In vitro and in vivo complementation of the Helicobacter pylori arginase mutant using an intergenic chromosomal site

BACKGROUND: Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. MATERIALS AND METHODS: A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203-hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). RESULTS: A rocF mutant unable to hydrolyze or consume l-arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3' ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. CONCLUSIONS: This complementation strategy should greatly facilitate genetic experiments in H. pylori.     

The CrdRS (HP1365-HP1364) Two-Component System Is Not Involved in pH-Responsive Gene Regulation in the <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and G27

The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease. However, urease-independent mechanisms are likely to contribute to acid adaptation. pH-responsive gene regulation in this organism is mediated by a two-component system (HP0166-HP0165) designated ArsRS and the metal-dependent regulators NikR and Fur. Recently, it was reported that another two-component system termed CrdRS (HP1365-HP1364) is required for pH-responsive regulation of the major acid-resistance systems in the H. pylori strain J99. By the analysis of crdRS null mutants of the H. pylori strains 26695 and G27, we show that low pH induction of both the urease and the amidase genes occurs in the absence of crdRS in these strains, suggesting substantial strain-specific differences in the regulation of a major virulence determinant.     

Effects of cholesterol on Helicobacter pylori growth and virulence properties in vitro

Background: Colonization of the gastric mucosa by Helicobacter pylori is often associated with chronic gastric pathologies in humans. Development of disease correlates with the presence of distinct bacterial pathogenicity factors, such as the cag type IV secretion system (cag‐T4SS), the vacuolating cytotoxin (VacA), or the ability of the bacteria to acquire and incorporate cholesterol from human tissue. Materials and Methods: The in vitro growth of H. pylori requires media (Brucella broth) complemented with vitamins and horse serum or cyclodextrins, prepared as blood agar plates or liquid cultures. Liquid cultures usually show a slow growth. Here, we describe the successful growth of H. pylori strains 26695, P217, P12, and 60190 on serum‐free media replacing serum components or cyclodextrins with a commercially available cholesterol solution. Results: The effects of cholesterol as a substitute for serum or cyclodextrin were rigorously tested for growth of H. pylori on agar plates in vitro, for its general effects on bacterial protein synthesis (the proteome level), for H. pylori’s natural competence and plasmid DNA transfer, for the production of VacA, and the general function of the cag‐pathogenicity island and its encoded cag‐T4SS. Generally, growth of H. pylori with cholesterol instead of serum supplementation did not reveal any restrictions in the physiology and functionality of the bacteria except for strain 26695 showing a reduced growth on cholesterol media, whereas strain 60190 grew more efficient in cholesterol‐ versus serum‐supplemented liquid medium. Conclusions: The use of cholesterol represents a considerable option to serum complementation of growth media for in vitro growth of H. pylori.     

The complex methylome of the human gastric pathogen Helicobacter pylori

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.     

Identification of the Genes That Contribute to Lactate Utilization in Helicobacter pylori

Helicobacter pylori are Gram-negative, spiral-shaped microaerophilic bacteria etiologically related to gastric cancer. Lactate utilization has been implicated although no corresponding genes have been identified in the H. pylori genome. Here, we report that gene products of hp0137–0139 (lldEFG), hp0140–0141 (lctP), and hp1222 (dld) contribute to D- and L-lactate utilization in H. pylori. The three-gene unit hp0137–0139 in H. pylori 26695 encodes L-lactate dehydrogenase (LDH) that catalyzes the conversion of lactate to pyruvate in an NAD-dependent manner. Isogenic mutants of these genes were unable to grow on L-lactate-dependent medium. The hp1222 gene product functions as an NAD-independent D-LDH and also contributes to the oxidation of L-lactate; the isogenic mutant of this gene failed to grow on D-lactate-dependent medium. The parallel genes hp0140–0141 encode two nearly identical lactate permeases (LctP) that promote uptake of both D- and L-lactate. Interestingly an alternate route must also exist for lactate transport as the knockout of genes did not completely prevent growth on D- or L-lactate. Gene expression levels of hp0137–0139 and hp1222 were not enhanced by lactate as the carbon source. Expression of hp0140–0141 was slightly suppressed in the presence of L-lactate but not D-lactate. This study identified the genes contributing to the lactate utilization and demonstrated the ability of H. pylori to utilize both D- and L-lactate.     

Positive Selection Scanning Reveals Decoupling of Enzymatic Activities of Carbamoyl Phosphate Synthetase in Helicobacter pylori

In an effort to detect factors which may be under positive selection, a survey for such genes in two pathogenic strains of Helicobacter pylori (J99 and 26695) was performed. Based on an analysis of synonymous and nonsynonymous substitutions, we identified 19 candidate genes under positive selection. A search for homologues with known crystallographic structures revealed Escherichia coli carbomoyl phosphate synthetase as a homologue of H. pylori carbamoyl phosphate synthetase. Carbamoyl phosphate synthetase as isolated from E. coli is a heterodimeric enzyme that possesses two different but coupled functionalities and is involved in the first committed step in the separate biosynthetic pathways for arginine and pyrimidine nucleotides. In this study, we provide evidence indicating that one of these functionalities appears to be under selective pressure. Reports from previously published site-directed mutagenesis studies point to a decoupling of amidotransferase and synthetase activities. Implications of these findings for a metabolic enzyme under positive selection are discussed in terms of the mechanisms of H. pylori pathogenesis. Received: 11 June 2001 / Accepted: 12 September 2001     

Lipopolysaccharide structures of Helicobacter pylori wild-type strain 26695 and 26695 HP0826::Kan mutant devoid of the O-chain polysaccharide component

We describe a re-investigation of the structure of the lipopolysaccharide (LPS) from Helicobacter pylori genomic strain 26695 and its corresponding HP0826::Kan mutant lacking the O-chain component based on the in-depth NMR analysis of the oligosaccharide products obtained through the use of various degradation procedures performed on the purified LPS from both strains, as well as CE–MS data. New structural evidence indicates the presence of the linear arrangement of glucan and heptan portions of the LPS attached through -6-α-ddHep-3-α-l-Fuc-3-β-GlcNAc- fragment to the inner core dd-heptose residue. This structure differs from previously reported structures of the H. pylori 26695 LPS in several aspects.     

A Single Nucleotide Change Affects Fur-Dependent Regulation of sodB in H. pylori

Helicobacter pylori is a significant human pathogen that has adapted to survive the many stresses found within the gastric environment. Superoxide Dismutase (SodB) is an important factor that helps H. pylori combat oxidative stress. sodB was previously shown to be repressed by the Ferric Uptake Regulator (Fur) in the absence of iron (apo-Fur regulation) [1]. Herein, we show that apo regulation is not fully conserved among all strains of H. pylori. apo-Fur dependent changes in sodB expression are not observed under iron deplete conditions in H. pylori strains G27, HPAG1, or J99. However, Fur regulation of pfr and amiE occurs as expected. Comparative analysis of the Fur coding sequence between G27 and 26695 revealed a single amino acid difference, which was not responsible for the altered sodB regulation. Comparison of the sodB promoters from G27 and 26695 also revealed a single nucleotide difference within the predicted Fur binding site. Alteration of this nucleotide in G27 to that of 26695 restored apo-Fur dependent sodB regulation, indicating that a single base difference is at least partially responsible for the difference in sodB regulation observed among these H. pylori strains. Fur binding studies revealed that alteration of this single nucleotide in G27 increased the affinity of Fur for the sodB promoter. Additionally, the single base change in G27 enabled the sodB promoter to bind to apo-Fur with affinities similar to the 26695 sodB promoter. Taken together these data indicate that this nucleotide residue is important for direct apo-Fur binding to the sodB promoter.     

Gene fusion in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>: making the ends meet

Fusion genes have been reported as a means of enabling the development of novel or enhanced functions. In this report, we analyzed fusion genes in the genomes of two Helicobacter pylori strains (26695 and J99) and identified 32 fusion genes that are present as neighbours in one strain (components) and are fused in the second (composite), and vice-versa. The mechanism for each case of gene fusion is explored. 28 out of 32 genes identified as fusion products in this analysis were reported as essential genes in the previously documented transposon mutagenesis of H. pylori strain G27. This observation suggests the potential of the products of fusion genes as putative microbial drug targets. These results underscore the utility of bacterial genomic sequence comparisons for understanding gene evolution and for in silico drug target identification in the post-genomic era. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Type I Restriction–Modification Loci Reveal High Allelic Diversity in Clinical Helicobacter pylori Isolates

Background: A remarkable variety of restriction‐modification (R‐M) systems is found in Helicobacter pylori. Since they encompass a large portion of the strain‐specific H. pylori genes and therefore contribute to genetic variability, they are suggested to have an impact on disease outcome. Type I R‐M systems comprise three different subunits and are the most complex of the three types of R‐M systems. Aims: We investigated the genetic diversity and distribution of type I R‐M systems in clinical isolates of H. pylori. Material and methods: Sixty‐one H. pylori isolates from a Swedish hospital based case‐control study and 6 H. pylori isolates of a Swedish population‐based study were analyzed using polymerase chain reaction for the presence of the three R‐M systems' subunits. Representative gene variants were sequenced. Results: Although the hsdM and hsdR genes appeared conserved in our clinical H. pylori isolates, the sequences of the hsdS loci were highly variable. Despite their sequence diversity, the genes per se were present at high frequencies. We identified a number of novel allelic hsdS variants, which are distinct from corresponding hsdS loci in the sequenced H. pylori strains 26695, J99 and HPAG1. In analyses of paired H. pylori isolates, obtained from the same individuals with a 4‐year interval, we observed genetic modifications of hsdS genes in patients with atrophic gastric mucosa. Discussion: We propose that the genetic variability of hsdS genes in a bacterial population will give rise to new specificities of these enzymes, which might lead to adaptation to an ever‐changing gastric environment.     

Nucleotide Correspondence between Protein-Coding Sequences of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains 26695 and J99

Comparison of ORFs between H. pylori strains 26695 and J99 showed that transitions (more than 3%) prevail over transversions (less than 1%). The predominance of transitions was explained by the high rates of cytosine replacement by thymine in the coding (3.5–5.3%) and noncoding (2.9–3.9%) DNA strands. The proportion of transversion-type correspondences (A → C, A → T, C → A, C → G, G → C, G → T, T → A, and T → G) did not exceed 0.84%. The highest proportion (28.3%) was observed for correspondences between C and T in ACGT-ATGT, the target site of active methyltransferase of H. pylori J99 (M.Hpy99XI). It was assumed that C → T mutations due to cytosine methylation-deamination are prevalent in H. pylori.     

Expression of Helicobacter pylori cag12 gene in Lactococcus lactis MG1363 and its oral administration to induce systemic anti-Cag12 immune response in mice

To develop an oral vaccine against Helicobacter pylori infection, we have expressed the H. pylori cag12 (HP0532) gene, encoding the outer membrane protein Cag12 (31 kDa), in a live delivery vehicle Lactococcus lactis. The cag12 gene was amplified by polymerase chain reaction (PCR) using the genomic DNA of H. pylori K51 isolated from Korean patients. DNA sequence analysis revealed that the cag12 gene of H. pylori K51 has 98.1 and 97.4% identity with individual cag12 genes of the H. pylori 26695 and J99, respectively. The GST–Cag12 fusion protein, produced using the Escherichia coli expression system, was used to raise a rat polyclonal anti-Cag12 antibody. The PCR-amplified cag12 gene of H. pylori K51 was cloned in the E. coli–L. lactis shuttle vector (pMG36e) and transformed into L. lactis. Western blot analysis demonstrated that the Cag12 protein was expressed in the L. lactis transformant, with a maximum level at the log phase without extracelluar secretion. The oral administration of the transformant into mice resulted in the generation of the anti-Cag12 antibody in serum in two out of five cases. These results suggest that the recombinant L. lactis, which expresses Cag12, may be applicable as an oral vaccine to induce protective immunity against H. pylori.     

Inhibition of the alpha- and beta-carbonic anhydrases from the gastric pathogen Helycobacter pylori with anions

The gastric pathogen Helicobacter pylori encodes two carbonic anhydrases (CAs, EC 4.2.1.1), an α- and a β-class one, hpαCA and hpβCA, crucial for its survival in the acidic environment from the stomach. Sulfonamides, strong inhibitors of these enzymes, block the growth of the pathogen, in vitro and in vivo. Here we report the inhibition of the two H. pylori CAs with inorganic and complex anions and other molecules interacting with zinc proteins. hpαCA was inhibited in the low micromolar range by diethyldithiocarbamate, sulfamide, sulfamic acid, phenylboronic acid, and in the submillimolar one by cyanide, cyanate, hydrogen sulfide, divanadate, tellurate, perruthenate, selenocyanide, trithiocarbonate, iminodisulfonate. hpβCA generally showed a stronger inhibition with most of these anions, with several low micromolar and many submillimolar inhibitors detected. These inhibitors may be used as leads for developing anti-H. pylori agents with a diverse mechanism of action compared to clinically used antibiotics.     

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat _GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10⁻⁷ and 4.7 × 10⁻⁷ transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10⁻⁵ transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylorirecA ⁺ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future. Received: 22 April 1997 / Accepted: 4 November 1997     

The Bactericidal Activity of Carbon Monoxide–Releasing Molecules against Helicobacter pylori

Helicobacter pylori is a pathogen that establishes long life infections responsible for chronic gastric ulcer diseases and a proved risk factor for gastric carcinoma. The therapeutic properties of carbon-monoxide releasing molecules (CORMs) led us to investigate their effect on H. pylori. We show that H. pylori 26695 is susceptible to two widely used CORMs, namely CORM-2 and CORM-3. Also, several H. pylori clinical isolates were killed by CORM-2, including those resistant to metronidazole. Moreover, sub-lethal doses of CORM-2 combined with metronidazole, amoxicillin and clarithromycin was found to potentiate the effect of the antibiotics. We further demonstrate that the mechanisms underpinning the antimicrobial effect of CORMs involve the inhibition of H. pylori respiration and urease activity. In vivo studies done in key cells of the innate immune system, such as macrophages, showed that CORM-2, either alone or when combined with metronidazole, strongly reduces the ability of H. pylori to infect animal cells. Hence, CORMs have the potential to kill antibiotic resistant strains of H. pylori.     

Helicobacter pylori Colonization Ameliorates Glucose Homeostasis in Mice through a PPAR γ-Dependent Mechanism

Background

There is an inverse secular trend between the incidence of obesity and gastric colonization with Helicobacter pylori, a bacterium that can affect the secretion of gastric hormones that relate to energy homeostasis. H. pylori strains that carry the cag pathogenicity island (PAI) interact more intimately with gastric epithelial cells and trigger more extensive host responses than cag⁻ strains. We hypothesized that gastric colonization with H. pylori strains differing in cag PAI status exert distinct effects on metabolic and inflammatory phenotypes.

Methodology/Principal Findings

To test this hypothesis, we examined metabolic and inflammatory markers in db/db mice and mice with diet-induced obesity experimentally infected with isogenic forms of H. pylori strain 26695: the cag PAI wild-type and its cag PAI mutant strain 99–305. H. pylori colonization decreased fasting blood glucose levels, increased levels of leptin, improved glucose tolerance, and suppressed weight gain. A response found in both wild-type and mutant H. pylori strain-infected mice included decreased white adipose tissue macrophages (ATM) and increased adipose tissue regulatory T cells (Treg) cells. Gene expression analyses demonstrated upregulation of gastric PPAR γ-responsive genes (i.e., CD36 and FABP4) in H. pylori-infected mice. The loss of PPAR γ in immune and epithelial cells in mice impaired the ability of H. pylori to favorably modulate glucose homeostasis and ATM infiltration during high fat feeding.

Conclusions/Significance

Gastric infection with some commensal strains of H. pylori ameliorates glucose homeostasis in mice through a PPAR γ-dependent mechanism and modulates macrophage and Treg cell infiltration into the abdominal white adipose tissue.     

A Novel DNA-Binding Protein Plays an Important Role in Helicobacter pylori Stress Tolerance and Survival in the Host

The gastric pathogen Helicobacter pylori must combat chronic acid and oxidative stress. It does so via many mechanisms, including macromolecule repair and gene regulation. Mitomycin C-sensitive clones from a transposon mutagenesis library were screened. One sensitive strain contained the insertion element at the locus of hp119, a hypothetical gene. No homologous gene exists in any (non-H. pylori) organism. Nevertheless, the predicted protein has some features characteristic of histone-like proteins, and we showed that purified HP119 protein is a DNA-binding protein. A Δhp119 strain was markedly more sensitive (viability loss) to acid or to air exposure, and these phenotypes were restored to wild-type (WT) attributes upon complementation of the mutant with the wild-type version of hp119 at a separate chromosomal locus. The mutant strain was approximately10-fold more sensitive to macrophage-mediated killing than the parent or the complemented strain. Of 12 mice inoculated with the wild type, all contained H. pylori, whereas 5 of 12 mice contained the mutant strain; the mean colonization numbers were 158-fold less for the mutant strain. A proteomic (two-dimensional PAGE with mass spectrometric analysis) comparison between the Δhp119 mutant and the WT strain under oxidative stress conditions revealed a number of important antioxidant protein differences; SodB, Tpx, TrxR, and NapA, as well as the peptidoglycan deacetylase PgdA, were significantly less expressed in the Δhp119 mutant than in the WT strain. This study identified HP119 as a putative histone-like DNA-binding protein and showed that it plays an important role in Helicobacter pylori stress tolerance and survival in the host.     

Environmental Isolates of Aeromonas spp. Harboring the cagA-Like Gene of Helicobacter pylori

We investigated the presence of cagA-like gene of Helicobacter pylori in environmental isolates of Aeromonas spp. from different water samples of Calcutta, India, by colony hybridization using a cagA-specific DNA probe and by PCR with cagA-specific primers. Nucleotide sequencing of five PCR products revealed 97 to 98% homology to canonical cagA of H. pylori 26695 as well as to four clinical H. pylori strains from Calcutta. The cagA-like gene of the environmental isolates was unstable in laboratory conditions and tended to be lost upon subculturing.