title: Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni.
abstract: The inorganic polyphosphate (poly-P) is a key regulator of stress responses and virulence in many bacterial pathogens including Campylobacter jejuni. The role of exopolyphosphatases/guanosine pentaphosphate (pppGpp) phosphohydrolases (PPX/GPPA) in poly-P homeostasis and C. jejuni pathobiology remains unexplored. Here, we analyzed deletion mutants (∆ppx1, ∆ppx2) and the double knockout mutant (dkppx), all ∆ppx mutants exhibited increased capacity to accumulate poly-P; however only ∆ppx1 and dkppx mutants showed decreased accumulation of ppGpp, an alarmone molecule that regulates stringent response in bacteria, suggesting potential dual role for PPX1/GPPA. Nutrient survival defect of ∆ppx mutants was rescued by the supplementation of specific amino acids implying that survival defect may be associated with decreased ppGpp and/ or increased poly-P in ∆ppx mutants. The ppk1 and spoT were upregulated in both ∆ppx1 and ∆ppx2 suggesting a compensatory role for SpoT and Ppk1 in poly-P and ppGpp homeostasis. The lack of ppx genes resulted in defects in motility, biofilm formation, nutrient stress survival, invasion and intracellular survival indicating that maintaining a certain level of poly-P is critical for ppx genes in C. jejuni pathophysiology. Both ppx1 and ppx2 mutants were resistant to human complement-mediated killing; however, the dkppx mutant was sensitive. The serum susceptibility did not occur in the presence of MgCl 2 and EGTA suggesting an involvement of the classical or lectin pathway of complement mediated killing. Interestingly, the chicken serum did not have any effect on the ∆ppx mutants' survival. The observed serum susceptibility was not related to C. jejuni surface capsule and lipooligosaccharide structures. Our study underscores the importance of PPX/GPPA proteins in poly-P and ppGpp homeostasis, two critical molecules that modulate environmental stress responses and virulence in C. jejuni.
authors.affiliation: Food Animal Health Research Program; Department of Veterinary Preventive Medicine; College of Veterinary Medicine; The Ohio State University; Wooster, OH USA.
authors.forename: Anandkumar
authors.initials: A
authors.lastname: Malde
authors.affiliation: Food Animal Health Research Program; Department of Veterinary Preventive Medicine; College of Veterinary Medicine; The Ohio State University; Wooster, OH USA.
authors.forename: Dharanesh
authors.initials: D
authors.lastname: Gangaiah
authors.affiliation: Food Animal Health Research Program; Department of Veterinary Preventive Medicine; College of Veterinary Medicine; The Ohio State University; Wooster, OH USA.
authors.forename: Kshipra
authors.initials: K
authors.lastname: Chandrashekhar
authors.affiliation: Food Animal Health Research Program; Department of Veterinary Preventive Medicine; College of Veterinary Medicine; The Ohio State University; Wooster, OH USA.
authors.forename: Ruby
authors.initials: R
authors.lastname: Pina-Mimbela
authors.affiliation: Department of Microbial Infection and Immunity; Center for Microbial Interface Biology; The Ohio State University; Columbus, OH USA.
authors.forename: Jordi B
authors.initials: JB
authors.lastname: Torrelles
authors.affiliation: Food Animal Health Research Program; Department of Veterinary Preventive Medicine; College of Veterinary Medicine; The Ohio State University; Wooster, OH USA.
authors.forename: Gireesh
authors.initials: G
authors.lastname: Rajashekara
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms; D002169:Campylobacter Infections / Q000276:immunology / Q000382:microbiology*; D016123:Campylobacter jejuni / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D003165:Complement System Proteins / Q000276:immunology; D006801:Humans; D011755:Pyrophosphatases / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003165:Complement System Proteins; D017766:Acid Anhydride Hydrolases; D011755:Pyrophosphatases; C020137:exopolyphosphatase; C082499:guanosine-5'-triphosphate, 3'-diphosphate pyrophosphatase
keywords: Campylobacter jejuni; GPPA; PPX; poly-P; ppGpp; stress response