Trichomonas Vaginalis and SLPI Mucosal Defenses

阴道毛滴虫和SLPI粘膜防御。这笔赠款的长期目标是了解健康女性生殖道的粘膜环境，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)和炎症反应的非免疫保护机制。实验将集中在分泌性白细胞蛋白酶抑制物(SLPI)提供的保护机制上，SLPI是阴道上皮中发现的一种主要的丝氨酸蛋白酶抑制剂，并确定阴道毛滴虫蛋白水解酶(TVP)对SLPI功能的有害影响。我们推测阴道毛滴虫(TV)半胱氨酸蛋白酶的作用是：1)确定SLPI对包括阴道毛滴虫(TV)、淋球菌(GC)和沙眼衣原体(CT)在内的性病生物的正常抗菌功能。2)测定SLPI在TV不同发酵液生长代谢条件下的抑菌作用，包括有氧和厌氧生长，不同浓度的游离铁、人转铁蛋白和乳铁蛋白，以及半胱氨酸蛋白酶抑制剂E-的存在和不存在时的抑菌效果。3)分离和鉴定导致SLPI降解的特异性TVP活性。通过诱导上清液的离子交换层析分离出三个TVP(220、92和68 kDa)，并通过SDS-PAGE和Western印迹检测它们对SLPI的单独降解效果。4)用气相微测序技术对得到的多肽片段进行测序。TVP的裂解位点和多肽将被映射到已知的序列数据和功能结构域。5)检测多肽的残留抗菌功能；6)检测青春期女性生殖道正常分泌物和感染分泌物中SLPI水平。14-19岁的青春期女孩将采集阴道液样本，并用ELISA法测定SLPI水平。这些患者将被纵向跟踪一年以上，并将SLPI水平与皱纹乳杆菌携带率进行比较。这里阐明的机制将与我们对自然阴道内稳态的机制以及感染简单或复杂生殖道疾病的机制的理解有关。这些发现也可能与其他粘膜性病病原体的感染机制有关，包括艾滋病毒感染和其他寄生虫病。

TRICHOMONAS VAGINALIS AND SLPI MUCOSAL DEFENSES. Deborah Draper, Ph.D. The long term objectives of this grant are to understand the mucosal environment of the health 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 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 over 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.