The genus Chlamydia consists of four species all of which are obligate intracellular parasites of eukaryotic cells and tissues. The human-specific pathogens are members of the species C. trachomatis and C. pneumoniae. Genomic sequencing projects have been completed for three representatives of C. trachomatis (i.e. Serovar D, Serovar L2, and Serovar MoPn) and C. pneumoniae (strain CWL029, strain AR39, and strain J138) allowing for genomic comparisons to be made within and across species boundaries. Comparative genomics may help identify specific gene-products that play a role in the different diseases caused by these species and serovariants. The most striking finding of these studies is the high degree of synteny found within the genus. Differences between the species and serovars are found predominantly within genetic regions of the chromosomes that define the 'termination of replication regions' and have been termed 'plasiticity zones' based on the high level of genetic rearrangement and recombination found.We have originated studies to more closely analyze the global gene expression patterns of these pathogens to understand both the underlying themes of controlled gene expression that result in the developmental cycle associated with chlamydial growth as well the differences in gene expression that may contribute to the nature and severity of infections giving rise to the various disease sequelae. Microarray slides have been generated that represent the entire chromosome of C. trachomatis serovar D (901 ORFs, including 8 ORFs encoded on the conserved small plasmid). The ORFs were amplified by PCR using oligonucleotides designed from the completed genome sequence to amplify entire genes. The ORFs were spotted, in triplicate, on glass slides and are being used in DNA:DNA and DNA:mRNA hybridization experiments to determine both gene carriage (among the different C. trachomatis serovariants) and gene expression (of Serovar D during the course of infection).To determine the nature of the interaction of C. trachomatis serovar D with the host cell (i.e. HeLa cells) during the course of the developmental cycle, we have chosen to purify total RNA from infected cells and fractionate the samples into bacterial and host cell-specific. The aforementioned bacterial microarray will be used to analyze chlamydial gene expression while the purified host cell mRNA (amplified and biotin-labelled) will be sent to colleagues at NCI where host cell expression patterns will be determined using commercially available, human, microarray chips (i.e. HG-U95A, representing ca. 12,000 sequences that have been characterized with respect to function or disease association) manufactured by Affymetrix. These experiments will establish the patterns of gene expression associated with normal growth of C. trachomatis in transformed human cell lines. We plan to use these analyses to provide a comparative basis for the investigation of chlamydial growth in relevant models of human disease.
