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FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection.

ABSTRACT: Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ?fdhU ?fdhA double mutant were similar to those of a ?fdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ?fdhU and ?fdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.

SUBMITTER: Pryjma M

PROVIDER: S-EPMC3416569 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection.

Pryjma Mark M Apel Dmitry D Huynh Steven S Parker Craig T CT Gaynor Erin C EC

Journal of bacteriology 20120525 15

Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. De ...[more]

PMID: 22636777

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Project description:Chemotaxis is an important virulence factor in some enteric pathogens, and it is involved in the pathogenesis and colonization of the host. However, there is limited knowledge regarding the environmental signals that promote chemotactic behavior and the sensing of these signals by chemoreceptors. To date, there is no information on the ligand molecule that directly binds to and is sensed by Campylobacter jejuni Tlp1, which is a chemoreceptor with a dCache-type ligand-binding domain (LBD). dCache (double Calcium channels and chemotaxis receptor) is the largest group of sensory domains in bacteria, but the dCache-type chemoreceptor that directly binds to formate has not yet been discovered. In this study, formate was identified as a direct-binding ligand of C. jejuni Tlp1 with high sensing specificity. We used the strategy of constructing a functional hybrid receptor of C. jejuni Tlp1 and the Escherichia coli chemoreceptor Tar to screen for the potential ligand of Tlp1, with the binding of formate to Tlp1-LBD being verified using isothermal titration calorimetry. Molecular docking and experimental analyses indicated that formate binds to the membrane-proximal pocket of the dCache subdomain. Chemotaxis assays demonstrated that formate elicits robust attractant responses of the C. jejuni strain NCTC 11168, specifically via Tlp1. The chemoattraction effect of formate via Tlp1 promoted the growth of C. jejuni, especially when competing with Tlp1- or CheY-knockout strains. Our study reveals the molecular mechanisms by which C. jejuni mediates chemotaxis toward formate, and, to our knowledge, is the first report on the high-specificity binding of the dCache-type chemoreceptor to formate as well as the physiological role of chemotaxis toward formate. IMPORTANCE Chemotaxis is important for Campylobacter jejuni to colonize favorable niches in the gastrointestinal tract of its host. However, there is still a lack of knowledge about the ligand molecules for C. jejuni chemoreceptors. The dCache-type chemoreceptor, namely, Tlp1, is the most conserved chemoreceptor in C. jejuni strains; however, the direct-binding ligand(s) triggering chemotaxis has not yet been discovered. In the present study, we found that the ligand that binds directly to Tlp1-LBD with high specificity is formate. C. jejuni exhibits robust chemoattraction toward formate, primarily via Tlp1. Tlp1 is the first reported dCache-type chemoreceptor that specifically binds formate and triggers strong chemotaxis. We further demonstrated that the formate-mediated promotion of C. jejuni growth is correlated with Tlp1-mediated chemotaxis toward formate. Our work provides important insights into the mechanism and physiological function of chemotaxis toward formate and will facilitate further investigations into the involvement of microbial chemotaxis in pathogen-host interactions.

Project description:Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Dataset Information

FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection.

Publications

FdhTU-modulated formate dehydrogenase expression and electron donor availability enhance recovery of Campylobacter jejuni following host cell infection.

Similar Datasets

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