Complement-independent role of C4 binding protein in gonococcal survival from human neutrophils

项目摘要 淋病奈瑟菌（Gc）是一种革兰氏阴性细菌，导致性传播疾病 淋病据估计，全世界每年有7800万淋病病例， 对所有推荐抗生素的耐药性，以及缺乏保护性疫苗，N.淋病是一个突出的 对人类健康的威胁越来越大。Gc感染的一个标志是中性粒细胞的流入，但这种炎性 在清除感染方面反应不成功。Gc上的相位可变不透明度相关（Opa）蛋白 中性粒细胞表面介导非调理吞噬作用。Opa蛋白如菌株OpaD的表达 FA 1090通过与癌胚抗原相关的吞噬受体结合来激活中性粒细胞。 细胞粘附分子（CEACAM）-1和CEACAM-3，在存在下显著降低Gc存活 体外人中性粒细胞。中性粒细胞对OpaD+ Gc的反应是产生活性氧， 有毒颗粒内容物细胞外，并内化细菌进入吞噬溶酶体，在那里它们被杀死 通过蛋白酶和抗菌肽。尽管在体外被中性粒细胞杀死，但表达Opa的Gc 在富含嗜中性粒细胞淋病渗出液中存活甚至占优势。作为一种解释， 不一致的发现，我们发现，与人血清的调理作用，这是发现在炎症 分泌，增强Opa+ Gc从原代人中性粒细胞的存活，并抑制中性粒细胞 氧化爆发通过离子交换色谱和质谱，我们鉴定了C4结合 蛋白质（C4 BP）作为血清组分，与Opa+ Gc结合以限制中性粒细胞活化并增加Gc 从中性粒细胞中存活。值得注意的是，这些作用独立于补体沉积或补体 裂解活性，提示C4 BP结合调节中性粒细胞功能的新机制。在这 建议，我们将通过定义分子相互作用来定义C4 BP如何介导Opa+ Gc的这些效应 C4 BP和Gc表面之间的联系以及限制人类中性粒细胞反应的细胞机制 至Opa+ Gc。这项工作的完成将揭示Opa+ Gc以一种以前未被认识的方式在植物中生存。 分泌物中富含嗜中性粒细胞，使其能够继续定居以及传播到新的宿主。与此 我们预测，干扰C4 BP与Gc结合的治疗方法的发展将增加 除了增强补体溶解活性之外，还增强了中性粒细胞抗淋球菌活性的功效，从而 改善淋病的预后

PROJECT SUMMARY Neisseria gonorrhoeae (Gc) is a Gram-negative bacterium that causes the sexually transmitted disease gonorrhea. With an estimated 78 million cases of gonorrhea annually worldwide, increasing frequency of resistance to all recommended antibiotics, and the lack of a protective vaccine, N. gonorrhoeae is a prominent and growing threat to human health. A hallmark of Gc infection is the influx of neutrophils, but this inflammatory response is unsuccessful in clearing infection. Phase-variable opacity-associated (Opa) proteins on the Gc surface mediate non-opsonic phagocytosis by neutrophils. Expression of Opa proteins like OpaD of strain FA1090, which activates neutrophils by binding to the phagocytic receptors carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-1 and CEACAM-3, significantly decreases Gc survival in the presence of human neutrophils in vitro. Neutrophils respond to OpaD+ Gc by generating reactive oxygen species, releasing toxic granule contents extracellularly, and internalizing the bacteria into a phagolysosome where they are killed by proteases and antimicrobial peptides. Despite being killed by neutrophils in vitro, Opa-expressing Gc survive and even predominate among Gc in neutrophil-rich gonorrheal exudates. As one explanation for these discordant findings, we have found that opsonization with human serum, which is found in inflammatory secretions, enhances Opa+ Gc survival from primary human neutrophils and suppresses the neutrophil oxidative burst. Through ion-exchange chromatography and mass spectrometry, we identified C4-binding protein (C4BP) as the serum component that binds to Opa+ Gc to limit neutrophil activation and increase Gc survival from neutrophils. Remarkably, these effects are independent of complement deposition or complement lytic activity, suggesting a novel mechanism by which binding of C4BP modulates neutrophil functions. In this proposal, we will define how C4BP mediates these effects on Opa+ Gc, by defining the molecular interactions between C4BP and the Gc surface and the cellular mechanisms that limit the response of human neutrophils to Opa+ Gc. Completion of this work will reveal a previously unappreciated way in which Opa+ Gc survives in neutrophil-rich secretions to enable its continued colonization as well as transmission to new hosts. With this knowledge, we predict that the development of therapies that interfere with C4BP binding to Gc will increase the efficacy of neutrophil antigonococcal activity, in addition to enhancing complement lytic activity, thereby improving outcomes of gonorrhea.