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    DISCOVERY OF GENETIC CORRELATES IMPORTANT FOR CHLAMYDIA INFECTION AND PATHOGENESIS

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    Issue Date
    2017-08-31
    Author
    Harrison, Kelly Sue
    Publisher
    University of Kansas
    Format
    188 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Molecular Biosciences
    Rights
    Copyright held by the author.
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    Abstract
    Chlamydia species are responsible for over 1.2 million reports of bacterial sexually transmitted infections in the United States; a number that has been steadily increasing for the past decade. Worldwide, a cumulative 131 million new cases of Chlamydia trachomatis are estimated among individuals between ages 15-49. In most individuals, chlamydial infections are asymptomatic, resulting in long-term sequelae such as pelvic inflammatory disease, salpingitis and infertility. Along with genitourinary infections, Chlamydia is also the leading cause of blinding trachoma, affecting nearly 1.9 million people across 42 different countries. While current treatment with antibiotics remains successful in combating infections, evidence of persistent infections, acquisition of antibiotic resistances, and recurring exposure intensifies the necessity for enhanced prophylactic approaches, including the development of a vaccine. In order to develop these advances, species-specific targets, as well as mechanisms the bacterium uses to establish infection must be identified. My research focuses on two forward-genetic approaches to identifying these correlates of pathogenicity: homologous recombination and transposon mutagenesis. Majority of information pertaining to Chlamydia’s pathogenicity in a mammalian host has been derived from the mouse-adapted species, Chlamydia muridarum. This species readily infects the vaginal canals of female mice and ascends to the upper genital tract— the primary site for causing disease in women. Conversely, the human-adapted strain, C. trachomatis, is incapable of ascension in the mouse model of infection and rapidly cleared. Even with the drastic differences in pathogenicity, C. muridarum and C. trachomatis share ~98% genome sequence identity, with most of the genetic differences occurring within the plasticity zone. In our efforts to understand which genetic components are responsible for ascension, in vitro recombination between C. trachomatis and C. muridarum was completed, enriching for recombination around this plasticity zone. I used molecular approaches to characterize regions of crossover among the hybrid progeny, focusing on hybrids containing gyrA-2 from C. muridarum as an indicator for genetic exchange near the plasticity zone. Among the 35 hybrids assessed, only three showed integration of the large cytotoxins specific to C. muridarum: 4357, 4406, and 4537. These three recombinants integrated only two of the cytotoxins, TC0438 and TC0439; none of the samples showed integration of the third cytotoxin, TC0437. To assess whether TC0438 and TC0439 are responsible for C. muridarum’s ability to ascend to the UGT, I used the mouse vaginal model of infection with these hybrids. Three mice each were infected with the hybrids and uterine horns isolated for assessment of ascension. Seven days post-infection, only one animal, infected with isolate 4406, showed detectable Chlamydia in the uterine horns. No other animals from any of the hybrids showed detectable organisms in the uterine horns, suggesting these two cytotoxins are not sufficient to cause a gain-of-function. The remaining 32 hybrids showed recombination at the same intergenic site between the C. trachomatis trpA and C muridarum TC0440. This region may indicate a recombination hotspot or chi site within Chlamydia or may highlight the selective pressure against C. trachomatis to harbor the cytotoxins. Secondly, the development of a transposon system functional in Chlamydia permitted the analysis of single-gene disruptions and their roles in growth and development. I used Tn libraries generated in both C. trachomatis and C. muridarum to characterize in vitro and in vivo phenotypes. All 13 Tn mutants from C. trachomatis were single insertions while three of the 26 C. muridarum mutants showed double inserts. One mutant from C. trachomatis, CT696::Tn, had a significant defect in growth in vitro resulting in lack of inclusion formation and aggregation of RBs. In vivo, two mutants from each library, CT148::Tn and CT868::Tn from C. trachomatis, and TC0657::Tn and TC685::Tn from C. muridarum showed decreases in bacterial burden in the uterine horns of mice. In addition, I performed in silico analysis of the Tn insert within the hypothetical protein CT339 from C. trachomatis. I ran BLAST analysis to identify the conserved competence motif then generated multiple sequence alignments and hydropathy predictions for structure. These indicated CT339 is a multi-pass transmembrane protein with structural homology to ComEC from Bacillus. I then used PCRs from three loci dispersed around the genomes of recombinant progeny to show that a disruption in CT339 prevents lateral gene transfer between C. trachomatis mutants and tetracycline-resistant C. suis R19. All recombinants with a C. suis OmpA seroreactivity incorporated the β-lactamase from CT339::Tn. No progeny showed tetracycline-resistance transfer into CT339::Tn. Experiments in which transformations were attempted with CT339::Tn mutants also showed no ability to uptake DNA, supporting the role of CT339 as both a structural and functional homolog of ComEC. Together, these two approaches provide the ability to assess large genetic loci and single- gene roles in growth and infectivity of chlamydial species. Homologous recombination allows for generation of both gain- and loss-of-function mutants for the determination of genetic regions essential for pathogenesis; narrowing the genome for assessment of individual components within each region. Transposon mutagenesis offers the advantage of single-gene disruptions for direct genotype-phenotype correlations, as demonstrated by reduced bacterial burdens in mammalian infections and my discovery of CT339 as a homolog to ComEC.
    URI
    http://hdl.handle.net/1808/26477
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    • Molecular Biosciences Dissertations and Theses [273]
    • Dissertations [4321]

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    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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