Heterologous expression of Chlamydia trachomatis polymorphic membrane proteins for in vitro studies.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease worldwide. Not only does Chlamydia target urogenital tissue that can lead to host infertility, it is also responsible for a high occurence of blinding trachoma in the developing world. Complete sequencing analysis of the C. trachomatis genome revealed a unique family of Polymorphic membrane proteins (Pmps) that had previously remained undiscovered. Nine to twenty-one pmp genes were found across the varied Chlamdia species, encoding for large proteins all phylogetically related to one of six basic subtypes. C. tractomatis encodes nine of these proteins termed PmpA - Pmpl. At present, information about the Pmps is limited with the vast majority of data derived from genomic analysis. Such studies have suggested that the Pmps act as virulence factors possibly offering antigenic variation to evade the host defenses. Although this has been displayed genotypically it is yet to be confirmed phenotypically. One of the major problems for studying such proteins in this bacterium, is that Chlamydia reside within a host cell and differentiate between two very different morphological forms during infection and the Pmps appear to be expressed at different stages within this biphasic life cycle. Additional theories have suggested that the Pmps act as adhesions, possibly displaying tissue specific tropism in attachment to host cells, an essential step in Chlamydial growth and survival. This has previously been shown for specific regions of Pmp D and Pmp G homologues belonging to C.pneumoniae but has not been repeated with other Pmps. Furthermore, bioinformatic data has proposed that the Pmp proteins are autotransporter proteins, a secretory protein that forms a beta-barrel pore within the outer membrane to allow translocation of the functional protein to the bacterial outer surface. The aim of this study was to gain a better understanding of the Pmps proteins. Specifically to clone, express and purify full length or truncated recombinant Pmps (rPmp) for secondary structural and functional analysis to test the theory that Pmps are putative adhesins. Expressing membrane proteins in a heterologous system poses many obstacles and the expression of rPmp proteins was hindered by the formation of insoluble aggregates and low yields. Several rPmps were produced and three truncated rPmps were expressed as soluble proteins with the use of nanodiscs to mimic the lipid bilayer. Purification of these domains resulted in a substantial loss of protein and subsequently secondary structural analysis could not be carried out on these proteins. However protein interactions were tested with urogenital cell lines using chemical cross-linking, immunocytochemistry, surface plasmon resonance and ellipsometry. Truncated rPmp domains of Pmp D, Pmp G and Pmp I were shown to interact with endometrial cells, supporting the theory that Pmps are involved in attachment. Also, putative proteolytic cleavage was observed, possibly indicating Pmpl is cleaved and released from the Chlamydial surface.