘要

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.

描述（由申请人提供）：淋病奈瑟氏菌（GC）引起性传播感染淋病，这是美国第二普遍可报告的细菌感染。由于GC对多种抗生素的耐药性，淋病仍然是一个重大的公共卫生问题，并且在2011年发现了完全抗药性的分离株。急性淋病的临床标志是在感染部位募集中性粒细胞。尽管中性粒细胞具有多种抗菌疗法，但GC在存在中性粒细胞的情况下生存。我的实验室正在研究GC用来抵抗嗜中性粒细胞清除率的细胞和分子机制。中性粒细胞用来清除细胞外细菌的一种方法是中性粒细胞外陷阱（NETS）的释放。网由染色质与相关的阳离子抗菌蛋白组成。我们的研究小组发现，GC将具有同源性的核酸酶分泌为金黄色葡萄球菌热核酸葡萄球菌，称为NUC。与母体或NUC补体GC相比，当原代人中嗜中性粒细胞感染了原代人性中性粒细胞时，我们发现净完整性增加。暴露于嗜中性粒细胞后，回收了较少的Nuc突变细菌，并通过添加源性DNase增强了NUC突变体的恢复。从这些观察结果中，我们假设GC NUC是一种新型的毒力因子，使GC能够逃脱网。在此提案中，我们将研究GC NUC如何增强网络中的细菌恢复。在特定的目标1中，我们将测试重组NUC，表达NUC的VS. NUC突变GC及其上清液的能力，以降低由原代人性中性粒细胞释放的净DNA。我们还将检查急性淋病个体的渗出液中是否存在网，以及它们是否容易受到NUC活性的影响。特定的目标2将检查NUC的表达如何允许GC逃脱网。我们将确定NUC表达如何影响GC的GC共定位与在体内和人类中形成的网络。我们将使用读取单个细菌的生存能力来确定网络是否为GC的杀菌性以及NUC表达如何影响GC细胞外活力的染料。该提案中概述的实验将撤销NUC促进GC发病机理的机制。通过揭示细胞外GC如何使用NUC来抵抗先天免疫系统的清除，这些研究可能会突出核心作为针对多药耐药性淋病的治疗干预的新靶标。