Isolation and partial characterization of a Chlamydia tracomatis cytotoxin.

Adele Cheddie, Bronwyn Joubert

Abstract


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.