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

项目总结 淋球菌(GC)是引起性传播疾病的革兰氏阴性细菌 淋病。据估计，全世界每年有7800万淋病病例，越来越频繁的 对所有推荐的抗生素都有耐药性，而且缺乏保护性疫苗，淋病奈瑟菌是突出的 对人类健康的威胁与日俱增。GC感染的一个特征是中性粒细胞的涌入，但这种炎症性 在清除感染方面反应不成功。GC上的相位可变不透明相关(OpA)蛋白 表面通过中性粒细胞介导非调理吞噬作用。菌株Opad等OpA蛋白的表达 FA1090，它通过与吞噬细胞受体癌胚抗原相关的结合来激活中性粒细胞 细胞黏附分子(CEACAM)-1和CEACAM-3显著降低胃癌存活率 体外培养的人中性粒细胞。中性粒细胞对Opad GC的反应是通过产生活性氧物种，释放 有毒颗粒内容物在细胞外，并将细菌内化到吞噬溶酶体中，在那里它们被杀死 通过蛋白水解酶和抗菌肽。尽管在体外被中性粒细胞杀死，但表达OpA的GC 在富含中性粒细胞的淋病分泌物中存活，甚至在GC中占优势。作为对这些问题的一种解释 不一致的发现，我们发现人血清的调理作用，这是在炎症中发现的 分泌，增强原代人中性粒细胞的Opa GC存活并抑制中性粒细胞 氧化爆发。通过离子交换层析和质谱分析，我们鉴定了C4结合 蛋白质(C4BP)作为血清成分与OPA GC结合以限制中性粒细胞激活并增加GC 从中性粒细胞存活下来。值得注意的是，这些效应与补体沉积或补体无关。 溶血活性，提示了C4BP结合调节中性粒细胞功能的新机制。在这 提案中，我们将通过定义分子相互作用来定义C4BP如何在Opa GC中调节这些影响 C4BP和GC表面之间的关系以及限制中性粒细胞反应的细胞机制 致奥帕GC。这项工作的完成将揭示Opa GC以前不为人知的生存方式 富含中性粒细胞的分泌物，使其能够继续定居并传播给新的宿主。有了这个 我们预测，干扰C4BP与GC结合的治疗方法的开发将会增加 中性粒细胞抗淋球菌活性的有效性，除了增强补体溶解活性外，从而 改善淋病的转归。

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.