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Functional Analysis of Inorganic Polyphosphate and its Associated Enzymes in Campylobacter jejuni

Gangaiah, Dharanesh Mahimapura
http://rave.ohiolink.edu/etdc/view?acc_num=osu1298486377

Abstract Details

2011, Doctor of Philosophy, Ohio State University, Veterinary Preventive Medicine.
The Gram-negative bacterium Campylobacter jejuni is a frequent cause of gastroenteritis in humans and accounts for up to 15% of all diarrheal cases worldwide. Despite its significance as a major foodborne pathogen, our understanding of the molecular mechanisms underlying C. jejuni pathogenesis and survival under stress is limited. Inorganic polyphosphate (poly P), a biological macromolecule composed of multiple phosphate residues, plays important roles in bacterial survival under conditions of stress and virulence. Polyphosphate kinase 1 (PPK1) is the principal enzyme involved in poly P synthesis; while polyphosphate kinase 2 (PPK2) utilizes poly P to generate GTP. Exopolyphosphatases (PPX) degrade poly P to inorganic phosphate. The genome of C. jejuni possesses homologs of ppk1, ppk2 and 2 ppx/gppa genes. In the present study, we explored the significance of poly P and its associated enzymes to C. jejuni biology using deletion mutagenesis approach. In the first part of this study, we demonstrated that the deletion of ppk1 encoding polyphosphate kinase 1 compromised C. jejuni ability to- accumulate poly P, survive under stress conditions and form viable-but-nonculturable cells under acid stress. In addition, the Δppk1 mutant also showed decreased frequency of natural transformation and increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. As the second part of this dissertation, we explored the importance of PPK2 in C. jejuni functions. Findings from this study showed that PPK2 is important for poly P-dependent generation of GTP as well as maintaining intracellular nucleotide balance in C. jejuni. Deletion of ppk2 resulted in decreased ability of C. jejuni to tolerate osmotic, nutrient, aerobic, and antimicrobial stresses. However, the Δppk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Δppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as chicken colonization. Finally, we determined the functional significance of PPX/GPPA enzymes to C. jejuni patho-physiology. Findings from this study demonstrated that PPX2/GPPA enzymes play a role in C. jejuni motility and nutrient stress tolerance. Despite their defect in motility, Δppx1/gppa and Δppx2/gppa mutants showed similar capacity to form biofilm under static conditions. Quantification of intracellular poly P by toluidine blue O method revealed that Δppx/gppa mutants accumulated more poly P than the wildtype strain, suggesting a role for PPX/GPPA enzymes in poly P degradation. Though the exact mechanisms by which poly P-associated enzymes mediate stress response and virulence in C. jejuni is unknown, this study adds to our understanding of the contribution of poly P-associated proteins in C. jejuni adaptation and survival under different stringencies in the absence of RpoS-mediated classical stress response mechanisms.
Gireesh Rajashekara, DVM, PhD (Committee Chair)
Yehia Saif, DVM, PhD (Committee Co-Chair)
John Gunn, PhD (Committee Member)
Jeff LeJeune, DVM, PhD (Committee Member)
Chang Won Lee, DVM, PhD (Committee Member)
291 p.
Microbiology
Campylobacter jejuni; polyphosphate; stress response; virulence

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Citations

  • Gangaiah, D. M. (2011). Functional Analysis of Inorganic Polyphosphate and its Associated Enzymes in Campylobacter jejuni [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1298486377

    APA Style (7th edition)

  • Gangaiah, Dharanesh. Functional Analysis of Inorganic Polyphosphate and its Associated Enzymes in Campylobacter jejuni. 2011. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1298486377.

    MLA Style (8th edition)

  • Gangaiah, Dharanesh. "Functional Analysis of Inorganic Polyphosphate and its Associated Enzymes in Campylobacter jejuni." Doctoral dissertation, Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1298486377

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.