Helicobacter pylori interstrain restriction-modification diversity prevents genome subversion by chromosomal DNA from competing strains

Helicobacter pylori, bacteria that colonize the human gastric mucosa, possess a large number of genes for restriction-modification (R-M) systems, and essentially, every strain possesses a unique complement of functional and partial R-M systems. Nearly half of the H.pylori strains studied possess an active type IIs R-M system, HpyII, with the recognition sequence GAAGA. Recombination between direct repeats that flank the R-M cassette allows for its deletion whereas strains lacking hpyIIRM can acquire this cassette through natural transformation. We asked whether strains lacking HpyII R-M activity can acquire an active hpyIIRM cassette [containing a 1.4 kb kanamycin resistance (aphA) marker], whether such acquisition is DNase sensitive or resistant and whether restriction barriers limit acquisition of chromosomal DNA. Our results indicate that natural transformation and conjugation-like mechanisms may contribute to the transfer of large (4.8 kb) insertions of chromosomal DNA between H.pylori strains, that inactive or partial R-M systems can be reactivated upon recombination with a functional allele, consistent with their being contingency genes, and that H.pylori R-M diversity limits acquisition of chromosomal DNA fragments of ≥1 kb.     

PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori.

DNA sequence diversity among 60 independent isolates of the gastric pathogen Helicobacter pylori was assessed by testing for restriction fragment length polymorphisms (RFLPs) in several PCR-amplified gene segments. 18 Mbol and 27 HaeIII RFLPs were found in the 2.4 kb ureA-ureB (urease) segment from the 60 strains; this identified 44 separate groups, with each group containing one to four isolates. With one exception, each isolate not distinguished from the others by RFLPs in ureA-ureB was distinguished by Mbol digestion of the neighboring 1.7 kb ureC-ureD segment. The 1.5 kb flaA (flagellin) gene, which is not close to ure gene cluster, was also highly polymorphic. In contrast, isolates from initial and followup biopsies yielded identical restriction patterns in each of the three cases tested. The potential of this method for detecting population heterogeneity was tested by mixing DNAs from different strains before amplification: the arrays of restriction fragments obtained indicated co-amplification from both genomes in each of the five pairwise combinations tested. These results show that H. pylori is a very diverse species, that indicate PCR-based RFLP tests are almost as sensitive as arbitrary primer PCR (RAPD) tests, and suggest that such RFLP tests will be useful for direct analysis of H. pylori in biopsy and gastric juice specimens.     

HobA--a novel protein involved in initiation of chromosomal replication in Helicobacter pylori

Replication of the bacterial chromosome is initiated by the binding of the DnaA protein to a unique DNA region, called oriC. Many regulatory factors in numerous species act by controlling the ability of DnaA to bind and unwind DNA, but the Helicobacter pylori genome does not contain homologues to any of these factors. Here, we describe HobA, a novel protein essential for initiation of H. pylori chromosome replication, which is conserved among, and unique to, epsilon proteobacteria. We demonstrate that HobA interacts specifically via DnaA with the oriC-DnaA complex. We postulate that HobA is essential for correct formation and stabilization of the orisome by facilitating the spatial positioning of DnaA at oriC. Consistent with its function, overexpression of hobA had no effect on growth of H. pylori, whereas depletion of HobA led to growth arrest and failure to initiate replication. In conclusion, HobA may be the first identified of a new group of initiation factors common to epsilon proteobacteria.     

Population genetic analysis of Helicobacter pylori by multilocus enzyme electrophoresis: extensive allelic diversity and recombinational population structure.

Genetic diversity and relationships in 74 Helicobacter pylori isolates recovered from patients assigned to distinct clinical categories were estimated by examination of allelic variation in six genes encoding metabolic housekeeping enzymes by multilocus enzyme electrophoresis. Seventy-three distinct allele profiles, representing multilocus chromosomal genotypes, were identified. All six loci were highly polymorphic, with an average of 11.2 alleles per locus. The mean genetic diversity in the sample was 0.735, a value that exceeds the level of diversity recorded in virtually all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles (lack of enzyme activity) was identified and warrants further investigation at the molecular level. Lack of linkage disequilibrium (nonrandom association (of alleles over loci) indicates that horizontal transfer and recombination of metabolic enzyme genes have contributed to the generation of chromosomal diversity in H. pylori. In this sample of isolates, there was no statistically significant association of multilocus enzyme electrophoretic types or cluster of related chromosomal types and disease category.     

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.     

Concerted evolution between duplicated genetic elements in Helicobacter pylori

The Helicobacter pylori genome includes a family of outer membrane proteins (OMPs) with substantial N and C-terminal identity. To better understand their evolution, the nucleotide sequences for two members, babA and babB, were determined from a worldwide group of 23 strains. The geographic origin of each strain was found to be the major determinant of phylogenetic structure, with strains of Eastern and Western origin showing greatest divergence. For strains 96-10 (Japan) and 96-74 (USA), the 5' regions of babB are replaced with babA sequences, demonstrating that recombination occurs between the two loci. babA and babB have nearly equivalent variation in nucleotide and amino acid identity, and frequencies of synonymous and non-synonymous substitutions. Both genes have segmental conservation but within the 3' segment, substitution patterns are nearly identical. Although babA and babB 5' and midregion segment phylogenies show strong interstrain similarity, the 3' segments show strong intrastrain similarity, indicative of concerted evolution. Within these 3' segments, the lower intrastrain than interstrain frequencies of nucleotide substitutions, which are below mean background H. pylori substitution frequencies, indicate selection against intrastrain diversification. Since babA/babB gene conversions likely underlie the concerted evolution of the 3' segments, in an experimental system, we demonstrate that gene conversions can frequently (10(-3)) occur in H. pylori. That these events are recA-dependent and DNase-resistant indicates their likely cause is intragenomic recombination.     

Mutation as an origin of genetic variability in Helicobacter pylori

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.     

Helicobacter pylori chromosomal DNA replication: current status and future perspectives

Helicobacter pylori causes gastritis, gastric ulcer and gastric cancer. Though DNA replication and its control are central to bacterial proliferation, pathogenesis, virulence and/or dormancy, our knowledge of DNA synthesis in slow growing pathogenic bacteria like H. pylori is still preliminary. Here, we review the current understanding of DNA replication, replication restart and recombinational repair in H. pylori. Several differences have been identified between the H. pylori and Escherichia coli replication machineries including the absence of DnaC, the helicase loader usually conserved in gram-negative bacteria. These differences suggest different mechanisms of DNA replication at initiation and restart of stalled forks in H. pylori.     

Intrafamilial spread of Helicobacter pylori: a genetic analysis

Background. A high incidence of Helicobacter pylori among family members of children with H. pylori gastritis has previously been documented on biopsy material. The main objective of this study was the genetic clarification of H. pylori strains involved in intrafamilial dispersion. Materials and Methods. Formalin‐fixed, paraffin‐embedded material of antral mucosa from 32 members of 11 families was studied for the presence of genetic homogeneity. To achieve this goal, the entire genome of H. pylori was studied by the polymerase chain reaction (PCR)‐based random amplified polymorphic DNA (RAPD) fingerprinting method. Furthermore, the Urease A gene was analyzed using a multiplex PCR‐assay and an alternative mutation detection method based on the Hydrolink? analysis. Results. RAPD fingerprinting confirmed that closely related H. pylori strains were involved in the intrafamilial dispersion. Mutations and small deletions in Urease A gene were found in 22 out of 32 individuals. Conclusions. The homology of the H. pylori genome in members of the same family strongly supports the hypothesis of transmission of H. pylori from person‐to‐person or from a common source.     

Helicobacter spp. Other Than Helicobacter pylori

Non- H. pylori Helicobacter species (NHPHS) are associated with several important human and animal diseases. In the past year research into this group of bacteria has continued to gain attention, and novel species have been described in new niches owing to improvements in detection methods. Polymerase chain reaction and/or sequencing remain the gold standard for the detection of this genus. New insights into the pathogenesis of the NHPHS in hepatobiliary, gastric, and intestinal diseases were gained. In particular, data revealed interaction between hepatic steatosis and infectious hepatitis in the development of hepatocellular carcinoma. Evidence of an association between hepatitis C virus and Helicobacter spp. in hepatocarcinoma development was also provided; and male sex hormone signaling appeared to influence infectious hepatitis induced by Helicobacter hepaticus . More findings support an association between Helicobacter heilmannii and gastric adenocarcinoma; and in mice, mucins MUC4 and MUC5 but not MUC1 influence the colonization and pathogenesis of Helicobacter felis . Data indicated that the roles of the adaptive immune system in H. hepaticus -induced intestinal tumorigenesis are different in the small and large intestines, and environmental factors, such as bile acids may modulate H. hepaticus carcinogenic potential. New reports in the prevention and eradication of NHPHS showed a protective response against Helicobacter suis induced by vaccine administration, and a successful cross-foster rederivation method successfully eradicated Helicobacter spp. from contaminated mice litters. Overall, the studies provided insights into the pathophysiology of Helicobacter species other than Helicobacter pylori.     

Helicobacter pylori infection and gastric cardia cancer in Chaoshan region

Helicobacter pylori (H. pylori) infection represents the most important risk factor for gastric cancer, while its association with gastric cardia cancer (GCC) has not been recognized yet. In this current study, we aim to investigate the status of H. pylori infection in the gastric cardia tissue samples from high-risk populations in Chaoshan littoral region, and the relationship between H. pylori infection and chronic inflammation as well as the proliferative activity of the gastric cardia epithelial cells. A total of 706 gastric cardia biopsy specimens were obtained from 372 GCC cases and 334 tumor-free controls in Chaoshan littoral, a high-risk region for esophageal and gastric cardia cancer. Immunohistochemistry and Giemsa staining were employed for the verification of H. pylori infection. H. pylori infection rate was significantly higher in GCC (81.5%, P < 0.01) and gastric carditis (80.1%, P < 0.01) in comparison with that in the healthy group (34.8%). A significant higher prevalence of chronic inflammation was found in H. pylori+ samples (96.9%) than that in H. pylori− specimens (80.5%) (P < 0.01). To explore the possible role of H. pylori infection-related chronic inflammation in the GCC, we found that the expression of Ki-67 was progressively increased in tissues with chronic inflammation degrees from normal to severe inflammation (P < 0.01). Collectively, these results suggest that persistent H. pylori infection and the related chronic inflammation may contribute to the high incidence of GCC in Chaoshan littoral.     

Partial molecular genetic map of the rabbit VH chromosomal region

Thirty VH-containing cosmid clones, isolated from rabbit germ-line DNA libraries, were restriction mapped and shown to contain approximately 100 VH genes in 765-kb of DNA. Twenty-two of the cosmid clones were grouped into seven distinct clusters. The VH genes were separated by an average of 8 kb, although some were separated by less than 3 kb. Comparison of the nucleotide sequences of two of these VH genes with the sequences of another 11 VH genes showed that they were all generally more than 80% homologous suggesting that rabbit VH genes are members of one highly homologous gene family. Most rabbit Ig molecules have the VH allotypic specificities a1, a2, or a3 and are designated VHa-positive. A small number (less than 30%) of Ig molecules lack these VHa allotypic specificities and are designated VHa-negative. The VH containing cosmid clones were hybridized with synthetic oligomer probes designed to be specific for genes encoding VHa-positive or VHa-negative molecules. At least 50% of the germ-line VH genes hybridized with the VHa-negative oligomer and thus presumably encode VHa-negative molecules; as few as 15% of the genes could be identified as encoding VHa-positive molecules based on hybridization with the VHa-positive oligomer. Approximately 35% of the VH genes did not hybridize with either oligomer and could not be classified as VHa-negative or VHa-positive. We propose that the predominance of serum VHa-positive molecules, in contrast to the predominance of VHa-negative encoding germ-line genes, may reflect preferential usage of a few germline VH genes. The implications of this idea toward explaining the allelic inheritance of VHa allotypes are discussed.     

Helicobacter pylori in Barrett's esophagus.

Barrett's esophagus is an anatomicoclinical state in which, due to the prolonged action of gastroesophageal reflux, the squamous epithelium is replaced by columnar epithelium. Helicobacter pylori has been implicated in the pathogenesis of various gastrointestinal disorders and has occasionally been observed in Barrett's esophagus. The aim of this study is to determine the incidence of H. pylori in Barrett's esophagus and try to establish its role in the pathogenesis of this disorder. H. pylori was observed in 31 biopsies (44.3%) of the 70 studied, mainly when the epithelium is of the gastric atrophic-fundic type (p less than 0.01). Its presence shows no relation to the degree of inflammatory activity and does not seem, therefore, to play an important role in the pathogenesis of the lesion.     

DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting.

The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.     

Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.     

Helicobacter pylori and hormones.

Helicobacter pylori affects gastric acid secretion via several mechanisms. One of these is by changing gastric regulatory physiology. The infection elevates plasma gastrin levels and decreases gastric mucosal expression of the inhibitory peptide somatostatin. These changes may be due to products of H. pylori itself or inflammatory cytokines released in H. pylori infection: acid secretion is inhibited less by a low intra-gastric pH, infusions of cholecystokinin and gastric distention in infected persons. Eradication of H. pylori rapidly decreases basal acid secretion and gastrin-releasing, peptide-stimulated acid secretion. There are now reports that maximally-stimulated acid secretion, a measure of the parietal cell mass, falls significantly six and 12 months after eradication of H. pylori from duodenal ulcer patients. This might be due to withdrawal of the trophic effect of gastrin. However H. pylori can also decrease gastric acid secretion, both through the mechanisms described in Dr. Cave''s paper and by causing gastric mucosal atrophy with loss of parietal cells. The net effect on acid presumably depends on which mechanism predominates. The processes involved may be crucial determinants of clinical outcome. For example, infection with little atrophy and high acid secretion is associated with duodenal ulcers, while infection with atrophy and low acid secretion increases the risk of gastric cancer of the intestinal-type.     

Helicobacter pylori and apoptosis.

In an attempt to understand the diverse effects of infection with Helicobacter pylori on epithelial mucosal mass and consequent clinical outcome, the relationship between H. pylori infection and gastric epithelial cellular turnover has been investigated. Our results indicate that H. pylori increases epithelial cell proliferation and apoptosis in vivo, but that infection with bacteria of the cagA genotype leads to relatively more proliferation than apoptosis. This review explores the causes of the induction of apoptosis in gastric epithelial cells by H. pylori and the consequences of alterations in apoptosis to the maintenance of gastric mucosal homeostasis.