[show abstract][hide abstract] ABSTRACT: Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp., including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes with parental phenotypes and genotypes.
Each of the 190 examined recombination events was the product of homologous recombination, and no candidate targeting motifs were identified at recombination sites. There was a single deletion event in one recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome.
These experiments examined the process of recombination in C. trachomatis and identified tools that can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the hypothesis that particular nucleotide selected sequences are preferentially used for recombination in vitro. Selected phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.
[show abstract][hide abstract] ABSTRACT: In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify "Chicago-" or "Nichols -specific" differences. All but one of the 16 SNPs were "Nichols-specific", with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature.
[show abstract][hide abstract] ABSTRACT: A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.
Antimicrobial Agents and Chemotherapy 05/2012; 56(8):4296-302. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The human pathogen Chlamydia trachomatis exists as multiple serovariants that have distinct organotropisms for different tissue sites. Culture and epidemiologic data have demonstrated that serovar G is more prevalent, while serovar E is less prevalent, for rectal isolates from men having sex with men (MSM). The relative prevalence of these serovars is the opposite for isolates from female cervical infections. In contrast, the prevalence of serovar J isolates is approximately the same at the different tissue sites, and these isolates are the only C-class strains that are routinely cultured from MSM populations. These correlations led us to hypothesize that polymorphisms in open reading frame (ORF) sequences correlate with the different tissue tropisms of these serovars. To explore this possibility, we sequenced and compared the genomes of clinical anorectal and cervical isolates belonging to serovars E, G, and J and compared these genomes with each other, as well as with a set of previously sequenced genomes. We then used PCR- and restriction digestion-based genotyping assays performed with a large collection of recent clinical isolates to show that polymorphisms in ORFs CT144, CT154, and CT326 were highly associated with rectal tropism in serovar G isolates and that polymorphisms in CT869 and CT870 were associated with tissue tropism across all serovars tested. The genome sequences collected were also used to identify regions of likely recombination in recent clinical strains. This work demonstrated that whole-genome sequencing along with comparative genomics is an effective approach for discovering variable loci in Chlamydia spp. that are associated with clinical presentation.
Infection and immunity 03/2010; 78(6):2544-53. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: In syphilis research, the Nichols strain of Treponema pallidum, isolated in 1912, has been the most widely studied. Recently, important differences among T. pallidum strains emerged; therefore, we sequenced and annotated the Chicago strain genome to facilitate and encourage the use of this strain in studying the pathogenesis of syphilis.
Journal of bacteriology 03/2010; 192(10):2645-6. · 3.94 Impact Factor