Ancestral European roots of Helicobacter pylori in India

Analysis of genetic diversity in microorganisms normally reflects patterns of their own evolution although it is very rare that this can portray their hosts' evolution. Co-evolution between host and pathogens can be explained only if pathogens are not horizontally transmitted, and this supports a possible phylogenetic and evolutionary parallel of the host and pathogens. Sadly, in many cases frequent horizontal transmission separates the evolution of the bacterium from that of the host. However, for some pathogens, such as H. pylori [1–3], and JC viruses [4], transmission is faithfully restricted to families within specific communities. This phenomenon has in recent times provided evidence regarding patterns of human migration [2, 4, 5] in different continents.

The human gastric pathogen H. pylori is presumed to have co-evolved with its host [6] and established itself in the human stomach possibly millions of years ago [7]. It has been recognized recently as a reliable biological marker of host-pathogen co-evolution and ancient human migration based on sequence variation in select gene loci. H. pylori are genetically diverse to the extreme, providing about 1,400 informative sites within 3.5 to 4.5 kb of sequence from housekeeping genes, and their global genetic structure based on such sequence-haplotypes parallels that of humans [2]. Moreover, epidemiological studies have shown that transmission occurs predominantly within families [8–11]. H. pylori therefore, could provide a window into human origins and migration [1, 3] and the impact of religions and social systems on stratification of human ethnic groups [12].

A landmark study based on PCR based DNA motif analysis proposed that H. pylori jumped recently from animals to humans and, therefore, the acquisition of H. pylori by humans may be a recent phenomenon [13]. This study has been the basis for the idea of 'H. pylori free New World' [13]. However, several independent studies based on large-scale analyses of candidate gene polymorphisms contrasted the idea of recent acquisition and suggest that H. pylori might have co-evolved with humans [1, 6, 14].

Using the same set of Peruvian isolates described earlier by Kersulyte et al. [13], Devi et al. [3], from our group have suggested that the genetic make up of south American isolates could be an admixture of ancestral and modern lineages of H. pylori. They clearly highlighted presence of ancestral H. pylori in Peruvians that possibly survived influxes of Spanish strains from Iberian expansions in Peru about 500 years ago. Also, according to this study, the survival advantage of indigenous strains was possibly due to the acquisition of western type cag PAIs from newly arrived Spanish strains.

Previous genotyping studies on Indian isolates have largely targeted molecular epidemiological issues. However, Wirth et al. [12], for the first time, using H. pylori genotypes, addressed issues such as impact of two different religions and societal systems on stratification of human ethnic groups [12] in the remotest north eastern Ladakh area of India. In view of intriguing ideas on ancient origin of H. pylori, and the fact that ancient origins and arrival of H. pylori are hardly known in the context of the vast South Asian continent, additional evidences based on strains from different geographical regions of Asia are clearly needed.

In this study, we attempted to unravel population genetic structure and gene pool diversity of Indian isolates of H. pylori from culturally and linguistically diverse ethnic Indians. The main objective behinds the study has been to explore genetic features of the strains that might explain their ancestral origin and might help reconstruct different waves of pre-historic human migration in India. We also looked if it is possible to link some of the native strains to their ancestors in West Asia, Eurasia or Europe.

Although the Indian peninsula has seen many different waves of population migration [21], the Paleolithic archaeological evidence is not clearly visible to understand peopling of this country [22]. Nonetheless, the Indus Valley and Harappan civilizations portray footprints of Neolithic period [23] suggestive of the arrival of Indo-European speakers who established the caste system, an anthropologically significant prehistoric event [24, 25]. The cultural and historical importance of the arrival and settlement of the Indo-Aryans is undisputed, but it is not clear if this was established through 'replacement of the existing people by outsiders' [22] or did the 'people already in India changed their habits and cultures?' [22]. Such questions have never been addressed in an unambiguous manner, even though the potential of polymorphic DNA markers in reconstruction of human migration and phylogeography [26, 27] has long been appreciated. It appears that even carefully planned geographic genomics studies remained largely speculative due to the lack of a universal 'gold standard' as the classical mitochondrial DNA markers offer too few informative polymorphisms and the newly developed Y – chromosome markers are even less polymorphic than mitochondrial hypervariable regions [2]. Lately, new genetic models were successfully harnessed based on parasites and pathogens that probably accompanied their human host during evolution and much of the human history including migrations and expansions [2, 4, 5] in different continents. Such approaches constitute an attractive alternative to reconstruct human origins and spreads, population dynamics and bottlenecks, wars and displacements, farming and plagues etc.

Our study was aimed at tracking ancient origins of the Indian H. pylori through a two-pronged approach to i) substantiate European link of the pathogen in India and ii) to prove that the pathogenicity island was also of European origin and this PAI has not been a 'recent' addition to the genome of Indian H. pylori. Our analyses, based on MLST and comprehensive genotyping of the cag PAI, linked about 100% of the Indian isolates to H. pylori sub-population hpEurope. This perhaps conveys the message that H. pylori was most probably introduced to the Indian subcontinent by ancient Indo-European nomadic people and our findings, therefore, are consistent with the idea of a possible gene flow into India with the arrival of Indo-Aryans.

Overall, based on the MLST data (Figure 2) and the cag PAI patterns (Figure 3), we suggest that H. pylori might have arrived in India probably at the same time when Indo-European language speaking people crossed into India (~4000–10,000 years before present). Alternatively, the unquestionable common origin of Indian strains with the European ones could be actually more ancient, following the upper Paleolithic spread of Homo sapiens in Eurasia, as suggested by mtDNA variability [18], and our data on H. pylori MLST do not rule out this possibility.

Present day India represents a 'genetic playground' with tremendous diversity of cultures, and languages. However, the people are largely stratified as tribals and nontribals [25]. Four main language families are spoken, the largest being, Indo European (IE), which is prevalent in North, and the second largest Dravidian (DR) group represents languages spoken in the South [28]. The other two language groups include Tibeto-Burman (TB) of the Sino-Tibetan and the Austro-Asiatic (AA) families, largely spoken in far North and the North-east India. While most of the IE speakers belong to castes, the majority of the tribal communities (>450) speak about 750 different dialects that fit within any one of the other three language families (DR, TB, AA) [25, 28]. Such an enormous cultural diversity might argue for many different populations and sub-populations of H. pylori. But until now, and including this study, H. pylori with genetic features of hpEurope have only been reported from India [29, 30]. Even the newly described sub-population hpAsia2 from Ladakh is also a variant of hpEurope and many Ladakhi strains that we looked at in this study, clustered with European H. pylori clade (Figure 2). Also, the cag A sequences from H. pylori belonging to tribal Oraon and Santhals were indistinguishable from those of the mainstream Indians and Europeans (Figure 4), indicating sweeping spread of a single H. pylori genotype across the Indian peninsula. Moreover, we did not document presence of any other H. pylori populations and sub-populations such as hspAmerind, hspMaori, hpAfrica and hpEast Asia in the limited, but representative culture collection that we looked at. However, the visible footprints of other migrations into India such as from the North Eastern corridor and the presence of phenotypic features resembling to Africans in the South, make it unwise to presume an 'H. pylori free India' at the time of arrival of Indo-European speaking invaders. This issue and the fact that H. pylori' s first association with humans traces back to millions of years before present, in Africa [6, 17], it is more realistic to hypothesize that H. pylori of African and Asian gene pool might have already been present in India. The predominance of a single H. pylori population might therefore, point to a distinct survival advantage conferred by a fully functional (western type) cag PAI. This analogy is consistent with the scenario we previously reported [3] for the South American, Amerindian strains, which were presumably out competed by their Spanish counterparts arriving with an intact and functional western cag PAI.

Finally, it is possible that phylogeny based on highly recombining gene loci [15, 29, 31–35] may not be completely foolproof to extract inheritance from different ancestral populations, especially when we use tools such as MEGA 3.0 [36], which do not support admixture analysis. Moreover, phylogenetic methods based on bifurcating trees, such as Neighbor joining analysis, may not be fully appropriate for analysis at the intra-species level [37, 38], especially in case of hypervariable genomic regions, where multiple homoplasy due to reversions, recurrent mutations etc., or polytomy may sometimes confound the phylogenetic interpretation. However, the housekeeping genes used here are selectively neutral and uniform as compared to virulence associated loci such as the flagellins and vac A [10], and therefore, recombinant and hybrid alleles that blur lineage inferences, could be a rare occurrence and not a routine. Partly in view of this assumption and due to our previous experiences on dissecting complex ancestry of native Peruvian isolates using phylogenetic methods [3] we did not attempt admixture analysis with complicated Bayesian statistics. However, to ensure that our conclusions did not represent shortcomings of a single method, we adopted an integrated phylogenetic approach combining MEGA/NETWORK based analyses and genotyping strategy based on full cag PAI and its left and right end sequences. Interestingly, these approaches unambiguously show the Indian H. pylori genotypes scattered among the European ones. Although this would be consistent with gene flow into India with the Indo-Aryans, or even more ancient origins following the Paleolithic expansion of humans in Eurasia, but also consistent with another scenario: migration from India to Europe. However, the later scenario becomes insignificant due to the unavailability of supporting archeological, linguistic and historical data. Nonetheless, an understanding of the time-scale would be helpful for choosing between such explanations, with the estimation of divergence times between the H. pylori sequences in the different human populations. These issues therefore need to be addressed in future.

In summary, we found significant overlap among genetic identities of Indian and European H. pylori based on core and flexible genome markers. This remarkable genetic similarity points to their possible common genetic origins and could therefore be potentially useful in understanding entry, survival, spread and adaptation of H. pylori in Indian stomachs. Also, this study is consistent with the hypothesis of co-evolution of H. pylori with H. sapiens and therefore, could form a reliable foundation to test and reconstruct gene flow into India with the arrival of Indo-Aryans or otherwise.