Functional Analysis of Inorganic Polyphosphate and its Associated Enzymes in Campylobacter jejuni
Year: 2011
Type: article
Abstract: Campylobacter jejuni, a Gram-negative microaerophilic bacterium, is a predominant cause of bacterial foodborne gastroenteritis in humans.Despite its importance as a major foodborne pathogen, our understanding of the molecular mechanisms underlying C. jejuni pathogenesis and survival under stress is limited.Of particular importance is the inorganic polyphosphate (poly P) which plays an important role in bacterial survival, stress responses, host colonization 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.However, to our knowledge, there have been no studies addressing the role of poly P and associated enzymes in C. jejuni patho-physiology.In the present study, we hypothesize that poly P and its associated enzymes might play an important role in C. jejuni biology.Findings from this study will have implications for developing strategies to control Campylobacter through identification of new targets for vaccines and antimicrobials.The work in this dissertation is broadly divided into three studies.iii In the first study, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival and adaptation using a deletion mutagenesis approach.Our findings demonstrate that C. jejuni ∆ppk1 mutant was deficient in poly P accumulation, which was associated with decreased ability to form viable but non-culturable cells (VBNC) under acid stress.The ∆ppk1 mutant also showed decreased frequency of natural transformation and increased susceptibility to various antimicrobials.Furthermore, the ∆ppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization.Complementation of the ∆ppk1 mutant with the wildtype copy of ppk1 restored the deficient phenotypes to levels similar to those of the wildtype.Our results suggest that poly P plays an important role in stress survival and adaptation and might contribute to genome plasticity, and spread and development of antimicrobial resistance in C. jejuni.In the second study, we determined the role of PPK2 in C. jejuni pathophysiology using a deletion mutant of ppk2.Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio.The ∆ppk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms.However, the ∆ppk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated.Importantly, the ∆ppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as chicken colonization.Taken together, we have I would like to gratefully and sincerely thank my advisor Dr. Rajashekara for his excellent guidance, understanding, patience, constructive criticism and persistent persuasion throughout my graduate studies at The Ohio State University.His mentorship has been of paramount importance in providing me with a well rounded experience.I thank you Dr. Rajashekara for everything you have done for me.
Author: Dharanesh Gangaiah
Institution: The Ohio State University
Language: English
Subfield: Food Science
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2301889659