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.

Is there a link between the lipopolysaccharide of Helicobacter pylori gastric MALT lymphoma associated strains and lymphoma pathogenesis?

The aim of this study was to investigate the Lewis antigen expression in Helicobacter pylori gastric MALT lymphoma associated strains in comparison to chronic gastritis only strains. Forty MALT strains (19 cagPAI (-) and 21 cagPAI (+)) and 39 cagPAI frequency-matched gastritis strains (17 cagPAI (-) and 22 cagPAI (+)) were included in this study. The lipopolyssacharide for each strain was extracted using a hot phenol method and the expression of Le(x) and Le(y) were investigated using Western Blot. The data were analyzed according to the strains' cagPAI status and vacA genotype. Le(x) was identified in 21 (52.5%) MALT strains and 29 (74.3%) gastritis strains. Le(y) was identified in 30 (75%) MALT strains and 31 (79.5%) gastritis strains. There was an association between cagPAI positivity and Le(x) expression among MALT strains (p<0.0001), but not in gastritis strains (p = 0.64). Among cagPAI (-) strains, isolates expressing solely Le(y) were associated with MALT with an odds ratio of 64.2 (95% CI 4.9-841.0) when compared to strains expressing both Le(x) and Le(y). vacA genotypes did not modify the association between Lewis antigen expression and disease status. In conclusion, cagPAI (-) MALT strains have a particular Lewis antigen profile which could represent an adaptive mechanism to the host response or participate in MALT lymphomagenesis.

A changing gastric environment leads to adaptation of lipopolysaccharide variants in Helicobacter pylori populations during colonization.

The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarcinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression.

Lipopolysaccharide diversity evolving in Helicobacter pylori communities through genetic modifications in fucosyltransferases.

Helicobacter pylori persistently colonizes the gastric mucosa of half the human population. It is one of the most genetically diverse bacterial organisms and subvariants are continuously emerging within an H. pylori population. In this study we characterized a number of single-colony isolates from H. pylori communities in various environmental settings, namely persistent human gastric infection, in vitro bacterial subcultures on agar medium, and experimental in vivo infection in mice. The lipopolysaccharide (LPS) O-antigen chain revealed considerable phenotypic diversity between individual cells in the studied bacterial communities, as demonstrated by size variable O-antigen chains and different levels of Lewis glycosylation. Absence of high-molecular-weight O-antigen chains was notable in a number of experimentally passaged isolates in vitro and in vivo. This phenotype was not evident in bacteria obtained from a human gastric biopsy, where all cells expressed high-molecular-weight O-antigen chains, which thus may be the preferred phenotype for H. pylori colonizing human gastric mucosa. Genotypic variability was monitored in the two genes encoding alpha1,3-fucosyltransferases, futA and futB, that are involved in Lewis antigen expression. Genetic modifications that could be attributable to recombination events within and between the two genes were commonly detected and created a diversity, which together with phase variation, contributed to divergent LPS expression. Our data suggest that the surrounding environment imposes a selective pressure on H. pylori to express certain LPS phenotypes. Thus, the milieu in a host will select for bacterial variants with particular characteristics that facilitate adaptation and survival in the gastric mucosa of that individual, and will shape the bacterial community structure.

Functional analysis of the M.HpyAIV DNA methyltransferase of Helicobacter pylori.

A large number of genes encoding restriction-modification (R-M) systems are found in the genome of the human pathogen Helicobacter pylori. R-M genes comprise approximately 10% of the strain-specific genes, but the relevance of having such an abundance of these genes is not clear. The type II methyltransferase (MTase) M.HpyAIV, which recognizes GANTC sites, was present in 60% of the H. pylori strains analyzed, whereof 69% were resistant to restriction enzyme digestion, which indicated the presence of an active MTase. H. pylori strains with an inactive M.HpyAIV phenotype contained deletions in regions of homopolymers within the gene, which resulted in premature translational stops, suggesting that M.HpyAIV may be subjected to phase variation by a slipped-strand mechanism. An M.HpyAIV gene mutant was constructed by insertional mutagenesis, and this mutant showed the same viability and ability to induce interleukin-8 in epithelial cells as the wild type in vitro but had, as expected, lost the ability to protect its self-DNA from digestion by a cognate restriction enzyme. The M.HpyAIV from H. pylori strain 26695 was overexpressed in Escherichia coli, and the protein was purified and was able to bind to DNA and protect GANTC sites from digestion in vitro. A bioinformatic analysis of the number of GANTC sites located in predicted regulatory regions of H. pylori strains 26695 and J99 resulted in a number of candidate genes. katA, a selected candidate gene, was further analyzed by quantitative real-time reverse transcription-PCR and shown to be significantly down-regulated in the M.HpyAIV gene mutant compared to the wild-type strain. This demonstrates the influence of M.HpyAIV methylation in gene expression.

An enzymatic ruler modulates Lewis antigen glycosylation of Helicobacter pylori LPS during persistent infection.

Helicobacter pylori persistently colonizes about half the human population and contributes to the development of peptic ulcer disease and gastric cancer. This organism has evolved means to structurally alter its surface characteristics to evade innate and adaptive immune responses. H. pylori produces LPS O-antigen units that can be posttranslationally fucosylated to generate Lewis antigens, structures also found on human epithelial cells. We demonstrate an extensive diversity of Lewis x and Lewis y expression in LPS O-antigen units, occurring over time and in different regions of the human stomach. Lewis expression patterns were correlated with the on/off status of the three fucosyltransferases (FucT), FutA, FutB, and FutC, which are regulated via slipped-strand mispairing in intragenic polyC tract regions of the corresponding genes. The alpha1,3-FucT, FutA and FutB, each contain a C-terminal heptad repeat region, consisting of a variable number of DD/NLRV/INY tandem repeats. Variations in the number of heptad repeats correlated to the sizes of O-antigen polymers to become decorated by fucose residues. Our data support a molecular ruler mechanism for how H. pylori varies its LPS fucosylation pattern, where one heptad repeat in the enzyme corresponds to one N-acetyl-beta-lactosamine unit in the O-antigen polysaccharide.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of HP1352, a putative DNA methyltransferase in Helicobacter pylori.

The putative methyltransferase gene HP1352 from Helicobacter pylori strain 26695 was heterologously expressed in Escherichia coli. The 359-amino-acid gene product was purified and crystallized. The crystals belong to space group I2(1)2(1)2(1) and show diffraction to at least 2.5 A resolution. The unit-cell parameters are a = 69.6, b = 86.6, c = 140.0 A. A greater than 90% complete native data set has been collected and structure determination using the molecular-replacement method is ongoing.