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 ( increment ppx1, increment ppx2) and the double knockout mutant (dkppx), all increment ppx mutants exhibited increased capacity to accumulate poly-P; however only increment 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 increment 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 increment ppx mutants. The ppk1 and spoT were upregulated in both increment ppx1 and increment 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 MgCl2 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 increment 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.