Trichomonas vaginalis is a human urogenital parasite and the causal agent of trichomoniasis, the most common non-viral, sexually transmitted disease in the United States. Much of the pathogenic properties of T. Vaginalis stem from cysteine proteases. Here, we present the results of several studies on one variant, TvCP2, including purification and characterization of its active form, gene regulation in response to iron and oxygen, and localization and trafficking. Homologous to human Cathepsin L, TvCP2 was hypothesized to function as a protease, presumably localize to lysosomes, and play a role in T. vaginalis pathogenesis that is distinct from TvCP4.
Levels of bacterially-expressed TvCP2 decreased faster in activation assays of lower pH. In Pichia pastoris, the amounts and form of TvCP2 expressed were variable, and protease activity was influenced by reducing agent and pH. Post-translational modifications may be in effect, or TvCP2 may be autocatalytic, however, actual autocatalytic processing remains to be determined. Consistent with previous reports, and contrary to TvCP4 regulation, TvCP2 mRNA levels were increased in T. vaginalis grown in media with reduced iron supplementation. Expression of processed TvCP2 protein increased, demonstrating post-transcriptional regulation and the potential for iron to influence processing of and/or proper sorting of TvCP2. Also, unlike TvCP4 expresion, which is unaffected by oxygen, both TvCP2 protein and mRNA were greatest under anaerobic conditions, suggestion transcriptional and translational regulation by oxygen, and that upon initial infection TvCP2 is not required immediately. Although overall immunofluorescent staining patterns were different between TvCP2 and TvCP4, hinting at distinct functions, both localized bto punctate vesicles, for which some colocalization was observed. Additionally, unlike TvCP4, TvCP2 did not colocalize with Vamp1/2 and did colocalize with legumain. These data suggest that TvCP2 is intracellular, targeted to lysosomes, and sorted independently from TvCP4. In conclusion, TvCP2 may play a unique role in the cell and is important for the life cycle and pathogenesis of T. vaginalis.
| Identifer | oai:union.ndltd.org:pacific.edu/oai:scholarlycommons.pacific.edu:uop_etds-5041 |
| Date | 01 January 2015 |
| Creators | Balayan, Shaina-Jill S. |
| Publisher | Scholarly Commons |
| Source Sets | University of the Pacific |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of the Pacific Theses and Dissertations |
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