Positive selection in the evolution of Helicobacter pylori outer membrane proteins

Homologous recombination in Helicobacter pylori has been extensively described to occur via Outer Membrane Proteins (OMPs), regulating protein expression and generating allelic diversity, while the importance of single nucleotide polymorphisms (SNP) remains little studied. We used an OMP-encoding gene, homC, as a model to evaluate the weight of positive selection in the evolution of H. pylori, by using G200 sequences obtained from strains collected worldwide. N-site and branch-site phylogenetic analysis by maximum likelihood models were used to identify specific codons that may be important in homC evolution, and to evaluate the impact of selective pressure on the geographic segregation of strains, respectively. The N-site overall analysis showed that 14 of the 742 (1.9%) homC codons are likely under positive selection (likelihood-ratio test (LRT), p < 10-61). Four of these codons are located in the most variable allelic gene middle region, probably reflecting recombination-derived hitchhiking events. On the other hand, eight codons are located in the more conserved 5¢and 3¢ gene regions, although the significance of this distribution remains to be clarified. Branch-site analysis revealed 36 codons (4.9%) under positive selection (LRT, p < 10-41), showing a non-random distribution, and 89% of these particular codons (p < 10-3) support the phylogenetic segregation of European strains from both African and East Asian strains. The lack of visible recombination within this segment suggests an important biological role of point mutations in the evolution of H. pylori OMPs. In conclusion, homC SNP analysis suggests that, besides recombination, positive selection contributes as well to the evolution of H. pylori OMPs

Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

Background: Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results: Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion: This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.. - New England Biolabs, Inc. (USA). - We thank Lurdes Monteiro and Sebastian Suerbaum for the H. pylori strains, Patricia Fonseca and Rui Moreira for critical review of the manuscript, and Afonso Cavaco, Antonio Belo and Dinis Pestana for helping on the logistic regression analysis. This work was partially supported by New England Biolabs, Inc. (USA)

Gastric Cancer: A Stem Cell Disease?

Gastric stem cells have been recently identified and are not yet fully characterized. Each gastric gland or unit is composed of different specialized cells and a small number of discrete stem cells. These gastric stem cells play key roles. They have self-renewal and multipotent properties and are the origin of specialized gastric epithelial cells. These properties are the basis for the stem cells’ role in tissue homeostasis, tissue repair, and cancer. In tumors, growing evidence indicates that a cell subpopulation with stem cell features, the so-called cancer stem cells (CSCs), represents the “fuel” for the tumor: they are at the origin of tumor initiation, growth, and dissemination, and they also display resistance to conventional chemotherapy treatments. The recent identification of CSCs in gastric carcinoma opens the door to the development of new therapeutic strategies targeting more specifically the CSCs at the origin of the disease, which is the third leading cause of cancer-related deaths worldwide

High worldwide conservation of a Helicobacter pylori outer membrane protein

The genetic diversity and evolution of homD, coding for Helicobacter pylori outer membrane protein (OMP) was investigated in a panel of approximately 200 clinical and reference strains, isolated from patients from different geographical origins and presenting different gastric diseases. PCR, sequencing and bioinformatics analyses were used. The homD gene was present in all strains, at a conserved locus, and showed a low genomic diversity, displaying high similarity at both nucleotide and amino acid level. A similarity plot analysis also showed a high level of sequence conservation, although a small region (~30 nucleotides) differed between Western strains and the other strains (East Asian/Ameridian and African). This region was also found in some allelic variants of another hom family member, the homC gene, suggesting the existence of recombination events between these two OMP encoding genes. Sequence analysis of the HomD predicted protein showed a N terminus region with a variable number of KP motif repeats (2 9 KP), with a correlation between the lowest number of KP motif repeats (£4 KP) and peptic ulcer disease and the highest number of repeats (£7 KP) and gastritis. In silico analysis of the HomD protein showed that the region of KP motif repeats exhibits a strong hydrophilicity and antigenicity and a high probability of being exposed to the bacterial surface, suggesting that HomD is immunogenic. These results suggest that homD gene is an important H. pylori antigen and, because of its high global conservation, it is likely to constitute a new vaccine target

Diversity and phylogeny of the Helicobacter pylori outer membrane protein-encoding gene homC

The genetic diversity and evolution of the homC gene was evaluated in a panel of approximately 200 clinical and reference strains, isolated from patients from different geographical origins and presenting different gastric diseases. PCR, sequencing and bioinformatics analyses were used. All the strains tested harboured a complete homC gene at a conserved locus. Phylogenetic reconstruction of homC showed a geographical segregation, with three predominant groups: Western, East Asian/Amerindian and African. A similarity plot analysis suggested a conserved profile of gene segmentation, where three segments were defined. In the first segment (5¢ end extremity), sequences were separated according to the geographical origin of the strain. A higher level of diversity (<50%) was observed in the middle segment, while the third segment (3¢ end extremity) was the most conserved (~90%). In the middle segment, eight allelic variants were identified, with geographic specificity regarding the most prevalent ones. The AI allele was predominant and exclusive of Western strains. The AII allele was predominant in African strains and was the only allele present in the three geographical groups. The AIV allele was predominant in East Asian/ Amerindian strains and was not observed in Western strains. The Western group showed greater molecular distance while the sequences from the East Asian/ Amerindian group were the closest. Overall, the regular presence of homC and its allelic variability suggest that this gene is a good candidate to be part of the pool of H. pylori outer membrane proteins involved in bacterial persistence

Dormant phages of Helicobacter pylori reveal distinct populations in Europe

Prophages of Helicobacter pylori, a bacterium known to co-evolve in the stomach of its human host, were recently identified. However, their role in the diversity of H. pylori strains is unknown. We demonstrate here and for the first time that the diversity of the prophage genes offers the ability to distinguish between European populations, and that H. pylori prophages and their host bacteria share a complex evolutionary history. By comparing the phylogenetic trees of two prophage genes (integrase and holin) and the multilocus sequence typing (MLST)-based data obtained for seven housekeeping genes, we observed that the majority of the strains belong to the same phylogeographic group in both trees. Furthermore, we found that the Bayesian analysis of the population structure of the prophage genes identified two H. pylori European populations, hpNEurope and hpSWEurope, while the MLST sequences identified one European population, hpEurope. The population structure analysis of H. pylori prophages was even more discriminative than the traditional MLST-based method for the European population. Prophages are new players to be considered not only to show the diversity of H. pylori strains but also to more sharply define human populations.University of Malaya-Ministry of Education (UM-MoE) High Impact Research (HIR) Grant UM.C/HIR/MOHE/13/5 (h-50001-00-A000033) and by the Fundação para a Ciência e a Tecnologia (FCT) project grant PTDC/EBB-EBI/119860/2010

Matrix-assisted laser-desorption/ionization BIOTYPER: experience in the routine of a University hospital

AbstractMatrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) is positioned at the forefront of bacterial identification in the future. Its performance needed to be evaluated in a routine Bacteriology laboratory to determine its true benefits. A prospective study was carried out in the Bacteriology laboratory of the Pellegrin University Hospital in Bordeaux, France, from April to May 2009. Bacterial isolates from clinical samples were identified by conventional phenotypic bacteriological methods [Phoenix (Becton-Dickinson) or API strips (bioMérieux)] and in parallel with a mass spectrometer (Ultraflex III TOF/TOF and the biotyper database from Bruker Daltonics). In case of a discrepancy between these results at the genus level, a 16S rRNA and/or rpoB gene sequencing was performed. Of the 1013 bacteria tested, 837 (82.6%) were correctly identified at the species level by MALDI-TOF mass spectrometry (MS) without extraction and 189 after extraction, i.e. 986 (97.3%) were correctly identified at the species level by MALDI-TOF MS, vs. 945 (93.2%) by phenotypic methods. Indeed, the extraction step was necessary for only 15% of the isolates. These results were even better when considering the genus, reaching almost 99% with MALDI-TOF MS and 98% with phenotypic methods. The performance of MALDI-TOF MS is very attractive considering its efficiency and rapidity, and the technique constitutes a precious tool for bacteriological identification in a routine laboratory

\u3cem\u3eHelicobacter pylori\u3c/em\u3e infection in Havana, Cuba. Prevalence and \u3cem\u3ecagA\u3c/em\u3e status of the strains

There is a great paucity of information about Helicobacter pylori infection in the countries of the Caribbean basin. Almost no studies have been performed to determine the prevalence, antibiotic resistance or virulence factors of the bacterium. To measure the prevalence of H. pylori infection among patients attending endoscopy in three clinics in Havana, Cuba, to evaluate clarithromycin resistance, and to determine the cagA status of the strains obtained. Endoscopy was performed and biopsies were obtained from 117 successive patients attending the Institute of Oncology, the Institute of Gastroenterology, and the Calixto Garcia Hospital in Havana, Cuba. Biopsies were maintained at –70 ºC before being cultured on three different media (two selective and one non-selective) and incubated for 7 days at 37 °C under a microaerobic atmosphere. The presence of H. pylori was identified by oxidase, catalase and urease activities. DNA was extracted, and PCR was performed with primers H2761676 which amplify a 397 bp fragment of the cagA gene. Clarithromycin susceptibility was measured by the gel diffusion method. The diagnoses of patients were: 1 gastric carcinoma; 19 duodenal ulcers; 8 gastric ulcers; and 89 non-ulcer dyspepsia, including (62) gastritis, (9) hiatal hernia,(2) biliary reflux, (1) gastric polyps, and (15) no abnormality. Among the 117 biopsies tested, 83 were H. pylori positive (70.9%). The cagA status determined for 35 cases gave a positive result in 31 cases (88.5%). Only 3% of the strains were resistant to clarithromycin. The prevalence of Helicobacter pylori infection in the symptomatic population of La Habana is the same as reported for other developing countries. Most strains were cagA positive and are likely harbour the cag pathogenicity island. The low resistance to clarithromycin in the strains studied probably reflects the low degree of use of the antibiotic in this population

Allelic diversity and phylogeny of homB, a novel co-virulence marker of Helicobacter pylori

<p>Abstract</p> <p>Background</p> <p>The <it>homB </it>gene is a <it>Helicobacter pylori </it>disease-marker candidate, strongly associated with peptic ulcer disease, while <it>homA</it>, its paralogue gene with 90% sequence identity, is correlated with non-ulcer dyspepsia. The HomB encoded outer membrane protein was shown to contribute to the proinflammatory properties of <it>H. pylori </it>and also to be involved in bacterial adherence.</p> <p>This study investigated the distribution of <it>homB </it>and <it>homA </it>genes in 455 <it>H. pylori </it>strains from East Asian and Western countries, and carried out sequence comparison and phylogenetic analyses.</p> <p>Results</p> <p>Both <it>homB </it>and <it>homA </it>genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains.</p> <p>Analysis of <it>homB </it>and <it>homA </it>sequences revealed diversity regarding the number of copies and their genomic localization, with East Asian and Western strains presenting different genotypes. Moreover, <it>homB </it>and <it>homA </it>sequence analysis suggests regulation by phase variation. It also indicates possible recombination events, leading to gene duplication or <it>homB</it>/<it>homA </it>conversion which may as well be implicated in the regulation of these genes. Phylogenetic reconstruction of <it>homB </it>and <it>homA </it>revealed clustering according to the geographic origin of strains. Allelic diversity in the middle region of the genes was observed for both <it>homB </it>and <it>homA</it>, although there was no correlation between any allele and disease. For each gene, a dominant worldwide allele was detected, suggesting that <it>hom</it>B/<it>hom</it>A allelic variants were independent of the geographical origin of the strain. Moreover, all alleles were demonstrated to be expressed <it>in vivo</it>.</p> <p>Conclusion</p> <p>Overall, these results suggest that <it>homB </it>and <it>homA </it>genes are good candidates to be part of the pool of <it>H. pylori </it>OMPs implicated in host-bacteria interface and also contributing to the generation of antigenic variability, and thus involved in <it>H. pylori </it>persistence.</p

Disease association with two Helicobacter pylori duplicate outer membrane protein genes, homB and homA

<p>Abstract</p> <p>Background</p> <p><it>homB </it>encodes a <it>Helicobacter pylori </it>outer membrane protein. This gene was previously associated with peptic ulcer disease (PUD) and was shown to induce activation of interleukin-8 secretion <it>in vitro</it>, as well as contributing to bacterial adherence. Its 90%-similar gene, <it>homA</it>, was previously correlated with gastritis. The present study aimed to evaluate the gastric disease association with <it>homB </it>and <it>homA</it>, as well as with the <it>H. pylori </it>virulence factors <it>cagA</it>, <it>babA </it>and <it>vacA</it>, in 415 <it>H. pylori </it>strains isolated from patients from East Asian and Western countries. The correlation among these genotypes was also evaluated.</p> <p>Results</p> <p>Both <it>homB </it>and <it>homA </it>genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains. In Western strains (n = 234, 124 PUD and 110 non-ulcer dyspepsia (NUD), <it>homB</it>, <it>cagA </it>and <it>vacA </it>s1 were all significantly associated with PUD (p = 0.025, p = 0.014, p = 0.039, respectively), and <it>homA </it>was closely correlated with NUD (p = 0.072). In East Asian strains (n = 138, 73 PUD and 65 NUD), <it>homB </it>was found more frequently than <it>homA</it>, and none of these genes was associated with the clinical outcome.</p> <p>Overall, <it>homB </it>was associated with the presence of <it>cagA </it>(p = 0.043) and <it>vacA </it>s1 (p < 0.001), whereas <it>homA </it>was found more frequently in <it>cagA</it>-negative (p = 0.062) and <it>vacA </it>s2 (p < 0.001) strains.</p> <p>Polymorphisms in <it>homB </it>and <it>homA </it>copy number were observed, with a clear geographical specificity, suggesting an involvement of these genes in host adaptation. A correlation between the <it>homB </it>two-copy genotype and PUD was also observed, emphasizing the role of <it>homB </it>in the virulence of the strain.</p> <p>Conclusion</p> <p>The global results suggest that <it>homB </it>and <it>homA </it>contribute to the determination of clinical outcome.</p