A virulence bacterium, Helicobacter pylori, evolved parallel to its host human, therefore, can work as a marker for tracing the human migration. We found H. pylori strains indigenous in the southernmost islands of Japanese Archipelago, Okinawa, and defined them as hspOkinawa and hpRyukyu. Genome data of the strains revealed that hspOkinawa diverged from other East Asian strains about 20,000 years ago, and that hpRyukyu diverged about 45,000 years ago. The closest strains of hpRyukyu were found from Afghanistan, Punjab, and Nepal, which suggest this strain originated in the central Asia and traveled across the Eurasian continent during Paleolithic era. The divergence date of hpRyukyu corresponds with human fossil records in Okinawa. Although it is controversial from human DNA analyses whether descendants of the Paleolithic migrants remain in the modern Japanese population, this study reveals that the bacterium of Paleolithic origin remains in the stomachs of current Japanese.

H. pylori adapted to the human stomach before the Out-of-Africa movement of anatomically modern humans around 70,000 years ago, and now approximately half of the global population carries H. pylori (Covacci et al., 1999). This bacterium transmits mainly from family members in childhood, and the infection continues throughout life unless eradicated (Kuipers et al., 2000). Because the evolution of H. pylori parallels that of humans, we can infer human migrations through genetic analyses of H. pylori (Linz et al., 2007; Moodley et al., 2009).

Conventionally, population and phylogenetic analyses of H. pylori have been conducted based on a multi-locus sequence typing (MLST) method utilizing concatenated sequences of seven housekeeping genes (atpA, efp, mutY, ppa, trpC, ureI, and yphC) (Maiden et al., 1998). Seven major bacterial populations of H. pylori (hpAfrica1, hpAfrica2, hpNEAfrica, hpEurope, hpAsia2, hpEastAsia, and hpSahul) (Falush et al., 2003b; Linz et al., 2007; Moodley et al., 2009) were identified, and the phylogenetic relationship of these seven populations illuminated the modern human dispersal from Africa to the Eurasian continent (Linz et al., 2007), to the Americas (Falush et al., 2003b; Moodley and Linz, 2009), and to Oceania (Moodley et al., 2009). HpEastAsia is further divided into three subpopulations: hspEAsia that distributes in the eastern area of the Eurasian continent, hspMaori that expanded from Taiwan to Oceania, and hspIndigenousAmericas (Moodley et al., 2021) that migrated to North and South America via the Bering Land Bridge.

In our previous study based on the MLST method, we found two H. pylori populations specific to Okinawa: one of the populations diverged earlier than any of the East Asian strains, and the other one diverged more recently but makes a distinct cluster within hpEastAsian strains (Matsunari et al., 2012). Okinawa area consists of the southmost islands of the Japanese Archipelago and ruled by Ryukyu dynasty before they were merged by the main island government in 1879. In this study, we name the group with the older divergence as hpRyukyu and the group with more recent divergence as hspOkinawa, regarding this group as a sub-population of hpEastAsia. HpRyukyu and hspOkinawa strains account for 16% and 14% of current Okinawa strains, respectively, and the rest belong to hspEAsia, the same group of strains on the main island of Japan.

Our previous study also revealed that hpRyukyu and hspOkinawa strains are less virulent than hspEAsia strains because of the difference of CagA, one of the major virulence genes of H. pylori (Matsunari et al., 2012). HpRyukyu strains possess CagA whose amino acid motif is similar to that of hpEurope strains. However, the CagA motif of hpRyukyu was originally found only from Okinawa but no other area. Therefore, it was named as J-Western CagA as “J” stands for “Japan.” HspOkinawa strains lack CagA, so the least virulent.

Mostly, all of strains in the main island of Japan belong to hspEAsia, which are also common in China and Korea. It was probably brought by people from the continent with rice cultivation. In contrast, the origin of hpRyukyu and hspOkinawa was enigmatic. We investigated virulence genes and seven housekeeping genes only in our previous study but advancement of DNA sequencers enabled us to obtain the whole genomes data of H. pylori. We investigated not only the whole genomes of Okinawa strains but also the genomes of global strains to speculate when and from where the host human population arrived at Japan.

Of the 88-sequence data, HP14065, UM211, UM139, and HP15060 were not complete genomes but contig sequences (Chua et al., 2019). First, we could find only one strain that is close to hpRyukyu, a Nepalian strain NP05-124, in the complete genome data, then we explored H. pylori contig sequences deposited in GenBank and found the above four strains. We asked the author of the paper about the origin of these strains and were informed that HP14065, UM211, UM139, and HP15060 were from Afghanistan, unknown origin, Punjab, and Nepal, respectively.

The hspOkinawa sub-branch was located between hspEAsia and hspIndigenousAmericas strains, as displayed by the colored squares on the right of the branches. The hspIndigenousAmericas strains were further divided into North (Alaska and Canada) and South (Peru, Colombia, and Venezuela) American sub-branches. Hokkaido strains, which were isolated from northern aboriginal Japanese, Ainu, belonged to the North American branch.

Figure 1 also shows the genotypes of cagA, one of the well-known H. pylori virulence genes. CagA is one of the member genes of cag pathogenicity island (cagPAI) and classified as Western cagA, East Asian cagA, Amerind cagA, and J-Western cagA (Figure S6). Because cagPAI is not essential for survival, some strains lack cagPAI (cagA (−)).

HspOkinawa strains are cagA (−) and so are the North American and Hokkaido strains. HpRyukyu has J-Western cagA whose amino acid motifs are similar to the Western type but discriminated by other features (Matsunari et al., 2012). The “J” of J-Western came from Japan because this genotype was first observed only in Okinawa, Japan. However, the Afghanistan and Nepal strains also have J-Western cagA, as well as some European and African strains. Our previous study on Nepalese strains reported that there were three bacterial populations: one was typical Asia2 strains, the second was recombinants between hpAsia2 and hpEurope strains, and the third was recombinants between hpAsia2 and Nepal-specific strains (Miftahussurur et al., 2015). The Nepal-specific strains may have relation with hpRyukyu. In the third population, strains containing high ratio of the Nepal-specific component was rare, which coincident with the fast that we investigated a lot of Nepalese strains but only a few of them were close to hpRyukyu.

We chose oki422 as the representative strain of hpRyukyu. In Figure 2, oki422 branched out after the Afghanistan strain, although Figure 1 shows that the four hpRyukyu strains formed a sub-branch outside the Afghanistan-Nepal cluster. In either case, hpRyukyu diverged earlier than Punjab and Nepal. This suggests that hpRyukyu did not come through the extension of the Punjab-Nepal line.