Polyphosphate Kinase 2: A Novel Determinant of Stress Responses and Pathogenesis in Campylobacter jejuni

Abstract

BackgroundInorganic 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 FindingsHere, 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 Δppk2 mutant exhibited a significant survival defect under osmotic, nutrient, aerobic, and antimicrobial stresses and displayed an enhanced ability to form static biofilms. However, the Δppk2 mutant was not defective in poly P and ppGpp synthesis suggesting that PPK2-mediated stress tolerance is not ppGpp-mediated. Importantly, the Δppk2 mutant was significantly attenuated in invasion and intracellular survival within human intestinal epithelial cells as well as in chicken colonization.Conclusions/SignificanceTaken 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.

Transcriptome analysis ofCampylobacter jejunipolyphosphate kinase (ppk1andppk2) mutants

“…Here, we conducted RNA-Seq analysis on C. jejuni 81-176 (parental strain) and its ppk1 and ppk2 mutants. [4][5] We found that both ppk1 and ppk2 affected the expression of multiple genes which can be linked to these pleiotropic phenotypes. Our results further highlight the importance of poly-P metabolism in the survival and adaptation of C. jejuni.…”

“…11 For example, we have shown that the impairment of the ppks (ppk1 and ppk2) can affect a wide range of phenotypes in C. jejuni, [4][5][6] including biofilm formation, and the resistance to osmotic, nutrient, and antimicrobial stresses. In addition, mutants show decreased ability to colonize chickens and infect human intestine epithelial cells in vitro.…”

“…4,5,[16][17] For example, 250 genes were up-regulated and more than 450 genes were down-regulated in a ppk1 mutant of Pseudomonas aeruginosa. 11 Another study showed that 81 genes were differentially expressed in a poly-P-deficient mutant of Pseudomonas sp.…”

“…[4][5] The strains were grown in Mueller-Hinton broth (MH, Fisher Scientific, Pennsylvania, USA) under microaerobic conditions (85% N 2 , 10% CO 2 , 5% O 2 ) at 42 C with shaking (200 rpm) and harvested at both log (8 h) and stationary (18 h) phases of growth. RNA was isolated from both phases of growth for each strain as described previously.…”

“…However, the Dppk2 showed decreased survival under aerobic stress, and the KatA has been shown to contribute to survival under aerobic stress. 5,34 Furthermore, the expression of the gene encoding DcuB (CJJ81176_RS03255), a protein that mediates aspartate transport under oxygen limitation and fumarate: succinate exchange during fumarate respiration, 35 was upregulated in the Dppk2 during log phase ( Table 2). It has been previously observed that dcuB was upregulated under acid shock conditions, and the authors speculated that dcuB might contribute to resisting cytoplasm acidification in C. jejuni.…”