Understanding chlamydial biology through genome sequencing and comparative genomics Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/zc77st02w

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  • The human pathogen Chlamydia trachomatis displays different phenotypes between serovars both in vivo and in vitro. The study of chlamydial biology has been hampered by the lack of a tractable genetic system. Here, chlamydial isolates with known phenotypes as well as strains generated though recombination were fully sequenced to address several aspects of chlamydial biology. The genomes of clonally isolated matched pairs of IncA-negative inclusion non-fusing strains and IncA-positive inclusion fusion strains from the same parents were very similar. In contrast, genome sequence of unmatched strains contained over 5,000 nucleotide polymorphisms. Additionally, transcriptional analysis, in vitro culture kinetics, and animal modeling show that the matched pairs are phenotypically more similar than the non-fusing strains isolated in absence of a serovar-matched wild-type strain. These results suggest that a change from an IncA positive strain to an IncA negative phenotype may involve multiple steps, including unidentified steps that lead to difference in growth rate and infectivity. Serovar G is proportionally more common in rectal isolates from men having sex with men, whereas serovar E is less prevalent. Genome sequencing and comparative genomics of isolates from rectal or cervical sites of serovar G, E, and J revealed several open reading frames that may be associated with the rectal tropism. Further PCR-based and statistical analyses of several clinical isolates show that ORFs CT144, CT154, and CT326 were highly associated with rectal tropism within the serovar G isolates. Previous studies have shown that chlamydia can exchange DNA both in vitro and in vivo. We produced several chlamydial recombinant strains where the parental strains had known inclusion fusion, attachment to host cell, and secondary inclusion forming phenotypes. Genome sequencing of the parents and the recombinant strains and genome-wide association analyses were used to identify genetic loci that could be associated with observed phenotypes. Results show that the correct identification of incA as being associated with inclusion fusion supports the use of this approach to studying aspects of chlamydial biology. Genome-wide association studies of these recombinants also revealed several loci that may be associated with the high attachment efficiency seen in lymphogranuloma strains, such as the Pmp genes. Analysis also revealed several loci that may be associated with secondary inclusion formation. Collectively, this work demonstrates that a combination of genome sequencing, comparative genomics, and the generation of recombinant chlamydial strains can function as a powerful tool for studying the biology of an organism that lacks classical genetic techniques.
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