Trichomonas Vaginalis and SLPI Mucosal Defenses

阴道毛滴虫和 SLPI 粘膜防御

• 批准号: 6345955
• 负责人: DEBORAH L DRAPER
• 金额: $ 3.57万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6345955
• 关键词: Chlamydia trachomatis; Lactobacillus; Neisseria gonorrhoeae; SDS polyacrylamide gel electrophoresis; Trichomonas vaginalis; adolescence (12-20); bacterial proteins; clinical research; cooperative study; cysteine endopeptidases; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; female; genital secretion; host organism interaction; human subject; ion exchange chromatography; iron; lactoferrin; mucosal immunity; protein sequence; serine proteinases; sexually transmitted diseases; transferrin; vagina; western blottings

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.

阴道毛滴虫与SLPI粘膜防御Deborah德雷珀博士这项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。实验将集中在分泌性白细胞蛋白酶抑制剂（SLPI）提供的保护机制，一种主要的丝氨酸蛋白酶抑制剂，发现在阴道上皮细胞和确定T。迷走蛋白酶（TVP）对SLPI功能的影响。我们假设阴道毛滴虫（TV）半胱氨酸蛋白酶切割：1）确定SLPI对STD微生物（包括阴道毛滴虫（TV）、淋病奈瑟菌（GC）和沙眼衣原体（CT））的正常抗菌功能。2)确定SLPI在TV的不同肉汤生长和代谢条件下的抗微生物功能，包括需氧和厌氧生长;不同浓度的游离铁、人转铁蛋白和乳铁蛋白;以及在存在和不存在半胱氨酸蛋白酶抑制剂E-64的情况下。3)分离并表征导致SLPI降解的特异性TVP活性。将通过诱导上清液的离子交换层析分离三种TVP（220、92和68 kDa），并通过SDS-PAGE和蛋白质印迹测试其对SLPI的单独降解作用。4)将通过气相微测序对所得肽片段进行测序。将TVP切割位点和肽映射到已知的序列数据和功能结构域。5)将测试肽的残余抗微生物功能。6）我们将测定来自青春期女性生殖道的正常和感染分泌物中的SLPI水平。将对14-19岁的青春期女孩进行阴道液采样，并通过ELISA测定确定SLPI水平。这些患者将纵向随访一年以上，并将SLPI水平与卷曲乳杆菌携带率进行比较。本文阐明的机制将有助于我们理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阴道内稳态机制。这些发现也可能与其他粘膜性病病原体（包括HIV感染）和其他寄生虫病的感染机制有关。