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Characterizing a Toxin-Antitoxin Locus in Shigella flexneri

SARPONG, DAVID DANIEL
http://orcid.org/0000-0001-7858-7807
http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1723568144699225

Abstract Details

2024, Doctor of Philosophy (PhD), Ohio University, Molecular and Cellular Biology (Arts and Sciences).
Members of the genus Shigella are Gram-negative, non-spore-forming bacilli that cause shigellosis, a severe bacillary dysentery in humans, mostly affecting children under the age of five, immunocompromised individuals, and people living in developing countries. With an estimated 27,000 annual cases of antibiotic-resistant Shigella infections in the US alone, and no success in vaccine development, Shigella infections pose a serious health concern, creating the need for more targeted therapeutics. Key to the development of novel therapeutics against Shigella is a comprehensive understanding of the diverse molecular strategies that underlie the pathogen’s physiology. An emerging phenomenon in bacterial gene expression is that of Toxin-antitoxin (TA) systems. TA systems are dual component genetic loci in bacteria, producing two genes, a toxin gene whose expression is lethal to the organism producing it, and an antitoxin which protects the organism from the unwanted expression of the toxin. There are currently VIII TA systems studied in bacteria, differing based on whether the toxin or antitoxin is a protein or an sRNA, as well as mechanism of action of either the toxin or antitoxin. This dissertation characterizes a Toxin-Antitoxin (TA) system in Shigella flexneri, focusing on the ryf locus, which includes the toxin-encoding ryfA gene and two small RNAs (sRNAs), ryfB and ryfB1, with distinct regulatory functions. The 305-nucleotide toxin RNA, ryfA, inhibits bacterial growth by inducing membrane lysis and ATP depletion. ryfB, approximately 100 nucleotides in length, neutralizes ryfA's toxicity via nucleic acid complementarity, without reducing its transcript abundance. ryfB1, although 77% identical to ryfB, does not function as an antitoxin but modulates global gene expression, mostly metabolism. Initial in silico analyses identified key genetic elements within the ryf locus, including promoters, open reading frames, and Shine-Dalgarno sequences, as well as potential targets of the genes. Some of these predictions were experimentally validated, revealing the regulatory mechanisms and physiological roles of the locus components. Key findings demonstrate that ryfA's toxicity depends on the expression of a conserved open reading frame and is neutralized by ryfB via complementary base-pairing. Environmental factors such as iron availability and exposure to deoxycholate (DOC) significantly upregulate ryfA expression, suggesting the ryfA-ryfB TA system's role in S. flexneri's adaptive response. Low-level expression of ryfA reduces plaque formation but has no effect on antibiotic resistance, biofilm formation, or protection from high osmolarity. RNA-seq analysis provided a comprehensive view of the ryf locus’s regulatory impact, highlighting its influence on multiple metabolic pathways and stress response mechanisms. Taken together, this thesis characterizes the ryfA-ryfB and ryfB1 locus in Shigella flexneri and offers insights into how it contributes to the organism’s physiology.
Erin Murphy (Advisor)
Tingyue Gu (Committee Chair)
Peter Coschigano (Committee Member)
Nathan Weyand (Committee Member)
196 p.
Bioinformatics; Biology; Biomedical Research; Microbiology; Molecular Biology
Toxin-Antitoxin, Type I, TA, System, Locus, Shigella flexneri, RyfA, RyfB, RyfB1, sRNA, Characterization

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Citations

  • SARPONG, D. D. (2024). Characterizing a Toxin-Antitoxin Locus in Shigella flexneri [Doctoral dissertation, Ohio University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1723568144699225

    APA Style (7th edition)

  • SARPONG, DAVID. Characterizing a Toxin-Antitoxin Locus in Shigella flexneri. 2024. Ohio University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1723568144699225.

    MLA Style (8th edition)

  • SARPONG, DAVID. "Characterizing a Toxin-Antitoxin Locus in Shigella flexneri." Doctoral dissertation, Ohio University, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1723568144699225

    Chicago Manual of Style (17th edition)

ohiou1723568144699225
© 2024, all rights reserved.
This open access ETD is published by Ohio University and OhioLINK.