Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni.

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.