Survival of Neisseria gonorrhoeae after primary human neutrophil challenge

项目摘要/摘要 淋病奈瑟菌(GC)是人类的一种主要致病菌，也是淋病的病因。GC是一种 主要的公共卫生问题因为病例数量仍然很高，它已经获得了对多个 抗生素，不存在疫苗，感染与虚弱的终身负面后果有关，如 不孕症和异位妊娠。GC在粘膜的定植推动中性粒细胞的募集 白细胞(中性粒细胞或中性粒细胞)到感染部位并产生一种特征性的脓液 渗出物。尽管PMN具有强大的吞噬和抗菌活性，但感染性GC可以从 富含PMN的渗出物，表明GC具有逃避PMN杀伤的机制。PMN未能达到 清除GC不仅允许感染持续存在，而且还维持PMN的炎症反应，导致 导致不孕不育和其他后遗症的组织损伤。要确定GC用来抵抗 通过PMN的杀伤，我们开发了一种感染模型，使用贴壁的、经趋化因子处理的原代人PMN 接近中性粒细胞募集到粘膜表面的生理状态。我们用这个模型揭示了 GC在暴露于PMN后存活的两种主要方式。首先，GC表达的基因产物可以赋予 对中性粒细胞抗菌化合物，如蛋白酶和抗菌肽的耐药性。其次，GC各不相同 表达表面结构以调节中性粒细胞吞噬和随后暴露于 这些化合物。具体地说，不透明相关(OpA)蛋白表达时相变的GC 内化为未成熟的吞噬小体，在其中存活并抑制PMN的氧化爆发，而 表达与癌胚抗原相关细胞黏附分子相互作用的OpA蛋白的GC (CEACAMS)1和3被吞噬成成熟的、可降解的吞噬小体，并诱导有效的 氧化爆发。在这个更新方案中，我们将继续剖析GC生存的基础机制 在PMN挑战之后。目标1将定义OpA阴性GC是如何吞噬到PMN中的，而不会引发 氧化爆发或吞噬小体形成，并将评估表达OpA的GC的生存情况 不结合CEACAM3的蛋白质。我们将评估这些受体的长期后果- 介导的相互作用对GC和PMN活性的影响。目标2将以TN-SEQ的结果为基础 在上一个资助期进行的实验，以检查细菌外排泵和 脂寡糖变体有助于中性粒细胞来源的GC存活。所有实验都将使用 黏附性白介素8处理的中性粒细胞感染模型及对新鲜采集的人淋病的分析 渗出物。总而言之，这项研究的结果将揭示GC为生存而使用的不同方法 来自PMN，这确保了淋病在人类群体中的持续存在，即使在面临 中性粒细胞炎症反应。这项研究可以指出GC或PMN中可以利用的目标 寻找治疗抗药性淋病的新疗法，同时限制与PMN流入相关的宿主损害。

PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae (Gc) is an obligate human bacterial pathogen and the cause of gonorrhea. Gc is a major public health concern because numbers of cases remain high, it has gained resistance to multiple antibiotics, no vaccine exists, and infection is associated with debilitating, lifelong negative outcomes such as infertility and ectopic pregnancy. Mucosal colonization by Gc drives the recruitment of polymorphonuclear leukocytes (PMNs or neutrophils) to the site of infection and the production of a characteristic purulent exudate. Despite PMNs’ robust phagocytic and antimicrobial activities, infectious Gc can be cultured from PMN-rich exudates, implying Gc possesses mechanisms to evade killing by PMNs. The failure of PMNs to clear Gc not only allows the infection to persist, but also sustains the PMN inflammatory response, causing the tissue damage that underlies infertility and other sequelae. To identify the mechanisms used by Gc to resist killing by PMNs, we developed an infection model using adherent, chemokine-treated primary human PMNs to approximate the physiological state of PMNs recruited to mucosal surfaces. We used this model to uncover two major ways in which Gc survives after exposure to PMNs. First, Gc expresses gene products that confer resistance to PMN antimicrobial compounds, such as proteases and antimicrobial peptides. Second, Gc varies expression of surface structures to modulate the route of phagocytosis by PMNs and consequent exposure to these compounds. Specifically, Gc that have phase-varied off expression of opacity-associated (Opa) proteins is internalized into immature phagosomes in which it survives and suppresses the PMN oxidative burst, while Gc expressing an Opa protein that interacts with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) 1 and 3 is phagocytosed into a mature, degradative phagolysosome and induces a potent oxidative burst. In this renewal proposal, we will continue to dissect the mechanisms that underlie Gc survival after PMN challenge. Aim 1 will define how Opa-negative Gc is phagocytosed into PMNs without eliciting the oxidative burst or phagolyososome formation and will evaluate the survival profile of Gc expressing an Opa protein that does not bind CEACAM3. We will evaluate the long-term consequences of these receptor- mediated interactions on the viability of both Gc and PMNs. Aim 2 will build upon results from a Tn-seq experiment conducted in the previous funding period to examine how bacterial efflux pumps and lipooligosaccharide variants contribute to Gc survival from PMNs. All experiments will be conducted with the adherent, interleukin-8 treated PMN infection model alongside analysis of freshly collected human gonorrheal exudates. Together, the results from this research will reveal the diverse approaches used by Gc to survive from PMNs, which ensures the continued persistence of gonorrhea in the human population even when facing a neutrophilic inflammatory response. This research can point to targets in Gc or PMNs that can be exploited for new therapies to treat drug-resistant gonorrhea while limiting the host damage associated with PMN influx.