Survival of Neisseria gonorrhoeae after primary human neutrophil challenge

描述（由申请人提供）：淋病奈瑟菌（Gc）是一种专性人类细菌病原体，是性传播感染淋病的原因。Gc感染驱动中性粒细胞（pmn）的募集和产生一种特征性的化脓性渗出物。虽然pmn产生多种抗菌化合物，但淋病渗出液含有活的和传染性的Gc。这一观察结果表明，Gc已经进化出逃避PMN清除的机制，这有助于Gc在宿主内的持久性和传播以及向新个体的传播。迄今为止，Gc在pmn存在下生存的机制尚不清楚。确定这些机制可能会突出可用于治疗淋病的新疗法的靶点，鉴于耐多药Gc猖獗的事实，迫切需要这些靶点。研究Gc对pmn的耐药性一直受到阻碍，因为Gc不会自然感染除人类以外的动物，而且pmn衍生的细胞系无法产生许多抗菌蛋白（APs）。此外，悬液中人体pmn的Gc感染不能准确反映急性淋病中pmn的粘附能力，趋化因子启动状态。因此，我开发了一种体外系统，用于检测暴露于附着的、白细胞介素8处理的原代人PMNs后的Gc存活。在该系统中，高达70%的Gc在初始PMN暴露中存活，此后可存活的Gc数量增加。我提出了两个非排他的假设来解释Gc如何在PMN挑战中幸存下来，并且在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固有地抵抗PMN ap。其次，Gc主动破坏pmn释放ap。在这个应用中，我们将扩展我们的初步观察，以确定和表征支持Gc在PMN挑战后存活的细胞和分子机制。目的1将研究Gc感染后活性AP从颗粒中释放到吞噬体或PMN表面，以及调节AP释放如何影响Gc在PMN内部和附着的存活。我们将检测人类淋病渗出液中感染的pmn，直接测量体内与pmn相关的Gc活力以及细菌活力与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方法来鉴定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Gc IV型菌毛和不透明相关（Opa）蛋白，它们在PMN攻击后因未知原因影响Gc存活，以及已知的保护Gc免受纯化APs攻击的基因产物。综上所述，该应用的结果将揭示Gc在抗微生物pmn存在下使用的多种方法，这确保了淋病在人群中的持续存在。

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.