Production and characterisation of putative early effector proteins in Chlamydia trachomatis infection

Chlamydia trachomatis is commonly known as the most common bacterial sexually transmitted infection in the world, having affected approximately 3.1% of the global population in 2010, and being a major cause of infertility in men and women. However, what is less well known among the general population is that C. trachomatis also causes the blindness disease trachoma, the leading cause of preventable blindness globally. This project aimed to produce and investigate three proteins known to be involved in the early stages of the infection process: CT694, CT166 and PmpD. CT694 and CT166 are believed to affect actin fibre formation and breakdown, acting in concert with one another to facilitate engulfment of the bacterium by the host. PmpD is a protein which draws significant interest as a vaccination target, as it is present on the outer membrane of C. trachomatis and is conserved very highly among serovars.

This project focussed mainly on the breakdown product ΔCT694. Its gene was amplified from Chlamydial genomic DNA and inserted into a plasmid, which was used to transform an expression strain of Escherichia coli. Batches of protein were produced for characterisation and investigation, mainly commercial and customised crystallisation screens. Although no crystals were achieved, a promising microcrystalline precipitate formed, and a “best condition” has been recorded. In addition, the fragment PmpD F2 was produced regularly for vaccine studies by inclusion body preparation, and characterisation experiments were performed upon the protein. Work on PCR and gene cloning of PmpD fragments and CT166 was performed as well. Although the primary goal of producing X-ray diffractable crystals was not met, this project has laid the foundations within the group for development and future work.