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

Helicobacter pylori colonizes about half of the human population and is associated with several gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer [1, 2]. 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 [2–5].

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 [6, 7]. The cag A 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 [10, 11], 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 [14, 15].

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 [24, 25]. 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 [26, 27]. The analysis of the expression of MTases in other strains confirmed the high number of expressed enzymes, as well as their diversity among strains [27–31]. 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 [30, 31]. 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 [34–36], which also support other biological roles for R-M systems. These may include regulation of genetic exchange in the naturally competent H. pylori [37, 38] 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 [40, 41]. 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 [18, 23, 44]. These genes represent about 2% of the total number of genes [18, 20, 21], 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 [45, 46], these R-M systems also present a high diversity among strains [18, 24, 25, 27–29, 47], allowing them to be used as a typing system [30, 31]. Moreover, some R-M systems are more prevalent in H. pylori than others, resulting in rare, medium, and frequent R-M systems [29, 30, 48]. 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.

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 [18, 61]. 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 [63, 65], 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 [6, 8, 69]. 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 [2, 3] 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.