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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.

SUBMITTER: Gangaiah D

PROVIDER: S-EPMC2923150 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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

Gangaiah Dharanesh D Liu Zhe Z Arcos Jesús J Kassem Issmat I II Sanad Yasser Y Torrelles Jordi B JB Rajashekara Gireesh G

PloS one 20100817 8

<h4>Background</h4>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 t ...[more]

PMID: 20808906

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Project description:Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, relatively little is known about C. jejuni's precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric organisms such as Escherichia coli and Salmonella spp. Altered expression of phosphate genes in a C. jejuni stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly-P), and virulence in other bacteria, led us to investigate the role of poly-P in C. jejuni stress survival and pathogenesis. All sequenced C. jejuni strains harbor a conserved putative polyphosphate kinase 1 predicted to be principally responsible for poly-P synthesis. We generated a targeted ppk1 deletion mutant (Deltappk1) in C. jejuni strain 81-176 and found that Deltappk1, as well as the DeltaspoT stringent response mutant, exhibited low levels of poly-P at all growth stages. In contrast, wild-type C. jejuni poly-P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the Deltappk1 mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with ppk1 deletion in other bacteria (i.e., motility and oxidative stress) were unaffected in the C. jejuni Deltappk1 mutant, which also displayed an unexpected increase in biofilm formation. The C. jejuni Deltappk1 mutant was also defective for the virulence-associated phenotype of intraepithelial cell survival in a tissue culture infection model and exhibited a striking, dose-dependent chick colonization defect. These results indicate that poly-P utilization and accumulation contribute significantly to C. jejuni pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly-P likely plays both similar and unique roles in C. jejuni compared to its roles in other bacteria and that poly-P metabolism is linked to stringent response mechanisms in C. jejuni.

Project description:Campylobacter jejuni is a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that a C. jejuni strain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required for C. jejuni pathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaF and lgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed DeltagalT and DeltacstII mutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (DeltawaaF and DeltalgtF but not DeltagalT or DeltacstII mutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (DeltacstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active against C. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The DeltawaaF mutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect of C. jejuni pathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of the C. jejuni LOS in host colonization. Collectively, this study has uncovered novel roles for the C. jejuni LOS, highlights the dynamic nature of the C. jejuni cell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

Dataset Information

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

Publications

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

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