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

项目摘要 阴道毛滴虫是一种单细胞，能动的原生动物寄生虫，负责第三个最常见的 性传播感染在美国和世界各地。而T.迷走神经感染是 无症状，感染的症状可以从阴道炎和泡沫分泌物，男性和女性， 不孕症、早产、恶性宫颈癌发病率增加以及艾滋病毒传播增加。T. 迷走神经炎最近在美国被列为一种被忽视的感染，因为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，或是否建立免疫记忆 感染后。抗药性的增加和疫苗的缺乏要求对寄生虫-宿主进行进一步的研究 相互作用：具体而言，对寄生虫的有效免疫需要什么。很久以前就知道免疫- 一种叫做中性粒细胞的细胞对T细胞的免疫清除至关重要。流浪，然而，直到最近， 发现中性粒细胞使用一种以前未知的抗菌机制，称为胞间刺胞（trogo= to 蚕食）来杀死这种相对较大的、能动的病原体。T.流浪汉被发现是一个 接触依赖性过程，其中中性粒细胞包围寄生虫，并内化多个片段 （“bites”）of T.寄生虫死亡前的迷走神经然而，介导中性粒细胞-寄生虫细胞的分子- 细胞接触引发胞啃作用的机制尚不清楚，中性粒细胞“蚕食”的细胞机制也不清楚。 并降解寄生虫“叮咬”，导致寄生虫死亡。由于中性粒细胞需要人血清 T.因此，我们假设人血清因子与寄生虫表面抗原交联， 嗜中性粒细胞上的免疫受体，以建立细胞-细胞接触并启动胞啃作用。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中 介导啃噬界面，而寄生虫“叮咬”的溶酶体降解是维持 啃噬以杀死寄生虫我们将使用一系列遗传功能丧失来测试这些假设 实验中，使用CRISPR-Cas9功能性删除细胞系（HL-60 s）中的候选基因，该细胞系是 中性粒细胞的发育前体，然后将细胞分化为嗜中性粒细胞样细胞，以进行功能性 试验.我们的初步数据表明，HL-60细胞是一个合适的和易处理的模型，用于研究的trogocytickilling T.流浪汉我们还将在溶酶体标记物存在的情况下进行成像实验， 在溶酶体抑制剂存在下的胞啃试验。总之，这些研究将概述 人类免疫细胞用来有效清除T细胞的亚细胞和分子机制。迷走神经， 这些信息对疫苗设计非常重要。此外，这些研究将有助于 关于新型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.