Genomes of Helicobacter pylori from native Peruvians suggest admixture of ancestral and modern lineages and reveal a western type cag-pathogenicity island

Abstract

Background: Helicobacter pylori is presumed to be co-evolved with its human host and is a highly diverse gastric pathogen at genetic levels. Ancient origins of H. pylori in the New World are still debatable. It is not clear how different waves of human migrations in South America contributed to the evolution of strain diversity of H. pylori. The objective of our 'phylogeographic' study was to gain fresh insights into these issues through mapping genetic origins of H. pylori of native Peruvians (of Amerindian ancestry) and their genomic comparison with isolates from Spain, and Japan.

Results: For this purpose, we attempted to dissect genetic identity of strains by fluorescent amplified fragment length polymorphism (FAFLP) analysis, multilocus sequence typing (MLST) of the 7 housekeeping genes (atpA, efp, ureI, ppa, mutY, trpC, yphC) and the sequence analyses of the babB adhesin and oipA genes. The whole cag pathogenicity-island (cagPAI) from these strains was analyzed using PCR and the geographic type of cagA phosphorylation motif EPIYA was determined by gene sequencing. We observed that while European genotype (hp-Europe) predominates in native Peruvian strains, approximately 20% of these strains represent a sub-population with an Amerindian ancestry (hsp-Amerind). All of these strains however, irrespective of their ancestral affiliation harbored a complete, 'western' type cagPAI and the motifs surrounding it. This indicates a possible acquisition of cagPAI by the hsp-Amerind strains from the European strains, during decades of co-colonization.

Conclusion: Our observations suggest presence of ancestral H. pylori (hsp-Amerind) in Peruvian Amerindians which possibly managed to survive and compete against the Spanish strains that arrived to the New World about 500 years ago. We suggest that this might have happened after native Peruvian H. pylori strains acquired cagPAI sequences, either by new acquisition in cag-negative strains or by recombination in cag positive Amerindian strains.

Figure 1

A. FAFLP analysis of H. pylori strains analyzed from native Peruvians (n = 27). The phylogenetic tree was developed based on various amplitypes generated for individual isolates after allele scoring and generation of similarity profiles in the form of binary tables. Genetic relationships in the form of a tree were deduced using MEGA 3.0 software using bootstrapping method at 10000 bootstrap trials. Two different lineages observed in the tree are colored as per the previous conventions [11, 20]. Six isolates from among those represented in the FAFLP tree (highlighted with circles) were also analyzed subsequently by MLST. B. Phylogenetic analysis of representative hsp-Amerind sequences from our isolates (SJM 83, 92) and those of other Amerind and non-Amerind (western) H. pylori sequences previously described by Ghose et al. from Caracas and Puerto Ayacucho in Venezuela [16]. SJM 83 and 92 formed a separate branch (maroon color) only with Amerind isolates (right). Both the Amerindian and western lineages (green color) are differentially colored as per previous conventions [11, 20]. Sequence alignment (left) of these Amerind sequences revealed significant conservation at the level of nucleotides.

Figure 2

MLST analysis based on concatenated gene sequences of 7 housekeeping genes of H. pylori (Kimura-2 parameter). The phylogenetic tree was based on a total of 19 sequence records (concatenated) obtained under this study (SJM, HUPB, HU, CPY) while incorporating other ~400 sequence records from pubMLST database (pubmlst.org) which were specific to different genotypes in the world (Courtesy, Daniel Falush). Different genetic populations (Hp) and subpopulations (hsp) or genotypes are named and differentially colored after previous conventions [11, 20]. All SJM isolates, Amerindians (SJM23 and 92-highlighted) and non-Amerindians (arrowheads) analyzed by us from Peru are highlighted in bold face black fonts with green twigs indicating presence of a western type cagPAI.

Figure 3

A. (Top) PCR based analysis of whole cagPAI of hsp-Amerind isolates from Peru. Overlapping primers spanning all of the constituent genes (see methods) amplified all the corresponding PCR products in the expected size range as described previously [30]. M indicates molecular weight marker (100 bp ladder). (Bottom) Amino acid signature of cagA (phosphorylation motifs – colored) – characteristic of modern cagA EPIYA (EPIYA C) as observed for all the SJM isolates from Peru. B. Pictorial depiction (right) of different types of cagA-EPIYA motif types prevalent in different H. pylori populations and their distribution in our isolates (boxes on the left).