Cysteine-rich proteins of Chlamydia

Watson, Mark Wayne (1992) Cysteine-rich proteins of Chlamydia. University of Southampton, Doctoral Thesis.

Abstract

This work was undertaken to characterize the 9kDa and 60kDa cysteine-rich proteins (CrPs) of the genus Chlamydia. These two proteins were believed to be located in the chlamydial outer envelope and appeared to be involved with disulphide cross linkage with other envelope components including the chlamydial major outer membrane protein. This disulphide-linked protein matrix has been implicated in maintaining the structural integrity of the cell in the absence of a peptidoglycan layer. Prior to this study only two 60kDa CrPs had been cloned and characterized at the molecular level, both from C.trachomatis LGV strains. In this study the 60kDa and 9kDa CrPs were cloned, sequenced and expressed from a trachoma strain of C.trachomatis, C.psittaci (EAE/A22/M) and C.pneumoniae (IOL-207). The results showed both areas of conserved and variable amino acid sequence with a clear conservation of cysteine residues. The 9kDa CrP was extremely rich in cysteine for a protein of this size. Both the gene and amino acid sequences are quite unique with no significant homology to other proteins. Expressed protein was used to raise polyclonal serum in rabbits which was utilized for immuno-gold labelling of chlamydial cell surfaces, and showed that the 60kDa CrP was not exposed at the chlamydial cell surface. The function of these CrPs is still not clear but the conservation of cysteine and close genetic relationship strengthens the hypothesis that they are structural proteins involved in cysteine cross linkage. The gene sequences of the 60kDa CrPs were used to develop a PCR test for Chlamydia, which was found to work with all chlamydial strains tested to date. The 9kDa and 60kDa CrPs are in close genetic proximity being transcribed together from a single promoter as polycistronic mRNA late in the growth cycle, though the 9kDa CrP may be transcribed independently. The use of reverse transcriptases with ribonuclease H activity for transcript characterization is discussed. As the 60kDa CrPs did not appear to be surface exposed their ability to elicit neutralizing immunological responses was expected to be poor and may not be of use in development of subunit vaccines. The 60kDa CrP is however a potent immunogen and antibody titres are raised toward it very early in the infection process making it a prime target for diagnostic tests. The expressed proteins described here could be used to develop an ELISA based diagnostic test to detect anti-60kDa CrP antibodies.

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