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感染促使中性粒细胞（PMN）的募集和特征性脓性渗出物的产生。虽然中性粒细胞产生各种各样的抗菌化合物，淋病渗出液含有活性和感染性的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在宿主中的持久性和传播以及向新个体的传播。迄今为止，Gc在中性粒细胞存在下生存的机制知之甚少。确定这些机制可能会突出可以用于治疗淋病的新疗法的目标，鉴于多药耐药Gc猖獗的事实，迫切需要这些目标。研究Gc对PMN的耐药性受到以下事实的阻碍：Gc不会自然感染除人类以外的动物，以及PMN衍生的细胞系无法产生许多抗菌蛋白（AP）。此外，悬浮液中的人PMNs的Gc感染不能准确地反映急性淋病中PMNs的粘附能力、趋化因子致敏状态。因此，我开发了一种体外系统，用于检查接触后，附着的，白细胞介素-8处理，原代人中性粒细胞的Gc生存。在这个系统中，高达70%的Gc存活初始PMN暴露，与可行的Gc的数量增加之后。我提出了两个非排他性的假设来解释Gc如何在PMN挑战中生存，在K99/R 00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。第二，Gc主动颠覆PMN释放AP。在本申请中，我们将扩展我们的初步观察，以确定和表征的细胞和分子机制，支持Gc的生存后，中性粒细胞的挑战。目的1将研究释放活性AP颗粒进入吞噬体或在PMN表面感染Gc后，以及如何调节AP释放影响Gc内的生存和附着到PMN。我们将检查感染的人淋病渗出液中的中性粒细胞，以直接测量体内与中性粒细胞相关的Gc的活力以及细菌活力和颗粒释放之间的关系。在目标2中，我们将结合联合收割机靶向和全基因组的方法，以确定细菌的基因产物，保护Gc后，中性粒细胞暴露，并确定保护发生的机制。这些基因产物包括Gc IV型皮利和不透明相关（Opa）蛋白，其在PMN激发后影响Gc存活，原因未知，以及已知保护Gc免受纯化AP的基因产物。总之，本申请的结果将揭示Gc在抗菌PMN存在下生存所使用的多种方法，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.