Helicobacter pylori colonizes about half of the human population and is associated with several gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer 12. The similar pattern of human and H. pylori geographic diversity and distribution suggests a co-evolution between bacteria and man, which can be used to understand human migrations 2. The H. pylori distribution pattern follows the human migration roots, which suggests that the colonization of the human stomach occurred before modern man left East Africa 2345.

Several H. pylori gene alleles present different prevalence rates among the world H. pylori population. This is the case for vacA that presents allelic diversity of the s-, m- and i-region 67. The cagA gene DNA motifs also presents a clear geographic association, and five types of deletion, insertion, and substitution motifs were found at the 3' end of the H. pylori cag pathogenicity island associated with different human populations 8. Another study confirms that the candidate virulence factors, vacA, cagA and iceA, cluster according to geographic region 9. Interestingly, iceA has two known alleles, iceA1 and iceA2 1011, with the locus iceA1 encoding a protein with 52% identity with the restriction endonuclease NlaIII 12. Likewise, the rpoB gene, which codes for RNA polymerase β subunit, presents allelic diversity between Asian and non-Asian strains at the amino acid threonine, which is present only in Asian strains (two thirds of the Asian strains), while it is substituted with alanine in strains of western origin 13. Allelic diversity according to the geographic distribution was also found for the babA and babB genes, which code for outer membrane proteins 1415.

The transposable element ISHp60 presents a non-random geographic distribution, being more frequent in Latin America and rarer in East Asia 16. The hopQ (omp27) alleles show high genetic variability, and type I alleles from Western and Asian H. pylori strains were similar and markedly different from type II hopQ. Type II hopQ alleles were frequently identified in Western H. pylori strains, but rarely in East Asian strains 17.

One class of highly variable genes in the H. pylori genome is the restriction and modification (R-M) systems 18. R-M systems usually comprise both a restriction endonuclease (REase) that recognizes a specific DNA sequence and cuts both strands and a cognate DNA methyltransferase (MTase) that methylates the same DNA sequence, thus protecting it from being cleaved by the companion REase 19. The sequenced H. pylori strains, strain 26695 20, strain J99 18, strain HPAG1 21, and strain G27 22, revealed 26 putative restriction and modification (R-M) systems in the first two strains, and 31 and 34 in the last two 23. Only a reduced number of the expressed MTases in strains J99 and 26695 are common 2425. A small fraction of the potential type II R-M systems in strains J99 and 26695 appear to be fully functional, but different sets of these R-M genes are functionally active in each strain 2627. The analysis of the expression of MTases in other strains confirmed the high number of expressed enzymes, as well as their diversity among strains 2728293031. Likewise, non-pylori Helicobacter spp. appears to express a high number of MTases, as it was previously determined for H. pylori 32. It has been proposed that the diversity of R-M systems in H. pylori is high enough to be used as a typing method 3031. Takata et al. studied the genomic methylation status in 122 H. pylori strains from several world regions, by performing hydrolysis with 14 REases. This study confirmed the diversity and the high number of expressed MTases, but did not reveal any significant association with the H. pylori geographic origin 29.

The biological function of R-M systems has yet to be ascertained. Typically, R-M systems function like an immune system to protect bacteria against invasion of foreign DNA, especially of bacteriophages 33. However there is a limited number of reports on H. pylori phages 343536, which also support other biological roles for R-M systems. These may include regulation of genetic exchange in the naturally competent H. pylori 3738 or promotion of homologous recombination between DNA fragments produced by incomplete REase digestion 39. The linkage of R-M genes allows for simultaneous loss of R and M genes, while physical separation of their gene products permits the hydrolysis of the genomic DNA by the residual REase present in daughter cells, leading to postsegregational killing. This occurs because when cells divide, the daughter cells lose the ability to protectively methylate all recognition sites in the newly synthesized chromosome, causing the cleavage of unmethylated sites by the residual REase still present in the bacterial cytoplasm 4041. The stability of the expression appears to be rather stable (94.9%) in strains isolated from the same patient at different times 30.

In the present study, the majority of strain specific genes with known function, e. g., those that code for restriction and modification (R-M) systems 18, were evaluated for their association with the geographic origin of the H. pylori strains. Since H. pylori co-evolved with man 2, it is important to understand if these strain specific genes (restriction and modification genes) reflect a similar geographic distribution between man and bacteria characteristic of isolated population. The expression of 29 MTases was assessed by hydrolysis of genomic DNA with the cognate REases in 221 H. pylori strains from Africa, America, Asia and Europe. Data were statistically analysed using independence tests as well as multiple and multinomial logistic regression models. Here, we present a geographic pattern for 10 MTases expressed in H. pylori and two conserved MTases expressed in all strains tested. We further explored the association of these MTases with geographic clusters of H. pylori populations to determine if the divergence of regional H. pylori populations is associated with its human host migrations and genetic/cultural habits.

The considerable genetic diversity among strains of H. pylori 42 has already been used to discriminate between closely related human populations, that could not be discriminated by human genetic markers. H. pylori sequence analysis has the potential to distinguish short term genetic changes in human populations 43.

Most methyltransferases genes are part of restriction and modification systems in H. pylori genome 182344. These genes represent about 2% of the total number of genes 182021, a very high proportion when compared with the mean percentage of methyltransferase (M) genes per sequenced genome in Bacteria (0.50%) 23. The average number of R-M genes present in H. pylori sequenced genomes is 30, an extremely high value considering all sequenced bacterial genomes, with an average of 4.3 R-M systems per genome 23. In addition to the high number of R-M systems present in H. pylori genome, which represent more than half of the strain-specific genes 4546, these R-M systems also present a high diversity among strains 18242527282947, allowing them to be used as a typing system 3031. Moreover, some R-M systems are more prevalent in H. pylori than others, resulting in rare, medium, and frequent R-M systems 293048. The present study confirms the high number of M genes expressed per H. pylori strain, independently of its geographic origin and suggests that MTase expression is clearly associated with strain origin. To date, there is only one study 29 specifically designed to characterize MTases expression from different geographic origins. This report confirmed the diversity and the high number of expressed MTases, but did not reveal any significant MTase association with the geographic origin of H. pylori 29. The difficulty in finding an association with geographic origin, may be due to the low number of strains analysed (122 strains),, which included only 3 strains from Africa as well as the limited number of MTases tested (14 REases). Table 2 summarizes MTases that present statistically significant geographic association. The odds ratio may present small differences for the same MTase, given analysis by several logistic regression models. Regardless, the values are always significant for an association between MTase and strain origin.

Our results suggest that the pattern of some H. pylori MTases is geographically defined, which may indicate that it is the result of geographic isolation of its human host or of the co-divergence of H. pylori MTases with host since the migration of modern human out of Africa. R-M systems present a lower G+C content than the total genome (Table 3), which has been considered as evidence for horizontal gene transfer 495051. Frequently, genes coding for R-M systems are within or adjacent to insertions with long target duplications, which suggests a similar transposon insertion with longer duplications, in agreement with an horizontal gene transfer 52. Horizontal gene transfer of H. pylori MTases could favour the geographic isolation hypothesis. However, if we consider that phase variation does not seem to appear in R-M systems 53, and that temporal analysis of gene expression appears to be rather stable 30, MTases are likely not that mobile among genomes. Even though R-M systems may be mainly acquired by horizontal gene transfer, the fact that their expression appears to be stable after acquisition 3053, arguing for a post segregational killing effect 415455, and that H. pylori transmission occurs mainly within the same nuclear family or community 565758, supports the concept of conservation of some R-M systems since the diaspora out of Africa 59, and the acquisition of other R-M genes later on, in specific geographic areas. Finally, the existence of MTases common to all geographic groups, M. NaeI and M. HhaI, is consistent with the hypothesis of H. pylori and Homo sapiens co-evolution after the human out-of-Africa movement 23. It is assumed that modern humans appeared first in Africa, then in Asia, and from this continent they settled in three neighbouring regions: Oceania, Europe and America 4. All H. pylori strains express the MTases M. HhaI and M. NaeI, suggesting that they have been present in the genome since the beginning of human dispersion from the Africa continent. Moreover, M. HhaI is an isoschizomer of M. Hpy99III, M. HpyORF1059P and M. HpyAVIII, which are MTases identified in H. pylori strains J99 (coded by hpy99IIIM), HPAG1 (coded by hpyHORF1059MP), 26695 (coded by hpyAVIIIM), and Shi470 (coded by HPSH_05770), respectively 2360. The identification of M. HhaI isoschizomers in three sequenced strains is in agreement with the hypothesis of these MTases being present in the H. pylori genome since the beginning of the human migrations.

It has been proposed that genes coding for R-M system were acquired recently, by horizontal gene transfer, with new systems being constantly acquired while old ones are inactivated or eliminated 27. Our results support the hypothesis that at least some R-M systems were acquired since human migration out of Africa, while others were obtained later by geographically isolated bacterial populations. It is likely that the first MTases to be stably acquired by H. pylori genome were M. HhaI and M. NaeI, while the others were added later (Figure 1).

The other MTases showing a significant geographic association have probably been acquired at a later stage, depending on the H. pylori geographic localization. Thus, African strains are associated with M. HpyCH4III and M. MspI; Asian strains with M. BstUI, M. DraI, M. FauI, M. FokI and M. Hpy188I; European strains with M. AseI; and, finally, American strains with M. HpyCH4III, M. Hpy99I, M. Hpy188I e M. FokI (Figure 1).

Some MTases are common to more than one continent of origin, as is the case for M. FokI and M. Hpy188I, being both associated with Asia and America. Human migrations from Asia to America could provide some clues to this observation. The presence of both MTases supports the hypothesis of H. pylori and man co-evolution since the settlement of modern man (and H. pylori) in Asia, and posterior settlement in American land between 10,000 and 25,000 years ago, when a land bridge (the Bering Strait) connected Siberia and Alaska during the last ice age 4. The expression of M. HpyCH4III in African and American strains, but not in the Asian ones, suggests that this MTase was acquired later and that the corresponding strains gained access to America in a second wave of migration of African slaves 4. It should be pointed out that most of the American strains analysed are from South and Central American countries, which also experienced human traffic of African origin, although to a smaller extent when compared with other countries, such as Brazil. FokI's resistance to cleavage, which in the present study associated with Asian and American strains, has been previously reported for American and Asian strains, but not European and African 29, which is in agreement with the present study. Likewise, M. Hpy99I and M. Hpy188I associated with the American strains and were first isolated from the strains J99 and J188, also of American origin 1861. Only M. HpyCH4III, which is associated with American strains and not with Asian strains in the present study, was first isolated from a Chinese strain 31. However, only strains from Singapore where considered in the Asian group, which is a limitation of this study. Nine out of ten of the Singapore strains (strains id. 33, 35-37, 39, 40, 42-43, available at Helicobacter pylori MLST Databases 62) were common with the study of Falush et al. After the sequence of eight genes (including vacA, which presents allelic diversity) the majority of these strains were assigned to the hspEastAsia subpopulation 5. This is in accordance with present results, since both studies clearly isolate these strains. For European, American and African strains, the dimension and diversity of these subgroups is higher, yielding more robust results.

It is thought that intra-familiar transmission plays a more important role in urban families than in rural families, where the horizontal transmission (for instance via non-parental caretakers) is predominant 63. H. pylori appears to have established a long lasting colonization of its human host 64, probably being transmitted to subsequent human hosts by close human contact, e.g. between family or community members 6365, depending on the genetic and social-cultural habits of each population. Our study supports previous reports on the co-evolution of H. pylori and man, in the sense that H. pylori reflects human migrations 3, being transmitted among individuals who have close contact with each other, but are not necessarily family members.

The biologic role of R-M systems is not completely understood. It remains an enigma if the increased number of MTases present in the H. pylori genome provides selective advantage to the bacteria, or if it is simply the result of the natural competence and postsegregational killing effects. Methylation has implications in gene expression 66, and H. pylori associates with several pathologies that may result from different sets of expressed genes 67. For instance, DNA methylation by M. HpyAIV was shown to alter transcription of the catalase gene (katA) in H. pylori 68. Further evidence is needed to understand if the high number and diversity of MTases expressed among H. pylori strains is beneficial for the bacteria and/or plays any role in pathogenicity.

In conclusion, there is a clear association of some MTases with geographic groups of H. pylori strains, making them useful as geomarkers (Table 2). Indeed, other genes, as cagA or vacA, have allelic forms with particular geographic distributions 6869. Similar results are now observed for the majority strain-specific genes. M. HhaI and M. NaeI are common to all tested strains, which is consistent with the co-evolution theory of man and H. pylori 23 and suggests that their presence in bacterial genome preceded the human diaspora out of Africa. Finally, the association of MTases with geographical bacterial clusters may be observed in other bacterial species, and may reveal to be good geographic markers to trace bacterial evolution.

FV designed and performed research, analyzed data and prepared the manuscript. FM provided strain collection and contributed to the manuscript. JV designed research and contributed to the manuscript. All authors approved the final manuscript