Isolation and partial characterization of a Chlamydia tracomatis cytotoxin.

The main objective of this study is to isolate and identify an unknown cytotoxin secreted by C.trachomatis. Summary of study design: Cell lines: McCoy mouse fibroblasts, HaCaT human keratinocytes, ME-180 Human cervical cells. Bacterial strains: LGV serovars: L1 , L2, L3 and three stored clinical isolates. Oculogenital biovar - serovar E. Cultivation of C. trachomatis: C. trachomatis will be propagated and inoculum determined in McCoy mouse fibroblasts. Experiments will be conducted using HaCaT and ME-180 cell lines. MOI 1 and MOI 10 will be used. Tests: Screening for C. trachomatis Cytotoxin using TUNEL assay. Determination of optimal toxin production. Isolation of Toxin: SDS-PAGE and Centrifugal filteration tubes. Cytotoxicity of toxin: Caspase ELISA Characterization of isolated C. trachomatis cytotoxin: Effect of Temperature: 60 and 95?C Effect of salt concentration: The effect of various the salt concentrations on the toxin will be tested. Effect of Enzymes activity: Protease K Chemical Analysis: MALDI/ Mass Spectrometry. Controls: Positive control – Etoposide Negative control – Supernatant of uninfected and unexposed cells.