Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions

1. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions Rodolfo Hernandez-Gutierrez, Leticia Avila-Gonzalez, Jaime Ortega-Lopez,Fernando Cruz-Talonia, Guillermo Gomez-Gutierrez, and Rossana Arroyoa Mariska Petersen

2. Trichomonaisis • History • Background Info • Biological Mechanism • Experimentation • Data • Results • Conclusion

3. Trichomonaisis • Sexually transmitted infection • Discovered in 1836 • 5 million cases reported every year • 170 million people infected worldwide • Resides in genitourinary tract of males and females • 50% asymptomatic carriers

4. Transmission • Sexual intercourse • Mutual masturbation • Sharing sex toys • Mother to child during vaginal delivery

5. In females there is… inflammation of the vaginal canal vulvar itching leading to edema tenderness and chaffing redness yellow and green discharge painful urination pain during sex punctuate hemorrhages on the cervix known as colpitis macularis or strawberry cervix. In males there is… whitish discharge although rare pain and burning during urination Signs and Symptoms

6. Treatment Metronidazole (Flagyl) ~single two gram dose (85-92% cure rate) OR 250 mg three time daily for 7-10 days. Tinidazole is an alternate drug

7. Biological Mechanism • T. vaginalis – causitive agent • Reproduction- binary fission • Incubation period is 4-28 days • Facultative anerobic parasite • Adhesion proteins on surface of flagella • Ligand/Receptor Cytoadherence • 11-23 different CP’s (cysteine proteinases) • CP’s play an important role in the pathogenicity of the parasite.

8. Key Terms to know: • Cysteine Proteinase (CP) 39- an active surface proteinase secreted during the infection. • Western Blot- a technique in molecular biology, used to separate and identify proteins. • HeLa cells- Cervical Cancer cells • VEC - Vaginal Epithelial Cells • DU-145- Human prostatic cancer cells • CaCo cell line- Human colon cancer cell line

9. Characterization of CP39 (cysteine proteinase) found in the vagina of women with trichomoniasis.

10. Determination of proteinases in vaginal secretions CP39 binding to different cell types Presence of CP39 Heat stability and pH tolerance of CP39. Degradation of substrates by CP39 Localization of CP39 on T. vaginalis Presence of antibodies to CP39 in sera from patients with T. vaginalis Patients were diagnosed with T. vaginalis Healthy patients used as negative controls The samples (vaginal secretions, urinary sediment, and sera) obtained were tested for the T. vaginalis and trichomonad proteinases. Samples used in several tests including  Materials and Methods

11. Determination of proteinases in vaginal secretions • Figure on the left shows proteinases can be secreted by viable and metabolically active trichomonads under in vitro controlled conditions.

12. CP39 Cell Binding Figure above shows that CP39 bound to the surface of the HeLa and VEC. CP39 also bound to the prostatic DU-145 cell line, but not to the surface of the CaCo cells.

13. Presence of CP39 CP39 present in different T. vaginalis isolates. CP39 was present in all isolates varying in intensities (despite loading same amount of cells on the gel).

14. Heat stability & pH A- CP39 active in temperatures ranging from 4 to 50˚ C. B- CP39 has maximum activity at pH 7.0. C- CP39 range of activity increased from 3.6 to 7.0. (Most activity)

15. Degradation of Substrates CP39 analyzed on substrate gel electrophoresis using Collagen I,II, III, IV, human Fibronectin, Hemoglobin, IgA and IgG as subtrates. CP39 degraded the all proteins tested. CP39 was strongest on Collagen IV, IgA and IgG. Other tests were performed on vaginal cultures whereby CP39 degraded the IgA-containing cells. Data suggests that CP39 might be apart of immune invasion mechanisms used by the parasite.

16. Localization of CP39 on T. vaginalis CP39 was determined to be another trichomonad surface protein.

17. Conclusion • The characteristics of CP39 are: • It is present in vaginal secretions in patients with trichomoniasis • Its optimal temperature is 37˚C and a pH range of 3.6 to 7.0 • Suggested to be involved in tissue damage. • Has broad substrate specifity • Plays a role in parasite survival and immune evasion by degrading hemoglobin and immunoglobulins. • Indicative that CP39 plays a role in trichomonal infection

18. References • Burgess, Donald and J.R. Schwebke . Trichomoniasis. Clinical Microbiology Reviews. Volume 17 pp. 794-803. • Clenney, Timothy L. and M.K. Owen. Management of Vaginitis. American Family Physician. Volume 70 pp.2125-2132. http://search.epnet.com/login.aspx?direct=true&db=aph&an=15224416 • Hernandez, G.R. L.A. Gonzalez, J. O. Lopez,F. C. Talonia, G. G. Gutierrez, and R. Arroyoa 2004. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions. Experimental Parasitology Volume 107(3-4):125-135. • Singh B.N. 2004. Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells. Infect. Immunology. Volume 72(7):4151-4158 • Soper, David. Trichomoniasis: under control or undercontrolled?. American Journal of Obstetrics and Gynecology, Volume 190 pp. 281-290. • Unknown. Vaginitis. American Family Physician. Volume 70. p.1. http://search.epnet.com/login.aspx?direct=true&db=aph&an=15227627

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