TRICHOMONAS VAGINALIS AND SLPI MUCOSAL DEFENSES

这项拨款的长期目标是了解健康女性生殖道的粘膜环境，以了解其对阴道毛滴虫和其他性传播疾病（包括艾滋病毒感染）的非免疫保护机制，以及炎症反应。实验将重点研究分泌性白细胞蛋白酶抑制剂（SLPI）提供的保护机制，SLPI是一种在阴道上皮上发现的主要丝氨酸蛋白酶抑制剂，并确定阴道T.阴道蛋白酶（TVPs）对SLPI功能的有害影响。我们推测阴道毛滴虫（Trichomonas vaginalis， TV）的半胱氨酸蛋白酶可裂解分泌性白细胞蛋白酶抑制剂（SLPI）并破坏其粘膜抗菌防御功能。我们计划：1)测定SLPI对阴道毛滴虫（TV）、淋病奈瑟菌（GC）、沙眼衣原体（CT）等性病生物的正常抑菌功能。2)测定SLPI在不同肉汤生长和TV代谢条件下的抑菌功能，包括好氧和厌氧生长；不同浓度的游离铁、人体转铁蛋白和乳铁蛋白；以及是否存在半胱氨酸蛋白酶抑制剂E-64。3)分离鉴定导致SLPI降解的特定TVP活性。通过诱导上清的离子交换色谱分离三个tpv(220、92和68 kDa)，并通过SDS-PAGE和Western blot检测它们对SLPI的降解作用。4)产生的肽片段将通过气相微测序进行测序。TVP切割位点和肽将被映射到已知的序列数据和功能域。5)检测多肽的残留抗菌功能；6)测定青春期女性生殖道正常分泌物和感染分泌物中SLPI的水平。14-19岁的青春期女孩将被采集阴道液样本，并通过ELISA法测定SLPI水平。研究人员将对这些患者进行为期一年的纵向随访，并将SLPI水平与crispr乳杆菌携带率进行比较。本文阐明的机制将有助于我们了解阴道内稳态的天然机制，以及简单或复杂生殖道疾病感染期间的机制。这些发现也可能与其他粘膜性病病原体的感染机制有关，包括HIV感染和其他寄生虫病。

The long term objectives of this grant are to understand the mucosal environment of the healthy female genital tract in terms of its non-immune protective mechanisms against Trichomonas vaginalis and other STDs including HIV infection, and the inflammatory response. Experiments will focus on protective mechanisms provided by secretory leukocyte protease inhibitor (SLPI), a predominant serine protease inhibitor found on the vaginal epithelium and determine the deleterious effects of T. vaginalis proteases (TVPs) on SLPI function. We hypothesize that Trichomonas vaginalis (TV) cysteine proteases cleave secretory leukocyte protease inhibitor (SLPI) and impair its mucosal antimicrobial defense function. We plan to: 1) determine the normal antimicrobial function of SLPI against STD organisms including Trichomonas vaginalis (TV), Neisseria gonorrhoeae (GC), and Chlamydia trachomatis (CT). 2) determine the antimicrobial function of SLPI under different broth growth and metabolic conditions of TV including aerobic and anaerobic growth; different concentrations of free iron, and human transferrin and lactoferrin; and in the presence and absence of cysteine protease inhibitor E-64. 3) isolate and characterize the specific TVP activity which causes the degradation of SLPI. Three TVPs (220, 92 and 68 kDa) will be isolated by ion exchange chromatography of induction supernatants and tested for their individual degradative effects on SLPI by SDS-PAGE and Western blot. 4) Resulting peptide fragments will be sequenced by gas phase microsequencing. The TVP cleavage sites and peptides will be mapped to known sequence data and functional domains. 5) Residual antimicrobic function of peptides will be tested, 6) We will determine SLPI levels in normal and infected secretions from the adolescent female genital tract. Adolescent girls, ages 14-19, will have vaginal fluid samples taken and SLPI levels determined by ELISA assay. These patients will be followed longitudinally lover a year and SLPI levels compared to Lactobacillus crispatus carriage rates, The mechanisms elucidated here will be relevant to our understanding of native vaginal mechanisms of homeostasis and during infection with simple or complicated genital tract disease. These findings may also be relevant to mechanisms of infection of other mucosal STD pathogens, including HIV infection, and other parasitic diseases.