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

项目总结

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.