Determining the subcellular and molecular players in neutrophil trogocytic killing of the sexually-transmitted parasite Trichomonas vaginalis

项目总结 阴道毛滴虫是一种单细胞、能动的原生动物寄生虫，是第三常见的 性传播感染在美国和世界范围内。而大约一半的阴道毛滴虫感染是 无症状的感染症状可从阴道炎和泡沫分泌物，到男性和女性。 不孕不育、早产、恶性宫颈癌发病率增加以及艾滋病毒传播增加。T. 阴道肺炎最近在美国被归类为一种被忽视的感染，因为人们对其感染模式知之甚少。 发病机制，免疫系统如何清除寄生虫，或者是否建立了免疫记忆 在感染之后。不断增长的耐药性和疫苗的缺乏要求对寄生虫宿主进行进一步研究 相互作用：具体地说，对寄生虫的有效免疫需要什么。很久以前就知道免疫- 被称为中性粒细胞的细胞对于阴道毛滴虫的免疫清除是至关重要的，然而这只是最近才发生的。 发现中性粒细胞使用一种以前未知的抗微生物机制，称为巨噬细胞增多症(trogo=to 蚕食)，以杀死这种相对较大的、能动的病原体。阴道毛滴虫中性粒细胞巨噬细胞增多症 接触依赖的过程，中性粒细胞包围寄生虫，并内化多个片段 (“叮咬”)阴道毛滴虫寄生虫死亡前。然而，调节中性粒细胞-寄生虫细胞的分子- 启动巨噬细胞增多症的细胞接触尚不清楚，中性粒细胞用来“吞噬”的细胞机制也是未知的。 并降解寄生虫的“叮咬”，导致寄生虫死亡。因为中性粒细胞需要人血清 阴道毛滴虫滋养细胞增多症，我们假设人血清因子使寄生虫表面抗原与 中性粒细胞上的免疫受体，以建立细胞与细胞之间的联系，并启动巨噬细胞增多症。我们还假设 含有膜降解因子的中性粒细胞毒性颗粒被动员到中性粒细胞寄生虫上 介导性咬合界面，需要寄生虫“叮咬”的溶酶体降解才能持续 啃咬以杀死寄生虫。我们将使用一系列遗传功能丧失来检验这些假说 实验中，使用CRISPR-Cas9在一个细胞系(HL-60S)中功能性删除候选基因 中性粒细胞的发育前体，然后将细胞分化为中性粒细胞样细胞，以实现 测试。我们的初步数据表明，HL-60S是一种适合于研究巨噬细胞杀伤的简便模型。 阴道毛滴虫。我们还将在溶酶体标记物存在的情况下进行成像实验，并进行 在溶酶体抑制剂存在的情况下进行巨噬细胞增多症分析。总而言之，这些研究将概述 人类免疫细胞用来有效清除阴道毛滴虫的亚细胞和分子机制， 这些信息将对疫苗设计起到无价的指导作用。此外，这些研究将有助于 关于一种新的抗菌工艺的基础知识。

PROJECT SUMMARY Trichomonas vaginalis is a unicellular, motile protozoan parasite responsible for the 3rd- most common sexually-transmitted infection in the US and worldwide. While around half of T. vaginalis infections are asymptomatic, symptoms of the infection can range from vaginitis and frothy discharge, to male and female infertility, pre-terms births, increased incidences of malignant cervical cancers, and increased spread of HIV. T. vaginalis was recently classified as a neglected infection in the US, as very little is known about its modes of pathogenesis, how the immune system clears the parasite, or whether immunological memory is established following infection. Rising drug resistance and lack of a vaccine demand further research into the parasite- host interactions: specifically, what effective immunity to the parasite entails. It has long been known that immune- cells called neutrophils are crucial for immune- clearance of T. vaginalis, however it was only recently discovered that neutrophils use a previously unknown antimicrobial mechanism called trogocytosis (trogo= to nibble) to kill this relatively large, motile pathogen. Neutrophil trogocytosis of T. vaginalis was found to be a contact-dependent process, in which neutrophils surround the parasite, and internalize multiple fragments ("bites") of T. vaginalis prior to parasite death. However, the molecules that mediate neutrophil-parasite cell- cell contact to initiate trogocytosis are unknown, as are the cellular mechanisms that neutrophils use to "nibble" and degrade parasite "bites," causing death of the parasite. As human serum is required for neutrophil trogocytosis of T. vaginalis, we hypothesize that human serum factors crosslink parasite surface antigens to immune- receptors on neutrophils to establish cell-cell contact and initiate trogocytosis. We also hypothesize that neutrophil toxic granules containing membrane- degrading factors are mobilized to the neutrophil- parasite interface to mediate nibbling, and that lysosomal degradation of parasite "bites" is required for sustained nibbling to kill the parasite. We will test these hypotheses using a series of genetic loss- of- function experiments, using CRISPR-Cas9 to functionally delete candidate genes in a cell-line (HL-60s) that is a developmental precursor to neutrophils, and then differentiate the cells into neutrophil-like cells for functional tests. Our preliminary data show that HL-60s are a suitable and tractable model for studying trogocytic killing of T. vaginalis. We will also perform imaging experiments in the presence of lysosomal markers, and perform trogocytosis assays in the presence of lysosomal inhibitors. Altogether, these studies will outline the subcellular and molecular mechanisms that human- immune cells use to effectively clear T. vaginalis, information that will be invaluable in informing vaccine design. Furthermore, these studies will contribute foundational knowledge regarding a novel antimicrobial process.