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

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

Ancestral European roots of <i>Helicobacter pylori</i> in India

Background. The human gastric pathogen Helicobacter pylori is co-evolved with its host and therefore, origins and expansion of multiple populations and sub populations of H. pylori mirror ancient human migrations. Ancestral origins of H. pylori in the vast Indian subcontinent are debatable. It is not clear how different waves of human migrations in South Asia shaped the population structure of H. pylori. We tried to address these issues through mapping genetic origins of present day H. pylori in India and their genomic comparison with hundreds of isolates from different geographic regions. Results. We attempted to dissect genetic identity of strains by multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and phylogeographic analysis of haplotypes using MEGA and NETWORK software while incorporating DNA sequences and genotyping data of whole cag pathogenicity-islands (cagPAI). The distribution of cagPAI genes within these strains was analyzed by using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. All the isolates analyzed revealed European ancestry and belonged to H. pylori sub-population, hpEurope. The cagPAI harbored by Indian strains revealed European features upon PCR based analysis and whole PAI sequencing. Conclusion. These observations suggest that H. pylori strains in India share ancestral origins with their European counterparts. Further, non-existence of other sub-populations such as hpAfrica and hpEastAsia, at least in our collection of isolates, suggest that the hpEurope strains enjoyed a special fitness advantage in Indian stomachs to out-compete any endogenous strains. These results also might support hypotheses related to gene flow in India through Indo-Aryans and arrival of Neolithic practices and languages from the Fertile Crescent

Ancestral European roots of Helicobacter pylori in India

<p>Abstract</p> <p>Background</p> <p>The human gastric pathogen <it>Helicobacter pylori </it>is co-evolved with its host and therefore, origins and expansion of multiple populations and sub populations of <it>H. pylori </it>mirror ancient human migrations. Ancestral origins of <it>H. pylori </it>in the vast Indian subcontinent are debatable. It is not clear how different waves of human migrations in South Asia shaped the population structure of <it>H. pylori</it>. We tried to address these issues through mapping genetic origins of present day <it>H. pylori </it>in India and their genomic comparison with hundreds of isolates from different geographic regions.</p> <p>Results</p> <p>We attempted to dissect genetic identity of strains by multilocus sequence typing (MLST) of the 7 housekeeping genes (<it>atp</it>A, <it>efp</it>, <it>ure</it>I, <it>ppa</it>, <it>mut</it>Y, <it>trp</it>C, <it>yph</it>C) and phylogeographic analysis of haplotypes using MEGA and NETWORK software while incorporating DNA sequences and genotyping data of whole <it>cag </it>pathogenicity-islands (<it>cag</it>PAI). The distribution of <it>cag</it>PAI genes within these strains was analyzed by using PCR and the geographic type of <it>cag</it>A phosphorylation motif EPIYA was determined by gene sequencing. All the isolates analyzed revealed European ancestry and belonged to <it>H. pylori </it>sub-population, hpEurope. The <it>cag</it>PAI harbored by Indian strains revealed European features upon PCR based analysis and whole PAI sequencing.</p> <p>Conclusion</p> <p>These observations suggest that <it>H. pylori </it>strains in India share ancestral origins with their European counterparts. Further, non-existence of other sub-populations such as hpAfrica and hpEastAsia, at least in our collection of isolates, suggest that the hpEurope strains enjoyed a special fitness advantage in Indian stomachs to out-compete any endogenous strains. These results also might support hypotheses related to gene flow in India through Indo-Aryans and arrival of Neolithic practices and languages from the Fertile Crescent.</p

First multi-locus sequence typing scheme for Arcobacter spp.

<p>Abstract</p> <p>Background</p> <p><it>Arcobacter </it>spp. are a common contaminant of food and water, and some species, primarily <it>A. butzleri </it>and <it>A. cryaerophilus</it>, have been isolated increasingly from human diarrheal stool samples. Here, we describe the first <it>Arcobacter </it>multilocus sequence typing (MLST) method for <it>A. butzleri</it>, <it>A. cryaerophilus</it>, <it>A. skirrowii, A. cibarius </it>and <it>A. thereius</it>.</p> <p>Results</p> <p>A sample set of 374 arcobacters, including 275 <it>A. butzleri</it>, 72 <it>A. cryaerophilus</it>, 15 <it>A. skirrowii </it>and 8 <it>A. cibarius </it>isolates from a wide variety of geographic locations and sources, was typed in this study. Additionally, this sample set contained four strains representing a new <it>Arcobacter </it>species, <it>A. thereius</it>. The seven loci used in the four-species <it>Arcobacter </it>MLST method are the same as those employed previously in <it>C. jejuni</it>, <it>C. coli</it>, <it>C. helveticus </it>and <it>C. fetus </it>(i.e. <it>aspA</it>, <it>atpA</it>(<it>uncA</it>), <it>glnA</it>, <it>gltA</it>, <it>glyA, pgm </it>and <it>tkt</it>). A large number of alleles were identified at each locus with the majority of isolates containing a unique sequence type. All <it>Arcobacter </it>isolates typed in this study contain two <it>glyA </it>genes, one linked to <it>lysS </it>(<it>glyA1</it>) and the other linked to <it>ada </it>(<it>glyA2</it>). <it>glyA1 </it>was incorporated into the <it>Arcobacter </it>MLST method while <it>glyA2 </it>was not because it did not increase substantially the level of discrimination.</p> <p>Conclusion</p> <p>No association of MLST alleles or sequence types with host or geographical source was observed with this sample set. Nevertheless, the large number of identified alleles and sequence types indicate that this MLST method will prove useful in both <it>Arcobacter </it>strain discrimination and in epidemiological studies of sporadic <it>Arcobacter</it>-related gastroenteritis. A new <it>Arcobacter </it>MLST database was created <url>http://pubmlst.org/arcobacter/</url>; allele and ST data generated in this study were deposited in this database and are available online.</p