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.
Showing the most recent 10 out of 15 publications
