ABSTRACT
A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [14C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2. Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2. Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo. This is the first time that chemolithoautotrophic growth is described for a pathogen.
IMPORTANCE Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.
INTRODUCTION
Helicobacter pylori is the causative agent of gastric ulcers and chronic gastritis (1). It infects about 50% of the world population (2), and if the infection persists without treatment, it can lead to the development of gastric cancers (3). H. pylori inhabits the gastric mucosa, an environment in which energy and carbon sources are limited. Most of the colonizing organisms within the host stomach are not associated with gastric epithelial cells; rather, they are found in the mucosa, where oxidizable carbon substrates are known to be scarce (4, 5). While the highly diverse glycans present in mucus can be accessed by certain microbiota (6), this requires specialized enzymes that seem to be lacking in Helicobacter species (7). Furthermore, the bacterium lacks a mucinase that would liberate utilizable substrates (8). Still, H. pylori seems to thrive in the mucosal environment, achieving typical viable numbers of 102 to 104 CFU per g or ml of stomach mucus (9). Carbon sources usable by H. pylori include small organic acids, amino acids, and peptides (8, 10–13), but the extent of this metabolism in vivo is not well understood.
H. pylori can use H2 as a respiratory substrate (14). The gastric pathogen possesses a [Ni-Fe] uptake-type hydrogenase (15), with a Km for H2 of about 1.8 μM, which indicates that the enzyme is largely substrate saturated, as the concentration of dissolved H2 (believed to be produced by the colonic flora) is estimated to be 43 μM in the gastric mucosa of live mice (14). Hydrogenase activity, while detected under all growth conditions, is dramatically increased when cells are grown with molecular hydrogen (14). In addition, a hydrogenase mutant strain was shown to be much less efficient in colonizing mice than the wild-type strain, suggesting that H2 is used in the host to bolster the pathogen's metabolism and to help in its colonization (14). Taken together, these results suggest that colonic flora-produced H2 can be used as a source of energy for H. pylori. In Helicobacter hepaticus, H2-derived energy has been shown to drive amino acid transport (16). However, in H. pylori, the specific roles related to the proton motive force (PMF) generated by the H2 respiration have not been identified.
H. pylori requires elevated levels of CO2 to grow in the laboratory. In fact, several groups have shown that H. pylori can tolerate and even grow in the presence of “high” (atmospheric) O2 levels when the CO2 concentration is elevated (5 to 10% partial pressure and above), suggesting that H. pylori could be reclassified as a capnophile instead of a microaerophile (17–19). The CO2 concentrations encountered by H. pylori in human or animal models are not known, but they are expected to be high, due to (i) acidic conversion from bicarbonate (HCO3−), which is continuously produced by the surface epithelial cells and (ii) production of CO2 (from urea) by the H. pylori urease enzyme, the most abundant protein in the gastric pathogen (20). During H. pylori growth, both CO2 and HCO3− can be incorporated into bacterial organic molecules, for instance, into pyrimidine nucleotides (21). Fixation of CO2 might occur via one or more identified carboxylation enzymes, including acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), pyruvate ferredoxin oxidoreductase, and oxoglutarate ferredoxin oxidoreductase. In this study, we show that the addition of H2 resulted in increased levels of the enzyme ACC, concomitant with increased ACC activity, and in higher inorganic carbon incorporation than under the no H2-supplemented condition. We propose an H2-stimulated chemoautotrophy-like growth mode for H. pylori; this type of metabolism has not been described for a human pathogen.
MATERIALS AND METHODS
Strains and growth conditions.
H. pylori wild-type (WT) strains 26695 (7), ATCC 43504, and SS1 (22), as well as a 26695 ΔhydABCDE (Δhyd) hydrogenase mutant strain (23), were used. For the initial growth studies, the complex medium used was brain heart infusion (BHI) supplemented with 0.4% β-cyclodextrin (βc) and 1 μM NiCl2. H. pylori cells were grown for less than 24 h on brucella blood agar (BA) plates, harvested, resuspended in BHI-βc-Ni, and used to inoculate 10 ml of the same medium (starting optical density at 600 nm [OD600] of 0.03 to 0.05) in 165-ml bottles. Cells were grown at 37°C with shaking (200 rpm) under microaerophilic conditions (10% O2, 5% CO2, and N2 as the balance) with or without 10% H2 added to the gas mixture. A more defined growth medium (LK1) (Table 1), similar to the previously described CR-Hyd medium (24), was designed for the growth of H. pylori and used for all other experiments (growth, bicarbonate uptake, and ACC activity). Microaerophilic conditions were the same as described above. Growth experiments in LK1 medium were performed at least 3 times.
TABLE 1.
Composition of LK1 medium
| Component | Final concn |
|---|---|
| Bacto peptone | 0.5% |
| Casamino Acids | 0.2% |
| Thiamine | 0.001% |
| β-Cyclodextrin | 0.4% |
| MgCl2 | 1 mM |
| NiCl2 | 5 μM |
| ZnSO4 | 5 μM |
| FeCl3 | 50 μM |
| Hypoxanthine | 34.5 μM |
| Pyruvate | 1 mM |
| NaCl | 104 mM |
| KCl | 3 mM |
Cell culture and subcellular fractionation.
The soluble and membrane fractions were prepared as previously described (25) with minor modifications. Briefly, the WT (strain 26695) was grown on brucella agar (Difco) plates supplemented with 10% defibrinated sheep blood (BA plates). The plates were incubated at 37°C under constant microaerobic conditions (4% O2, 10% H2, 5% CO2, and N2 as the balance). After 36 h of growth, the cells were harvested and washed in phosphate-buffered saline (PBS) (pH 7.5) in the presence of a protease inhibitor (phenylmethylsulfonyl fluoride [PMSF]) and then were centrifuged at 15,000 × g for 10 min at 4°C. The cells were then resuspended in 3 ml of PBS and disrupted by passage through a French pressure cell at 18,000 lb/in2. The cell extracts were centrifuged at 15,000 × g for 10 min at 4°C to remove the unbroken bacterial cells. The supernatant was then collected and subjected to ultracentrifugation at 100,000 × g for 1 h. The supernatant was collected, and the membrane pellet was resuspended in PBS. The soluble and membrane protein fractions from WT bacteria are named WT-SP and WT-MP, respectively. The final protein concentration was determined using the BCA kit (Thermo Fisher Pierce, Rockford, IL, USA).
In-gel tryptic digestion.
Equal amounts of the soluble and membrane protein fractions were separated on a 4 to 20% TGX precast gel (Bio-Rad, Hercules, CA). Each lane was separated into 10 sections, and each section was cut into small cubes. The gel pieces were destained with a 50 mM NH4HCO3-50% acetonitrile (ACN) solution. After reduction using 10 mM dithiothreitol (DTT) and alkylation using 55 mM iodoacetamide, the proteins were digested overnight with modified trypsin (Promega, Madison, WI, USA) at 37°C. The tryptic peptides were extracted with a 50% ACN-0.1% formic acid (FA) solution. The digests were vacuum dried and stored at −20°C pending mass spectrometry analysis.
LC-MS/MS analysis.
The tryptic peptides were resuspended in a volume of 20 μl of 2% ACN-0.1% FA. Proteolyzed peptide samples were separated and analyzed on an Agilent 1100 capillary liquid chromatograph (LC) interfaced directly to an LTQ linear ion trap mass spectrometer (Thermo Fisher Pierce). The tandem mass spectrometry (MS/MS) instrument was set to acquire spectra for the nine most abundant precursor ions from each MS scan. Raw tandem mass spectra were converted to mzXML files and then to Mascot generic files (MGF) via the Trans-Proteomic Pipeline (Seattle Proteome Center, Seattle, WA). MGF files were searched against separate target and decoy databases using Mascot (Matrix Scientific, Boston, MA). The target database was the complete H. pylori 26695 TIGR protein database (v1.0), and the decoy database contained the reversed sequences of the proteins found in the target database. The Mascot settings were as follows: tryptic enzymatic cleavages allowing for up to 2 missed cleavages, peptide tolerance of 1,000 parts per million, fragment ion tolerance of 0.6 Da, fixed modification due to carboxyamidomethylation of cysteine (+57 Da), and variable modifications of the oxidation of methionine (+16 Da) and deamidation of asparagine or glutamine (+0.98 Da). The Mascot search results were combined with ProteoIQ (NuSep, Bogart, GA), which provided relative protein quantitation utilizing the spectral counting approach. Proteins with a <3% protein false discovery as estimated by ProteoIQ were deemed to be confidently identified.
Whole-cell [14C]bicarbonate uptake assay.
The protocol for the whole-cell [14C]bicarbonate uptake assay was as previously described (26), with minor modifications. Briefly, sealed 70-ml bottles containing 5 ml of defined medium LK1 (Table 1) were inoculated with H. pylori 26695 to an initial OD600 of 0.02, under microaerophilic conditions as described above, with or without 10% H2 added to the gas mixture. A bottle containing only LK1 medium (no bacteria) was used as a blank. The cells were allowed to grow at 37°C for 7 h; cells grown with or without H2 achieved similar growth. After 7 h, the bottles were sparged with N2, and then 5 mM NaHCO3 and 1.3 μCi of NaH14CO3 were added to each bottle. Cells were incubated for an additional 12 h at 37°C with shaking (200 rpm). For each bottle, three 1-ml aliquots were removed and added to scintillation vials containing 0.3 ml of 60% trichloroacetic acid (TCA). The vials were shaken and left for 48 h in a fume hood to dissipate most of the 14CO2 that was not fixed (26). Finally, 5 ml of scintillation fluid was added to each vial, the vials were shaken by hand, and the 14C contents were measured over a 1-min period on a Beckman LS6000TA scintillation counter. Counts obtained from reaction vials containing no cells (blank) were subtracted. The results are the means and standard deviations from biological triplicates, expressed as counts per minute per 108 H. pylori cells.
Acetyl-CoA carboxylase activity assay.
H. pylori cells were broken by sonication (with a Heat Systems Ultrasonics sonicator) in 3 sets of 10-s intervals (4-W output power and 40% duty cycle). The acetyl-CoA carboxylase activity of cell extracts was measured spectrophotometrically by using a coupled enzyme assay with pyruvate kinase and lactate dehydrogenase, as previously described (27). Briefly, each 0.5-ml reaction mixture contained 10 U of pyruvate kinase, 9.3 U of lactate dehydrogenase, 0.1 mM NADH, 1 mM acetyl-CoA, and 50 μM ATP, and the oxidation of NADH was followed at 340 nm. All reactions were conducted in a quartz cuvette with a 1-cm path length and were initiated by addition of 3 μl of cell extract that was diluted 1:1 with water. The spectrophotometric data were collected using a Cary 60 spectrophotometer interfaced with a computer with a data acquisition program. The initial velocities are expressed as specific activity (micromoles of ADP produced per minute per milligram of protein).
RESULTS AND DISCUSSION
When H. pylori WT cells were grown in a complex medium (BHI-βc-Ni), the addition of H2 caused significant growth yield enhancements over that for non-H2-containing atmospheres added to the cultures (Fig. 1). Indeed, we observed 1.7- to 2.1-fold increases in growth yields for cells grown in the presence of H2 over those grown without added H2, depending on the H. pylori strain. Since strain 26695 showed the biggest increase in response to H2 (2.1-fold) (Fig. 1), this strain was selected for subsequent studies. However, in order to assign roles to H2 in specific carbon assimilation processes, a more defined medium was needed. A defined medium (CR-Hyd) composed of small peptides and amino acids was previously used in H2 metabolism studies of Salmonella enterica serovar Typhimurium (17). For the purpose of the current study, we designed a similar defined medium supplemented with the essential metals and amino acids required for the growth of H. pylori (referred to as LK1) (Table 1). When H2 was added to the gas mixture, we observed a significant growth yield enhancement of H. pylori strain 26695 in LK1 compared to that under the no-H2 condition (Fig. 2). Furthermore, when a hydrogenase-negative mutant strain derived from strain 26695 was used as a control, the H2-mediated yield augmentation was abolished (data not shown). Taken together, the results from these growth experiment studies indicate that H2 use (through hydrogenase) supports H. pylori metabolism and growth, whether in rich or defined medium.
FIG 1.
H. pylori growth in the presence (+) or absence (−) of H2. H. pylori cells from three different wild-type strains were grown in a complex medium for less than 24 h under microaerophilic conditions (10% O2, 5% CO2, and N2 as the balance) with or without 10% H2 added to the gas mixture. The results shown are the means and standard deviations of OD600 values recorded after 22 to 24 h and represent 3 or 4 bottle replicates.
FIG 2.
H. pylori growth in a defined medium in the presence or absence of H2. H. pylori cells (strain 26695) were grown in a defined medium under microaerophilic conditions (10% O2, 5% CO2, and N2 as the balance) with or without 10% H2 added to the mixture. The data are from three independent experiments sampled in triplicate. The error bars represent the standard deviations.
Since the LK1 medium contains few oxidizable carbon substrates, we asked whether H2 use augments CO2 fixation in H. pylori. Thus, H. pylori WT cells (strain 26695) were grown in LK1 medium for 7 h under microaerophilic conditions, with or without H2 added to the gas mixture. After the headspace was flushed with N2, H14CO3 was added to the medium, and the cells were further incubated for an additional 12 h. Analysis of the 14C content of cells revealed 3-fold more uptake of H14CO3 (on a per cell basis) in cells grown in the presence of H2 than in those not given H2 (Fig. 3). This result suggests the operation of an H2-stimulated CO2 fixation mechanism, a hallmark of aerobic chemolithoautotrophic H2 oxidizers (26, 28).
FIG 3.
[14C]bicarbonate uptake by cells grown in the presence (+) or absence (−) of H2. H. pylori cells (strain 26695) were grown for 7 h in LK1 defined medium, under microaerophilic conditions, with or without H2 added to the gas mixture. The gas mixture was replaced with N2, and the cells were incubated overnight in the same growth medium supplemented with 5 mM NaHCO3 and 1.3 μCi of NaH14CO3. Samples were taken from each bottle, added to scintillation vials containing 0.3% TCA, and evaporated in a fume hood, and cell-associated 14C radioactivity was counted using a scintillation counter. The data are from two independent experiments sampled in triplicate. The error bars represent the standard deviations.
H. pylori contains several enzymes for CO2 fixation, including acetyl-CoA carboxylase (ACC), pyruvate ferredoxin oxidoreductase, and oxoglutarate ferredoxin oxidoreductase. To determine whether any of these proteins would be induced by H2 exposure, we conducted a proteomics study with cells grown in the presence or absence of H2. Interestingly, the biotin carboxylase subunit of ACC was among the most up-expressed proteins in H2-exposed cells (Table 2).
TABLE 2.
Membrane and soluble proteins with increased expression in WT cells when grown in the presence of H2
| Protein and HP no.a | Protein name | Function(s) | Fold increase |
|---|---|---|---|
| Membrane proteins | |||
| HP0402 | Phenylalanyl-tRNA synthetase, beta subunit (PheT) | Aminoacyl-tRNA biosynthesis | 6.7 |
| HP0763 | Cell division protein (FtsY) | Cell division protein, protein export | 6.3 |
| HP0576 | Signal peptidase I (LepB) | Protein export | 6.0 |
| HP0194 | Triosephosphate isomerase (TpiA) | Glycolysis | 4.0 |
| HP0380 | Glutamate dehydrogenase (GdhA) | Amino acid and nitrogen metabolism | 4.0 |
| HP0309 | Amidohydrolase | Putative C-N hydrolase | 4.0 |
| HP0632 | Hydrogenase large subunit (HydB) | [Ni-Fe]H2 uptake hydrogenase | 3.9 |
| HP0485 | Catalase-like protein | Superoxide radical degradation | 3.7 |
| HP1454 | Hypothetical protein | Unknown (secreted protein) | 3.6 |
| HP0468 | Conserved hypothetical protein | Unknown | 3.2 |
| HP0204 | Hypothetical protein | Unknown | 3.0 |
| HP0106 | Cystathionine gamma-synthase (MetB) | Amino acid metabolism; lyase | 3.0 |
| HP0492 | Neuraminyllactose-binding hemagglutinin precursor | Unknown | 2.9 |
| HP1262 | NADH-ubiquinone oxidoreductase (Nqo5) | NADH dehydrogenase (ubiquinone) | 2.9 |
| HP1346 | Glyceraldehyde-3-phosphate dehydrogenase (Gap) | Oxidoreductase | 2.9 |
| Soluble proteins | |||
| HP0515 | Heat shock protein (HslV) | Hydrolase; peptidase | 5.0 |
| HP1317 | 50S ribosomal protein L23 (RplW) | Protein synthesis | 5.0 |
| HP0370 | Biotin carboxylase (AccC) | Fatty acid biosynthesis; part of ACC complex | 4.9 |
| HP1152 | Signal recognition particle protein (Ffh) | Protein export | 4.0 |
| HP1460 | DNA polymerase III, alpha subunit (DnaE) | DNA replication and repair | 4.0 |
| HP1430 | Conserved hypothetical ATP-binding protein | RNA degradation; hydrolase | 3.5 |
| HP0163 | Delta-aminolevulinic acid dehydratase (HemB) | Metabolism of cofactors and vitamins | 3.5 |
| HP1458 | Thioredoxin 2 (TrxC) | Redox balance | 3.1 |
| HP0466 | Conserved hypothetical protein | Unknown | 3.1 |
| HP0306 | Glutamate-1-semialdehyde aminotransferase (HemL) | Metabolism of cofactors and vitamins | 3.0 |
| HP0138 | l-Lactate dehydrogenase complex protein (LldF) | Iron-sulfur protein | 2.8 |
| HP0088 | RNA polymerase sigma-70 factor (RpoD) | DNA binding, transcription | 2.8 |
| HP0742 | Ribose-phosphate pyrophosphokinase (PrsA) | Pentose phosphate pathway | 2.8 |
| HP1182 | tRNA 2-thiocytidine biosynthesis protein (TtcA) | tRNA processing | 2.8 |
HP no. refers to H. pylori sequenced strain 26695 (7).
ACC catalyzes the first committed step in fatty acid biosynthesis via two half-reactions. Biotin carboxylase (AccC, or HP0370 [7]) is one component of ACC and catalyzes the ATP-dependent carboxylation of biotin in the first half-reaction. The other components of ACC are the biotin carboxyl carrier protein AccB (HP0371), to which the biotin moiety is covalently attached, and the heterotetramer carboxyltransferase comprising AccA (HP0557) and AccD (HP0950) (7), which catalyzes the second half-reaction wherein the carboxyl group is transferred from biotin to acetyl-CoA to produce malonyl-CoA. The three components of ACC form a complex where the stoichiometry is unknown (29). However, formation of the ACC complex along with the substrate acetyl-CoA is required to detect biotin carboxylase activity as measured by the hydrolysis of ATP (29). Because the isolated biotin carboxylase subunit exhibits little to no enzymatic activity (29), the hydrolysis of ATP by the biotin carboxylase subunit in the protein complex is a direct measure of ACC activity.
The proteomics study showed an approximately 5-fold increase in the expression of ACC biotin carboxylase of cells exposed to H2 compared to that in those grown without H2 (Table 2). Interestingly, the hydrogenase large subunit, HydB, was also among the proteins up-expressed in the presence of H2, in agreement with previous results showing that hydrogenase is H2 transcriptionally activated (14). To determine whether an increase in ACC biotin carboxylase synthesis correlates with an increase in ACC activity, we performed ACC enzyme assays on cell extracts of cells grown with and without H2. There was a 3-fold stimulation of ACC activity in cells grown under an atmosphere containing H2 compared to that in cells grown without H2 (Fig. 4), indicating that ACC activity is indeed induced by H2.
FIG 4.
Acetyl-CoA carboxylase activity in cells grown in the presence (+) or absence (−) of H2. H. pylori cells (strain 26695) were grown in LK1 defined medium under microaerophilic conditions (10% O2, 5% CO2, and N2 as the balance) with or without 10% H2 added to the gas mixture. Cell extracts were used to measure acetyl-CoA carboxylase specific activity. The results shown are the means and standard deviations of assays done in triplicate and represent initial activities, expressed as micromoles of ADP produced per minute per milligram of total protein.
Taken together, our results show that the addition of H2 to the growth medium has a pleiotropic effect on H. pylori, including (i) up-expression of hydrogenase and ACC biotin carboxylase subunit, (ii) increased ACC activity, (iii) enhanced CO2 fixation, and (iv) better overall metabolism and growth, as shown by the higher growth yield. This suggests that H2-derived energy can be used by H. pylori to fix CO2 and that the gastric pathogen should be defined as having chemolithoautotrophic capacity.
Chemolithoautotrophs typically obtain a portion of their energy from the oxidation of inorganic compounds and all or a portion of their carbon from CO2 fixation. Examples of well-studied chemolithoautotrophs are nitrifying bacteria, sulfur oxidizers, and iron oxidizers (30). In bacteria capable of chemolithoautotrophic growth with O2 and H2 (oxyhydrogen mixture, or knallgas) or H2-oxidizing bacteria (such as Hydrogenobacter thermophilus and Hydrogenovibrio marinus), H2 use is usually coupled to energy-conserving respiratory chains that terminate with the reduction of molecular oxygen. The chemolithotrophic knallgas bacteria can be isolated from a wide range of ecosystems, and it is thus not surprising that they are a metabolically diverse group. For the vast majority of these microorganisms, CO2 fixation occurs via the Calvin-Benson cycle, more specifically through the activity of the enzyme ribulose bisphosphate carboxylase; however, it is worth noting that this enzyme is absent in H. pylori. Most H2 oxidizers that use CO2 are considered to be facultative with respect to this metabolism. In general, they are considered to be mixotrophs, assimilating organic carbon while also using carbon dioxide (29, 30). H. pylori can be considered mixotrophic as well. In summary, the results presented here indicate that even a pathogenic bacterium, namely, H. pylori, can use chemolithoautotrophy to bolster its metabolism.
ACKNOWLEDGMENT
This work was supported by the University of Georgia Foundation.
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