Survival of Neisseria gonorrhoeae after primary human neutrophil challenge

项目总结/摘要 淋病奈瑟氏菌（Neisseria gonorrhoeae，Gc）是一种专性人类细菌病原体，是淋病的致病菌。Gc为 主要的公共卫生问题，因为病例数量仍然很高，它已经获得了耐多 抗生素，没有疫苗存在，感染与衰弱，终身的负面后果，如 不孕症和宫外孕。Gc引起的粘多糖定殖驱动多形核菌的募集 白细胞（中性粒细胞或中性粒细胞）感染的网站和生产的特征化脓性 渗出物。尽管中性粒细胞具有强大的吞噬和抗微生物活性，但感染性Gc可以从 富含PMN的渗出物，这意味着Gc具有逃避PMN杀伤的机制。PMNs的失败 透明的Gc不仅使感染持续，而且维持PMN炎症反应，引起炎症反应。 导致不孕和其他后遗症的组织损伤。为了确定Gc用来抵抗 我们用粘附的、趋化因子处理的原代人中性粒细胞建立了一个感染模型， 接近于被募集到粘膜表面的中性粒细胞的生理状态。我们用这个模型来揭示 Gc在暴露于PMNs后存活的两种主要方式。首先，Gc表达基因产物， 对PMN抗微生物化合物如蛋白酶和抗微生物肽的抗性。其次，Gc变化 表达表面结构，以调节PMN的吞噬途径，并随后暴露于 这些化合物。具体而言，具有阶段变化的不透明相关（Opa）蛋白表达的Gc 被内化到未成熟的吞噬体中，在其中存活并抑制PMN氧化爆发， 表达与癌胚抗原相关细胞粘附分子相互作用的Opa蛋白的Gc （CEACAMs）1和3被吞噬成成熟的、降解的吞噬溶酶体，并诱导有效的 氧化爆发在这个更新建议中，我们将继续剖析Gc生存的机制 PMN挑战后。目的1将定义Opa阴性Gc是如何被吞噬到PMN中而不引起细胞凋亡的。 并将评估表达Opa的Gc的存活概况 不结合CEACAM 3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的活力都有影响。目标2将建立在Tn-seq的结果基础上 在上一个资助期进行的实验，以研究细菌外排泵和 脂寡糖变体有助于Gc从PMN中存活。所有实验将使用 白细胞介素-8处理的粘附性中性粒细胞感染模型以及对新鲜采集的人淋病标本的分析 渗出物总之，这项研究的结果将揭示Gc为生存所使用的各种方法 从中性粒细胞，这确保了持续存在的淋病在人群中，即使面对 一种嗜酸性炎症反应这项研究可以指出Gc或PMNs中可以利用的靶点 寻找新的疗法来治疗耐药淋病，同时限制与PMN流入相关的宿主损伤。

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.