PMID:24569519

Citation	Malde, A, Gangaiah, D, Chandrashekhar, K, Pina-Mimbela, R, Torrelles, JB and Rajashekara, G (2014) Functional characterization of exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) of Campylobacter jejuni. Virulence 5:521-33
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.
Links	PubMed PMC4063813 Online version:10.4161/viru.28311
Keywords	Acid Anhydride Hydrolases/genetics; Acid Anhydride Hydrolases/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biofilms; Campylobacter Infections/immunology; Campylobacter Infections/microbiology; Campylobacter jejuni/enzymology; Campylobacter jejuni/genetics; Campylobacter jejuni/physiology; Cell Line; Complement System Proteins/immunology; Humans; Pyrophosphatases/genetics; Pyrophosphatases/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:A0A0K2D0I8		GO:0004309: exopolyphosphatase activity	ECO:0000315:		F		to test the motility and biofilm formation of C.jejuni delta ppx mutants, the author assessed the motility on 0.4% soft agar at 42C for 24h. motility was quantified by measuring the swarming zone around the stabbed area. the result indicate that deleting the gene ppx in C.jejuni affect the amount of biofilm, therefore its affect the motility. also, ppx/gppa genes play a role in transmission related phenotypes such as motility and biofilm formation in C.jejuni.	complete CACAO 11488
9CAUD:A0A0K2D0I8		GO:0004309: exopolyphosphatase activity	ECO:0000317:	UniProtKB:904677	F		the phylogenetic analysis using MEGA-5 showed that C. jejuni PPX2 clustered with E.coli and mycobacterium tuberculosis species. (fig.S1) also HHpred show that gene TSARBOMBA_148 homologues to Mycobacterium tuberculosis with e-value= 8.8e-28 and probable 99.96.	complete CACAO 11490
CAMJJ:A0A0H3PES2		GO:0071978: bacterial-type flagellum-dependent swarming motility	ECO:0000315:		P		Figure 4. Motility and biofilm formation of C. jejuni ∆ppx mutants. (A) Motility was assessed on 0.4% soft agar at 42 °C for 24 h. (B) Motility was quantified by measuring the swarming zone (diameter in cm) surrounding the stabbed area. Each bar represents the average from 3 independent experiments with duplicate samples. *P ≤ 0.05 **P ≤ 0.001.	complete CACAO 11525

References

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