Allelic diversity and phylogeny of homB, a novel co-virulence marker of Helicobacter pylori

Abstract

Background: The homB gene is a Helicobacter pylori disease-marker candidate, strongly associated with peptic ulcer disease, while homA, its paralogue gene with 90% sequence identity, is correlated with non-ulcer dyspepsia. The HomB encoded outer membrane protein was shown to contribute to the proinflammatory properties of H. pylori and also to be involved in bacterial adherence.This study investigated the distribution of homB and homA genes in 455 H. pylori strains from East Asian and Western countries, and carried out sequence comparison and phylogenetic analyses.

Results: Both homB and homA genes were heterogeneously distributed worldwide, with a marked difference between East Asian and Western strains.Analysis of homB and homA sequences revealed diversity regarding the number of copies and their genomic localization, with East Asian and Western strains presenting different genotypes. Moreover, homB and homA sequence analysis suggests regulation by phase variation. It also indicates possible recombination events, leading to gene duplication or homB/homA conversion which may as well be implicated in the regulation of these genes. Phylogenetic reconstruction of homB and homA revealed clustering according to the geographic origin of strains. Allelic diversity in the middle region of the genes was observed for both homB and homA, although there was no correlation between any allele and disease. For each gene, a dominant worldwide allele was detected, suggesting that homB/homA allelic variants were independent of the geographical origin of the strain. Moreover, all alleles were demonstrated to be expressed in vivo.

Conclusion: Overall, these results suggest that homB and homA genes are good candidates to be part of the pool of H. pylori OMPs implicated in host-bacteria interface and also contributing to the generation of antigenic variability, and thus involved in H. pylori persistence.

Figure 1

Diversity in the number of copies and genomic localization of homB and homA in Western and East Asian Helicobacter pylori strains. The percentage indicates the frequency of each type of genotype among Western and East Asian strains. X represents the "empty" locus.

Figure 2

Phylogenetic analysis of 58 homB and 48 homA sequences, obtained from Helicobacter pylori clinical strains from different geographical regions. The branch length index is represented below the tree. Country of origin is located at the beginning of each strain designation (Pt, Portugal; Fr, France; Sw, Sweden; Gr, Germany; USA; Br, Brazil; Col, Colombia; Jp, Japan; Ko, Korea; BF, Burkina Faso) followed by the homB or homA status. Dotted circle, East Asian cluster; Full circle, Western cluster. The sequence of the homB and homA genes of the three H. pylori reference strains, 26695, J99 and HPAG1, were also included. The dotted line separates the homB and homA clusters. The numbers next to the main nodes are bootstrap values over 75% after 1000 iterations.

Figure 3

Similarity plot representation of homB (black lines) and homA (grey lines) genes of various Helicobacter pylori strains. The plot was generated by using 16 strains representative of each gene, with the Jukes-Cantor correction (1-parameter), a 200-bp window, a 20-bp step, without Gap Strip and the jhp870 gene sequence as reference (GenBank accession number NC_000921). The arrow delineates the region which discriminates between homB and homA genotypes. bp, base pair.

Figure 4

Phylogenetic analysis of (A) segment 1 (nucleotides 1 to 750) and (B) segment 3 (nucleotides 1000 to 2000) for the pairs of homB and homA genes of 24 Helicobacter pylori strains carrying one copy of each gene. The branch length index is represented below each tree. Country of origin is located at the beginning of each strain designation (Pt, Portugal; Br, Brazil; Col, Colombia; BF, Burkina Faso) followed by the homB or homA status. In Fig. 4A, the dotted line separates the homB and homA clusters. The numbers next to the main nodes are bootstrap values over 75% after 1000 iterations.

Figure 5

Amino acid alignment of 22 homB and homA allelic region fragments from segment 2 (720 to 1050 bp; predicted amino acids 240 to 350), showing the six allelic variants. The sequence of the homB product of the J99 strain was used as reference (Genbank accession number NP_223588). The dots refer to sites where the amino acids match those of the reference sequence, the hyphens represent deletions. The boxes are used to separate the 6 different allele groups named AI to AVI. Country of origin is located at the beginning of each strain designation (Pt, Portugal; Sw, Sweden; Gr, Germany; USA; Br, Brazil; Jp, Japan; BF, Burkina Faso). * Allelic variants exclusive of homB; ^† allelic variant exclusive of homA.