Polyphosphate kinase 2: a novel determinant of stress responses and pathogenesis in Campylobacter jejuni

Abstract

Background: Inorganic polyphosphate (poly P) plays an important role in stress tolerance and virulence in many bacteria. PPK1 is the principal enzyme involved in poly P synthesis, while PPK2 uses poly P to generate GTP, a signaling molecule that serves as an alternative energy source and a precursor for various physiological processes. Campylobacter jejuni, an important cause of foodborne gastroenteritis in humans, possesses homologs of both ppk1 and ppk2. ppk1 has been previously shown to impact the pathobiology of C. jejuni.

Methodology/principal findings: Here, we demonstrate for the first time that the deletion of ppk2 in C. jejuni resulted in a significant decrease in poly P-dependent GTP synthesis, while displaying an increased intracellular ATP:GTP ratio. The Deltappk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Deltappk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Deltappk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization.

Conclusions/significance: Taken together, we have highlighted the role of PPK2 as a novel pathogenicity determinant that is critical for C. jejuni survival, adaptation, and persistence in the host environments. PPK2 is absent in humans and animals; therefore, can serve as a novel target for therapeutic intervention of C. jejuni infections.

Figure 1. Poly P-dependent GTP and ATP synthesis in the Δppk2 mutant.

(A) The Δppk2 mutant is defective in poly P-dependent GTP synthesis. GTP synthetic activity was determined spectrophotometrically using a modified enzyme coupled assay. (B) The Δppk2 mutant exhibits increased intracellular ATP:GTP ratio. Poly P-dependent ATP synthesis was determined spectrophotometrically using a modified enzyme coupled assay. ATP synthesis was measured for up to 10 min. Only data for 5 and 10 min are shown. Both assays were repeated 3 times with 3 replicates in each assay and the data was expressed as mean±SE. * P≤0.05.

Figure 2. ppGpp and poly P levels in the Δppk2 mutant.

(A) The Δppk2 mutant shows similar ppGpp levels to that of WT under nutrient downshift. The amount of ppGpp accumulation was assessed by growing the bacterial strains in MH broth microaerobically to early log phase (OD₆₀₀ of 0.25) and labeling in nutrient poor medium (MOPS-MGS) with ³²P. Nucleotides were resolved by TLC and visualized using autoradiography. (B) Poly P accumulation in the WT and Δppk2 mutant at different growth phases. The amount of poly P in the cell was determined by toluidine blue O method. Each data point is the mean±SE of 3 independent experiments. ** P≤0.01.

Figure 3. Survival of the Δppk2 mutant under nutrient, osmotic and aerobic stresses.

(A) Survival of C. jejuni under nutrient downshift. Survival under nutrient stress was assessed by growing bacterial strains in MEM and determining the CFU at different time points. (B) Survival of C. jejuni under osmotic stress. Survival under osmotic stress in liquid culture was assessed in MH broth containing 0.25 M NaCl. (C) Survival of C. jejuni under osmotic stress in solid medium. Survival in solid medium was determined by spotting 5×10²–5×10⁵ CFU onto MH agar containing 0.17 M NaCl. (D) Survival of C. jejuni under aerobic stress. Survival of the C. jejuni 81–176 WT and Δppk2 mutant was assessed by determining the CFU of aerobically grown bacteria at different time points. For all assays, each data point represents the mean±SE of 3 independent experiments. * P≤0.05 and ** P≤0.01.

Figure 4. Culturability and viability of the C. jejuni strains after formic acid treatment.

Culturability was determined by plating formic acid treated bacteria on MH agar at different time points. Viability was assessed by measuring OD₆₀₀ after CTC staining. Each data point represents the mean±SE of 3 independent experiments. * P≤0.05.

Figure 5. Biofilm formation in the Δppk2 mutant.

(A) The Δppk2 mutant shows enhanced biofilm formation. Biofilm formation as assessed by crystal violet staining. (B) Quantification of biofilm using DMSO. The amount of biofilm formed was dissolved in 1 ml DMSO for 48 h and quantified by measuring absorbance at 570 nm. Each bar represents the mean±SE of 3 independent experiments. ** P≤0.01.

Figure 6. Quantitative RT-PCR analysis of the WT and Δppk2 mutant.

Fold differences in transcript levels were assessed from ΔΔCT after normalization using rpoA. Each bar represents the mean±SE of relative fold change in expression from 3 independent experiments. * P≤0.05.

Figure 7. Invasion and intracellular survival of the Δppk2 mutant in INT407 cells.

(A) The Δppk2 mutant displays a dose-dependent invasion defect in INT407 human intestinal epithelial cells. The data represents the average of 2 experiments with 3 replicates in each experiment. * P≤0.05. (B) The Δppk2 mutant is defective in intracellular survival in INT407 cells. INT407 cells were infected with 100∶1 MOI of bacteria. Intracellular survival of C. jejuni was determined after 24 h of incubation. The data represents the average of 2 independent experiments with 3 replicates in each experiment. ** P≤0.01.

Figure 8. Colonization of the Δppk2 mutant in chickens.

The Δppk2 mutant exhibits a dose-dependent colonization defect in day-old chicks. Eight days after inoculation, the chicks were sacrificed; cecum, feces and bursa were harvested and colonization level was assessed by determining the CFU/g of tissue. Each data point represents log₁₀ CFU/g of tissue. Average CFU for each dose is denoted by a line. The dotted line indicates minimum detection limit of 50 CFU. * P≤0.05 and ** P≤0.01.