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Random Mutagenesis for the Discovery of Obligate Intracellular Bacterial In vivo Virulence Genes

Bekebrede, Hannah S.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1574767897548976

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Ehrlichia spp. are emerging tick-borne obligatory intracellular bacteria that cause febrile diseases with abnormal blood cell counts and signs of hepatitis. Ehrlichia HF strain provides an excellent mouse disease model of fatal human ehrlichiosis. We recently established stable culture of Ehrlichia HF strain in DH82 canine macrophage cell line, and annotated its whole genome sequence. To identify genes required for in vivo virulence of Ehrlichia, we constructed random insertional HF strain mutants by using Himar1 transposon-based mutagenesis procedure. Of total 158 insertional mutants isolated, 84 insertions were within the non-coding regions, and 74 insertions were in the coding regions of 55 distinct protein coding genes including TRP120 and multi-copy genes, such as p28/omp-1, virB2, and virB6. Using limited dilution methods, nine stable clonal mutants that had no apparent defect for intracellular multiplication in DH82 macrophages, were obtained. Mouse virulence of seven mutant clones was similar to that of wild-type HF strain, whereas two mutant clones showed significantly retarded growth in blood, livers, and spleens, and the mice inoculated with them lived longer than mice inoculated with wild-type. The two clones contained mutations in genes conserved among Ehrlichia spp., but lacked homology to other bacterial genes. Inflammatory cytokine mRNA levels in the liver of mice infected with the two mutants were significantly diminished than those infected with HF strain wild-type, except IFN-γ, IL 1β, and IL-12 p40 in one clone. Thus, we identified two Ehrlichia genes responsible for non-macrophage infection-related virulence.
Yasuko Rikihisa, PhD (Advisor)
Amal Amer, MD, PhD (Committee Member)
Estelle Cormet-Boyaka, PhD (Committee Member)
Stephanie Seveau, PhD (Committee Member)
140 p.
Cellular Biology; Microbiology; Molecular Biology
Ehrlichia; HF strain; obligate intracellular bacteria; virulence; mutagenesis

Recommended Citations

Citations

  • Bekebrede, H. S. (2019). Random Mutagenesis for the Discovery of Obligate Intracellular Bacterial In vivo Virulence Genes [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574767897548976

    APA Style (7th edition)

  • Bekebrede, Hannah. Random Mutagenesis for the Discovery of Obligate Intracellular Bacterial In vivo Virulence Genes. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1574767897548976.

    MLA Style (8th edition)

  • Bekebrede, Hannah. "Random Mutagenesis for the Discovery of Obligate Intracellular Bacterial In vivo Virulence Genes." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574767897548976

    Chicago Manual of Style (17th edition)

osu1574767897548976
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© 2019, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.