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Functional Characterization of Exopolyphosphatase/ Guanosine Pentaphosphate Phosphohydrolase (PPX/GPPA) Enzymes of Campylobacter jejuni

Kumar, Anand
http://rave.ohiolink.edu/etdc/view?acc_num=osu1339085410

Abstract Details

2012, Master of Science, Ohio State University, Veterinary Preventive Medicine.
Campylobacter are small, curved, flagellated, non-spore forming, Gram-negative rods. Of 17 species within the genus Campylobacter, C. jejuni and C. coli are of most important public health concern. Campylobacter are considered to be most common causative agents of foodborne bacterial gastroenteritis and nearly, 90% of human Campylobacter infections are attributed to C. jejuni. The high prevalence of C. jejuni and its rare potential fatal sequelaes like Guillain Barre Syndrome, Miller Fisher Syndrome, and Reactive Arthritis have attracted considerable research interest. Efforts to reduce C. jejuni infection in humans are directly linked to the better understanding of its physio-biology which inturn explore the pathogenesis mechanism in host. Despite the availability of genomic information of different strains of C. jejuni and advanced genetic tools, the complete understanding of the virulence mechanism is still an ongoing effort. Hence, any research diverted towards better understanding of host-microbe interaction is of immense importance. In-organic polyphosphate (poly-P), homeostasis is known to be essential for the virulence related phenotypic traits in pathogenic bacteria including C. jejuni. Although many enzymes are explored and implicated for their role in poly-P metabolism, the exopolyphosphatases (PPX) that hydrolyze poly-P is a less studied group of enzymes. More importantly, C. jejuni encodes two dual-function exopolyphosphatases (PPX1/GPPA and PPX2/GPPA) which in addition to poly-P hydrolysis also hydrolyze guanosine pentaphosphate (pppGpp) to guanosine tetraphosphate (ppGpp), an intracellular signaling molecule or an alarmone in bacteria. Therefore, we hypothesize that PPX/GPPA enzymes might play a key role in poly-P metabolism and thus significantly impact C. jejuni’s ability to persist in both the host and environment. C. jejuni 81-176 encodes two potential dual-function exopolyphosphatases ppx1/gppa (CJJ81176_0377) and ppx2/gppa (CJJ81176_1251) and we generated deletion mutants (¿¿¿¿¿¿¿ppx1, ¿¿¿¿¿¿¿ppx2 and dkppx) and complement strains (Cppx1, Cppx2) to better understand the PPX/GPPA role in C. jejuni pathophysiology. The mutants (¿¿¿¿¿¿¿ppx1, ¿¿¿¿¿¿¿ppx2 and dkppx) exhibited defect in motility, invasion and intracellular survival, survival under nutrient limited condition. Furthermore they showed increased capacity to accumulate poly-P, however only ¿¿¿¿¿¿¿ppx1 and dkppx showed decreased accumulation of ppGpp compared to wildtype. Strikingly, the deletions of either ppx1 or ppx2 genes did not affect the biofilm formation, survival under osmotic and oxidative stresses; but on contrary deletion of both the genes (dkppx) did affect amount of biofilm formation and osmotic stress tolerance. Bactericidal assay to determine the role of PPX1/GPPA and PPX2/GPPA for resistant to complement mediated killing indicated that both ¿¿¿¿¿¿¿ppx1, and ¿¿¿¿¿¿¿ppx2 mutant were resistant to human complement mediated killing however, dkppx mutant was sensitive. In contrary, the chicken serum did not have any effect on the survival of ¿¿¿¿¿¿¿ppx1, ¿¿¿¿¿¿¿ppx2, and dkppx mutants. Interestingly, the expression of ppk1 and spoT genes, which are known to be interconnected with poly-P and ppGpp homeostasis were up regulated in the both ¿¿¿¿¿¿¿ppx1 and ¿¿¿¿¿¿¿ppx2 mutants compared to wildtype suggesting a compensatory role in poly-P and ppGpp homeostasis. In all the cases complemented strains (Cppx1, Cppx2) restored the phenotypic characters similar to wildtype. Our future studies will decipher the direct or indirect role of ppx/gppa genes in poly-P metabolism, investigate mechanism behind resistant of ¿¿¿¿¿¿¿ppx1, ¿¿¿¿¿¿¿ppx2 mutants to human complement factors, and study the functional redundancy of ppx/gppa genes in C. jejuni. In conclusion, we report that C. jejuni PPX1/GPPA protein has bifunctional activity capable of both poly-P and pppGpp hydrolysis, whereas PPX2/GPPA protein has monofunctional activity and mainly involved in poly-P hydrolysis. Thus, theses enzymes play critical role in poly-P metabolism and ppGpp homeostasis. Beside their role in stress related phenotypic traits they do have role in complement mediated resistance in human serum. Our study expands the multi-factorial regulation of poly-P and ppGpp metabolism in C. jejuni, may serve as unique model for other bacteria as well.
Gireesh Rajashekara, PhD (Advisor)
Yehia Saif, PhD (Committee Member)
Daral Jackwood, PhD (Committee Member)
Jordi Torrelles, PhD (Committee Member)
78 p.
Veterinary Services
ppGpp; PPX; poly-P

Recommended Citations

Citations

  • Kumar, A. (2012). Functional Characterization of Exopolyphosphatase/ Guanosine Pentaphosphate Phosphohydrolase (PPX/GPPA) Enzymes of Campylobacter jejuni [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339085410

    APA Style (7th edition)

  • Kumar, Anand. Functional Characterization of Exopolyphosphatase/ Guanosine Pentaphosphate Phosphohydrolase (PPX/GPPA) Enzymes of Campylobacter jejuni. 2012. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1339085410.

    MLA Style (8th edition)

  • Kumar, Anand. "Functional Characterization of Exopolyphosphatase/ Guanosine Pentaphosphate Phosphohydrolase (PPX/GPPA) Enzymes of Campylobacter jejuni." Master's thesis, Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1339085410

    Chicago Manual of Style (17th edition)

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