Survival of Neisseria gonorrhoeae after primary human neutrophil challenge

描述(申请人提供)：淋病奈瑟氏菌(GC)是人类的一种主要细菌病原体，也是性传播感染淋病的原因。GC感染促使中性粒细胞(PMN)募集，并产生典型的化脓性渗出物。虽然中性粒细胞产生各种各样的抗微生物化合物，但淋病渗出物中含有具有活性和传染性的GC。这一观察结果表明，GC已经进化出逃避PMN清除的机制，这促进了GC的持久性和在宿主内的传播，并传播给新的个体。到目前为止，GC用来在PMN存在下生存的机制还知之甚少。识别这些机制可能会突出可用于治疗淋病的新疗法的靶点，鉴于多药耐药GC猖獗的事实，淋病是迫切需要的。由于GC不会自然感染人类以外的动物，以及PMN来源的细胞株不能产生许多抗微生物蛋白(AP)，阻碍了GC对PMN耐药性的研究。此外，悬液中人中性粒细胞的GC感染不能准确反映急性淋病中中性粒细胞的黏附能力和趋化因子启动状态。因此，我开发了一个体外系统，用于检测贴壁的、经白细胞介素8处理的原代人中性粒细胞暴露后GC的存活率。在这个系统中，高达70%的GC在最初的PMN暴露中存活下来，此后存活的GC数量增加。我提出了两个非排他性假设来解释GC如何在PMN挑战中幸存下来，在K99/R00奖支持的研究中，我获得了支持这两个假设的证据。首先，GC对PMN AP具有固有的抵抗力。第二，GC主动颠覆PMN释放AP。在这一应用中，我们将扩展我们的初步观察，以确定和表征支持GC在PMN攻击后存活的细胞和分子机制。目的1研究GC感染后活性AP从颗粒释放到吞噬小体或在PMN表面，以及调节AP释放如何影响GC在PMN内和附着在PMN上的存活。我们将检测人类淋病分泌物中感染的PMN，以直接测量与PMN相关的GC在体内的活性，以及细菌活性和颗粒释放之间的关系。在目标2中，我们将结合靶向和全基因组的方法来识别在PMN暴露后保护GC的细菌基因产物，并确定保护发生的机制。这些基因产物包括GCIV型菌毛和不透明相关(OpA)蛋白，它们在未知原因的PMN攻击后影响GC的存活，以及已知的保护GC免受纯化AP攻击的基因产物。综上所述，这项应用的结果将揭示GC在抗菌剂PMN存在的情况下使用的各种生存方法，这确保了淋病在人类群体中的持续存在。

DESCRIPTION (provided by applicant): Neisseria gonorrhoeae (Gc) is an obligate human bacterial pathogen and the cause of the sexually transmitted infection gonorrhea. Infection with Gc drives the recruitment of neutrophils (PMNs) and production of a characteristic purulent exudate. Although PMNs produce a diverse array of antimicrobial compounds, gonorrheal exudates contain viable and infectious Gc. This observation indicates that Gc has evolved mechanisms to evade PMN clearance, which facilitate Gc persistence and dissemination within a host and transmission to new individuals. To date, the mechanisms used by Gc to survive in the presence of PMNs are poorly understood. Identifying these mechanisms may highlight targets that can be exploited for new therapies to treat gonorrhea, which are urgently needed given the fact that multidrug-resistant Gc is rampant. Investigating Gc resistance to PMNs has been hampered by the fact that Gc does not naturally infect animals other than humans and by the inability of PMN-derived cell lines to make many antimicrobial proteins (APs). Furthermore, Gc infection of human PMNs in suspension does not accurately reflect the adherence- competent, chemokine-primed state of PMNs in acute gonorrhea. Therefore, I developed an in vitro system for examining Gc survival after exposure to attached, interleukin-8 treated, primary human PMNs. In this system, up to 70% of Gc survives initial PMN exposure, with numbers of viable Gc increasing thereafter. I proposed two nonexclusive hypotheses to explain how Gc survives PMN challenge, and in research supported by a K99/R00 award, I gained evidence in support of both. First, Gc is inherently resistant to PMN APs. Second, Gc actively subverts PMNs from releasing APs. In this application, we will extend our preliminary observations in order to identify and characterize the cellular and molecular mechanisms that support Gc survival after PMN challenge. Aim 1 will investigate the release of active APs from granules into phagosomes or at the PMN surface after infection with Gc, and how modulating AP release affects the survival of Gc inside and attached to PMNs. We will examine infected PMNs from human gonorrheal exudates to directly measure the viability of Gc associated with PMNs in vivo and the relationship between bacterial viability and granule release. In Aim 2, we will combine targeted and genome-wide approaches to identify the bacterial gene products that protect Gc after PMN exposure and define the mechanisms by which protection occurs. These gene products include Gc type IV pili and opacity-associated (Opa) proteins, which affect Gc survival after PMN challenge for unknown reasons, as well as gene products known to defend Gc from purified APs. Taken together, the results from this application will reveal the diverse approaches used by Gc to survive in the presence of antimicrobial PMNs, which ensures the continued persistence of gonorrhea within the human population.