D-aspartate, an amino-acid important for human health, supports anaerobic respiration in several Campylobacter species.

Despite being classified as microaerophilic microorganisms, most Campylobacter species can grow anaerobically, using formate or molecular hydrogen (H2) as electron donors, and various nitrogenous and sulfurous compounds as electron acceptors. Herein, we showed that both L-asparagine (L-Asn) and L-aspartic acid (L-Asp) bolster H2-driven anaerobic growth in several Campylobacter species, whereas the D-enantiomer form of both asparagine (D-Asn) and aspartic acid (D-Asp) only increased anaerobic growth in C. concisus strain 13826 and C. ureolyticus strain NCTC10941. A gene annotated as racD encoding for a putative D/L-Asp racemase was identified in the genome of both strains. Disruption of racD in Cc13826 resulted in the inability of the mutant strain to use either D-enantiomer during anaerobic growth. Hence, our results suggest that the racD gene is required for campylobacters to use either D-Asp or D-Asn. The use of D-Asp by various human opportunistic bacterial pathogens, including C. concisus, C. ureolyticus, and also possibly select strains of C. gracilis, C. rectus and C. showae, is significant, because D-Asp is an important signal molecule for both human nervous and neuroendocrine systems. To our knowledge, this is the first report of pathogens scavenging a D-amino acid essential for human health.

The Campylobacter concisus BisA protein plays a dual role: oxide-dependent anaerobic respiration and periplasmic methionine sulfoxide repair.

IMPORTANCE: Campylobacter concisus is an excellent model organism to study respiration diversity, including anaerobic respiration of physiologically relevant N-/S-oxides compounds, such as biotin sulfoxide, dimethyl sulfoxide, methionine sulfoxide (MetO), nicotinamide N-oxide, and trimethylamine N-oxide. All C. concisus strains harbor at least two, often three, and up to five genes encoding for putative periplasmic Mo/W-bisPGD-containing N-/S-oxide reductases. The respective role (substrate specificity) of each enzyme was studied using a mutagenesis approach. One of the N/SOR enzymes, annotated as "BisA", was found to be essential for anaerobic respiration of both N- and S-oxides. Additional phenotypes associated with disruption of the bisA gene included increased sensitivity toward oxidative stress and elongated cell morphology. Furthermore, a biochemical approach confirmed that BisA can repair protein-bound MetO residues. Hence, we propose that BisA plays a role as a periplasmic methionine sulfoxide reductase. This is the first report of a Mo/W-bisPGD-enzyme supporting both N- or S-oxide respiration and protein-bound MetO repair in a pathogen.

Restoring and Enhancing the Potency of Existing Antibiotics against Drug-Resistant Gram-Negative Bacteria through the Development of Potent Small-Molecule Adjuvants.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

Copper toxicity towards Campylobacter jejuni is enhanced by the nickel chelator dimethylglyoxime.

The nickel (Ni)-chelator dimethylglyoxime (DMG) was found to be bacteriostatic towards Campylobacter jejuni. Supplementation of nickel to DMG-containing media restored bacterial growth, whereas supplementation of cobalt or zinc had no effect on the growth inhibition. Unexpectedly, the combination of millimolar levels of DMG with micromolar levels of copper (Cu) was bactericidal, an effect not seen in select Gram-negative pathogenic bacteria. Both the cytoplasmic Ni-binding chaperone SlyD and the twin arginine translocation (Tat)-dependent periplasmic copper oxidase CueO were found to play a central role in the Cu-DMG hypersensitivity phenotype. Ni-replete SlyD is needed for Tat-dependent CueO translocation to the periplasm, whereas Ni-depleted (DMG-treated) SlyD is unable to interact with the CueO Tat signal peptide, leading to mislocalization of CueO and increased copper sensitivity. In support of this model, C. jejuni ΔslyD and ΔcueO mutants were more sensitive to copper than the wild-type (WT); CueO was less abundant in the periplasmic fraction of ΔslyD or DMG-grown WT cells, compared to WT cells grown on plain medium; SlyD binds the CueO signal sequence peptide, with DMG inhibiting and nickel enhancing the binding, respectively. Injection of Cu-DMG into Galleria mellonella before C. jejuni inoculation significantly increased the insect survival rate compared to the control group. In chickens, oral administration of DMG or Cu-DMG decreased and even abolished C. jejuni colonization in some cases, compared to both water-only and Cu-only control groups. The latter finding is important, since campylobacteriosis is the leading bacterial foodborne infection, and chicken meat constitutes the major foodborne source.

A two-hybrid system reveals previously uncharacterized protein-protein interactions within the Helicobacter pylori NIF iron-sulfur maturation system.

Iron-sulfur (Fe-S) proteins play essential roles in all living organisms. The gastric pathogen Helicobacter pylori relies exclusively on the NIF system for biosynthesis and delivery of Fe-S clusters. Previously characterized components include two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein), and a dispensable Fe-S carrier, Nfu. Among 38 proteins previously predicted to coordinate Fe-S clusters, two proteins, HP0207 (a member of the Nbp35/ApbC ATPase family) and HP0277 (previously annotated as FdxA, a member of the YfhL ferredoxin-like family) were further studied, using a bacterial two-hybrid system approach to identify protein-protein interactions. ApbC was found to interact with 30 proteins, including itself, NifS, NifU, Nfu and FdxA, and alteration of the conserved ATPase motif in ApbC resulted in a significant (50%) decrease in the number of protein interactions, suggesting the ATpase activity is needed for some ApbC-target protein interactions. FdxA was shown to interact with 21 proteins, including itself, NifS, ApbC and Nfu, however no interactions between NifU and FdxA were detected. By use of cross-linking studies, a 51-kDa ApbC-Nfu heterodimer complex was identified. Attempts to generate apbC chromosomal deletion mutants in H. pylori were unsuccessful, therefore indirectly suggesting the hp0207 gene is essential. In contrast, mutants in the fdxA gene were obtained, albeit only in one parental strain (26695). Taken together, these results suggest both ApbC and FdxA are important players in the H. pylori NIF maturation system.

The nickel-chelator dimethylglyoxime inhibits human amyloid beta peptide in vitro aggregation.

One of the hallmarks of the most common neurodegenerative disease, Alzheimer's disease (AD), is the extracellular deposition and aggregation of Amyloid Beta (Aß)-peptides in the brain. Previous studies have shown that select metal ions, most specifically copper (Cu) and zinc (Zn) ions, have a synergistic effect on the aggregation of Aß-peptides. In the present study, inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the metal content of a commercial recombinant human Aß40 peptide. Cu and Zn were among the metals detected; unexpectedly, nickel (Ni) was one of the most abundant elements. Using a fluorescence-based assay, we found that Aß40 peptide in vitro aggregation was enhanced by addition of Zn2+ and Ni2+, and Ni2+-induced aggregation was facilitated by acidic conditions. Nickel binding to Aß40 peptide was confirmed by isothermal titration calorimetry. Addition of the Ni-specific chelator dimethylglyoxime (DMG) inhibited Aß40 aggregation in absence of added metal, as well as in presence of Cu2+ and Ni2+, but not in presence of Zn2+. Finally, mass spectrometry analysis revealed that DMG can coordinate Cu or Ni, but not Fe, Se or Zn. Taken together, our results indicate that Ni2+ ions enhance, whereas nickel chelation inhibits, Aß peptide in vitro aggregation. Hence, DMG-mediated Ni-chelation constitutes a promising approach towards inhibiting or slowing down Aß40 aggregation.

Highly Efficient Antimicrobial Activity of CuxFeyOz Nanoparticles against Important Human Pathogens.

The development of innovative antimicrobial materials is crucial in thwarting infectious diseases caused by microbes, as drug-resistant pathogens are increasing in both number and capacity to detoxify the antimicrobial drugs used today. An ideal antimicrobial material should inhibit a wide variety of bacteria in a short period of time, be less or not toxic to normal cells, and the fabrication or synthesis process should be cheap and easy. We report a one-step microwave-assisted hydrothermal synthesis of mixed composite CuxFeyOz (Fe2O3/Cu2O/CuO/CuFe2O) nanoparticles (NPs) as an excellent antimicrobial material. The 1 mg/mL CuxFeyOz NPs with the composition 36% CuFeO2, 28% Cu2O and 36% Fe2O3 have a general antimicrobial activity greater than 5 log reduction within 4 h against nine important human pathogenic bacteria (including drug-resistant bacteria as well as Gram-positive and Gram-negative strains). For example, they induced a >9 log reduction in Escherichia coli B viability after 15 min of incubation, and an ~8 log reduction in multidrug-resistant Klebsiella pneumoniae after 4 h incubation. Cytotoxicity tests against mouse fibroblast cells showed about 74% viability when exposed to 1 mg/mL CuxFeyOz NPs for 24 h, compared to the 20% viability for 1 mg/mL pure Cu2O NPs synthesized by the same method. These results show that the CuxFeyOz composite NPs are a highly efficient, low-toxicity and cheap antimicrobial material that has promising potential for applications in medical and food safety.

Influence of Protein Glycosylation on Campylobacter fetus Physiology.

Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

Molecular Hydrogen Metabolism: a Widespread Trait of Pathogenic Bacteria and Protists.

Pathogenic microorganisms use various mechanisms to conserve energy in host tissues and environmental reservoirs. One widespread but often overlooked means of energy conservation is through the consumption or production of molecular hydrogen (H2). Here, we comprehensively review the distribution, biochemistry, and physiology of H2 metabolism in pathogens. Over 200 pathogens and pathobionts carry genes for hydrogenases, the enzymes responsible for H2 oxidation and/or production. Furthermore, at least 46 of these species have been experimentally shown to consume or produce H2 Several major human pathogens use the large amounts of H2 produced by colonic microbiota as an energy source for aerobic or anaerobic respiration. This process has been shown to be critical for growth and virulence of the gastrointestinal bacteria Salmonella enterica serovar Typhimurium, Campylobacter jejuni, Campylobacter concisus, and Helicobacter pylori (including carcinogenic strains). H2 oxidation is generally a facultative trait controlled by central regulators in response to energy and oxidant availability. Other bacterial and protist pathogens produce H2 as a diffusible end product of fermentation processes. These include facultative anaerobes such as Escherichia coli, S Typhimurium, and Giardia intestinalis, which persist by fermentation when limited for respiratory electron acceptors, as well as obligate anaerobes, such as Clostridium perfringens, Clostridioides difficile, and Trichomonas vaginalis, that produce large amounts of H2 during growth. Overall, there is a rich literature on hydrogenases in growth, survival, and virulence in some pathogens. However, we lack a detailed understanding of H2 metabolism in most pathogens, especially obligately anaerobic bacteria, as well as a holistic understanding of gastrointestinal H2 transactions overall. Based on these findings, we also evaluate H2 metabolism as a possible target for drug development or other therapies.

Author Correction: Nickel chelation therapy as an approach to combat multi-drug resistant enteric pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nickel chelation therapy as an approach to combat multi-drug resistant enteric pathogens.

The nickel (Ni)-specific chelator dimethylglyoxime (DMG) has been used for many years to detect, quantitate or decrease Ni levels in various environments. Addition of DMG at millimolar levels has a bacteriostatic effect on some enteric pathogens, including multidrug resistant (MDR) strains of Salmonella Typhimurium and Klebsiella pneumoniae. DMG inhibited activity of two Ni-containing enzymes, Salmonella hydrogenase and Klebsiella urease. Oral delivery of nontoxic levels of DMG to mice previously inoculated with S. Typhimurium led to a 50% survival rate, while 100% of infected mice in the no-DMG control group succumbed to salmonellosis. Pathogen colonization numbers from livers and spleens of mice were 10- fold reduced by DMG treatment of the Salmonella-infected mice. Using Nuclear Magnetic Resonance, we were able to detect DMG in the livers of DMG-(orally) treated mice. Inoculation of Galleria mellonella (wax moth) larvae with DMG prior to injection of either MDR K. pneumoniae or MDR S. Typhimurium led to 40% and 60% survival, respectively, compared to 100% mortality of larvae infected with either pathogen, but without prior DMG administration. Our results suggest that DMG-mediated Ni-chelation could provide a novel approach to combat enteric pathogens, including recalcitrant multi-drug resistant strains.

Site-directed mutagenesis of Campylobacter concisus respiratory genes provides insight into the pathogen's growth requirements.

Campylobacter concisus is an emerging human pathogen found throughout the entire human oral-gastrointestinal tract. The ability of C. concisus to colonize diverse niches of the human body indicates the pathogen is metabolically versatile. C. concisus is able to grow under both anaerobic conditions and microaerophilic conditions. Hydrogen (H2) has been shown to enhance growth and may even be required. Analysis of several C. concisus genome sequences reveals the presence of two sets of genes encoding for distinct hydrogenases: a H2-uptake-type ("Hyd") complex and a H2-evolving hydrogenase ("Hyf"). Whole cells hydrogenase assays indicate that the former (H2-uptake) activity is predominant in C. concisus, with activity among the highest we have found for pathogenic bacteria. Attempts to generate site-directed chromosomal mutants were partially successful, as we could disrupt hyfB, but not hydB, suggesting that H2-uptake, but not H2-evolving activity, is an essential respiratory pathway in C. concisus. Furthermore, the tetrathionate reductase ttrA gene was inactivated in various C. concisus genomospecies. Addition of tetrathionate to the medium resulted in a ten-fold increase in cell yield for the WT, while it had no effect on the ttrA mutant growth. To our knowledge, this is the first report of mutants in C. concisus.

Noncatalytic Antioxidant Role for Helicobacter pylori Urease.

The well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protect Helicobacter pylori against the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureAB deletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protect H. pylori against oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purified H. pylori urease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCE Chronic Helicobacter pylori infection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid, H. pylori must survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge of H. pylori survival mechanisms.

Iron-sulfur protein maturation in Helicobacter pylori: identifying a Nfu-type cluster carrier protein and its iron-sulfur protein targets.

Helicobacter pylori is anomalous among non nitrogen-fixing bacteria in containing an incomplete NIF system for Fe-S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU-depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild-type (WT) strains. The hp1492 gene, encoding a putative Nfu-type Fe-S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe-2S] or [4Fe-4S] cluster/dimer, based on analytical, UV-visible absorption/CD and resonance Raman studies. A bacterial two-hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe-S-containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe-S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe-S clusters in H. pylori.

Click, Release, and Fluoresce: A Chemical Strategy for a Cascade Prodrug System for Codelivery of Carbon Monoxide, a Drug Payload, and a Fluorescent Reporter.

A chemical strategy was developed wherein a single trigger sets in motion a three-reaction cascade leading to the release of more than one drug-component in sequence with the generation of a fluorescent side product for easy monitoring. As a proof of concept, codelivery of CO with the antibiotic metronidazole was demonstrated.

Clinical and genetic heterogeneity in familial steroid-sensitive nephrotic syndrome.

BACKGROUND: Familial steroid-sensitive nephrotic syndrome (SSNS) is a rare condition. The disease pathophysiology remains elusive. However, bi-allelic mutations in the EMP2 gene were identified, and specific variations in HLA-DQA1 were linked to a high risk of developing the disease. METHODS: Clinical data were analyzed in 59 SSNS families. EMP2 gene was sequenced in families with a potential autosomal recessive (AR) inheritance. Exome sequencing was performed in a subset of 13 families with potential AR inheritance. Two variations in HLA-DQA1 were genotyped in the whole cohort. RESULTS: Transmission was compatible with an AR (n = 33) or autosomal dominant (AD, n = 26) inheritance, assuming that familial SSNS is a monogenic trait. Clinical features did not differ between AR and AD groups. All patients, including primary (n = 7) and secondary steroid resistant nephrotic syndrone (SRNS), (n = 13) were sensitive to additional immunosuppressive therapy. Both HLA-DQA1 variations were found to be highly linked to the disease (OR = 4.34 and OR = 4.89; p < 0.001). Exome sequencing did not reveal any pathogenic mutation, neither did EMP2 sequencing. CONCLUSIONS: Taken together, these results highlight the clinical and genetic heterogeneity in familial SSNS. Clinical findings sustain an immune origin in all patients, whatever the initial steroid-sensitivity. The absence of a variant shared by two families and the HLA-DQA1 variation enrichments suggest a complex mode of inheritance.

Structure-function analyses of metal-binding sites of HypA reveal residues important for hydrogenase maturation in Helicobacter pylori.

The nickel-containing enzymes of Helicobacter pylori, urease and hydrogenase, are essential for efficient colonization in the human stomach. The insertion of nickel into urease and hydrogenase is mediated by the accessory protein HypA. HypA contains an N-terminal nickel-binding site and a dynamic structural zinc-binding site. The coordination of nickel and zinc within HypA is known to be critical for urease maturation and activity. Herein, we test the hydrogenase activity of a panel of H. pylori mutant strains containing point mutations within the nickel- and zinc-binding sites. We found that the residues that are important for hydrogenase activity are those that were similarly vital for urease activity. Thus, the zinc and metal coordination sites of HypA play similar roles in urease and hydrogenase maturation. In other pathogenic bacteria, deletion of hydrogenase leads to a loss in acid resistance. Thus, the acid resistance of two strains of H. pylori containing a hydrogenase deletion was also tested. These mutant strains demonstrated wild-type levels of acid resistance, suggesting that in H. pylori, hydrogenase does not play a role in acid resistance.

Helicobacter Catalase Devoid of Catalytic Activity Protects the Bacterium against Oxidative Stress.

Catalase, a conserved and abundant enzyme found in all domains of life, dissipates the oxidant hydrogen peroxide (H2O2). The gastric pathogen Helicobacter pylori undergoes host-mediated oxidant stress exposure, and its catalase contains oxidizable methionine (Met) residues. We hypothesized catalase may play a large stress-combating role independent of its classical catalytic one, namely quenching harmful oxidants through its recyclable Met residues, resulting in oxidant protection to the bacterium. Two Helicobacter mutant strains (katAH56A and katAY339A) containing catalase without enzyme activity but that retain all Met residues were created. These strains were much more resistant to oxidants than a catalase-deletion mutant strain. The quenching ability of the altered versions was shown, whereby oxidant-stressed (HOCl-exposed) Helicobacter retained viability even upon extracellular addition of the inactive versions of catalase, in contrast to cells receiving HOCl alone. The importance of the methionine-mediated quenching to the pathogen residing in the oxidant-rich gastric mucus was studied. In contrast to a catalase-null strain, both site-change mutants proficiently colonized the murine gastric mucosa, suggesting that the amino acid composition-dependent oxidant-quenching role of catalase is more important than the well described H2O2-dissipating catalytic role. Over 100 years after the discovery of catalase, these findings reveal a new non-enzymatic protective mechanism of action for the ubiquitous enzyme.

Hydrogen Metabolism in Helicobacter pylori Plays a Role in Gastric Carcinogenesis through Facilitating CagA Translocation.

UNLABELLED: A known virulence factor of Helicobacter pylori that augments gastric cancer risk is the CagA cytotoxin. A carcinogenic derivative strain, 7.13, that has a greater ability to translocate CagA exhibits much higher hydrogenase activity than its parent noncarcinogenic strain, B128. A Δhyd mutant strain with deletion of hydrogenase genes was ineffective in CagA translocation into human gastric epithelial AGS cells, while no significant attenuation of cell adhesion was observed. The quinone reductase inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) was used to specifically inhibit the H2-utilizing respiratory chain of outer membrane-permeabilized bacterial cells; that level of inhibitor also greatly attenuated CagA translocation into AGS cells, indicating the H2-generated transmembrane potential is a contributor to toxin translocation. The Δhyd strain showed a decreased frequency of DNA transformation, suggesting that H. pylori hydrogenase is also involved in energizing the DNA uptake apparatus. In a gerbil model of infection, the ability of the Δhyd strain to induce inflammation was significantly attenuated (at 12 weeks postinoculation), while all of the gerbils infected with the parent strain (7.13) exhibited a high level of inflammation. Gastric cancer developed in 50% of gerbils infected with the wild-type strain 7.13 but in none of the animals infected with the Δhyd strain. By examining the hydrogenase activities from well-defined clinical H. pylori isolates, we observed that strains isolated from cancer patients (n = 6) have a significantly higher hydrogenase (H2/O2) activity than the strains isolated from gastritis patients (n = 6), further supporting an association between H. pylori hydrogenase activity and gastric carcinogenesis in humans. IMPORTANCE: Hydrogen-utilizing hydrogenases are known to be important for some respiratory pathogens to colonize hosts. Here a gastric cancer connection is made via a pathogen's (H. pylori) use of molecular hydrogen, a host microbiome-produced gas. Delivery of the known carcinogenic factor CagA into host cells is augmented by the H2-utilizing respiratory chain of the bacterium. The role of hydrogenase in carcinogenesis is demonstrated in an animal model, whereby inflammation markers and cancer development were attenuated in the hydrogenase-null strain. Hydrogenase activity comparisons of clinical strains of the pathogen also support a connection between hydrogen metabolism and gastric cancer risk. While molecular hydrogen use is acknowledged to be an alternative high-energy substrate for some pathogens, this work extends the roles of H2 oxidation to include transport of a carcinogenic toxin. The work provides a new avenue for exploratory treatment of some cancers via microflora alterations.