Evolution of cagA oncogene of Helicobacter pylori through recombination

Abstract

Helicobacter pylori is a gastric pathogen that infects half the human population and causes gastritis, ulcers, and cancer. The cagA gene product is a major virulence factor associated with gastric cancer. It is injected into epithelial cells, undergoes phosphorylation by host cell kinases, and perturbs host signaling pathways. CagA is known for its geographical, structural, and functional diversity in the C-terminal half, where an EPIYA host-interacting motif is repeated. The Western version of CagA carries the EPIYA segment types A, B, and C, while the East Asian CagA carries types A, B, and D and shows higher virulence. Many structural variants such as duplications and deletions are reported. In this study, we gained insight into the relationships of CagA variants through various modes of recombination, by analyzing all known cagA variants at the DNA sequence level with the single nucleotide resolution. Processes that occurred were: (i) homologous recombination between DNA sequences for CagA multimerization (CM) sequence; (ii) recombination between DNA sequences for the EPIYA motif; and (iii) recombination between short similar DNA sequences. The left half of the EPIYA-D segment characteristic of East Asian CagA was derived from Western type EPIYA, with Amerind type EPIYA as the intermediate, through rearrangements of specific sequences within the gene. Adaptive amino acid changes were detected in the variable region as well as in the conserved region at sites to which no specific function has yet been assigned. Each showed a unique evolutionary distribution. These results clarify recombination-mediated routes of cagA evolution and provide a solid basis for a deeper understanding of its function in pathogenesis.

Figure 1. CagA protein and its evolutional pathway.

(A) CagA typical of Western strains and East Asian strains. (B) Aligned amino acid sequences of a Western CagA (strain 26695, hpEurope) and an East Asian CagA (strain F32, Japanese). Red: positively selected amino acid changes. Black: EPIYA motif. Gray: CM sequence. (C) Organization of each type and proposed steps of evolution. (D) Alignment of sequence 2 substituted region between Western type and Amerind type. (E) Alignment of sequence 1 duplicated region between Amerind type and East Asian type. Sequences presumed to be involved in recombination are boxed. (F) Alignment of sequence 1 duplication.

Figure 2. Copy number variation of EPIYA segments by homologous recombination at the CM sequence.

(A) Deletion of the EPIYA-C segment. (B) DNA sequence alignment. (C) Duplication of the EPIYA-C segment. (D) DNA sequence alignment. Black: EPIYA motif. Gray: CM sequence.

Figure 3. Generation of a chimeric EPIYA segment by recombination between EPIYA motifs.

(A) A chimera of EPIYA-B_L/EPIYA-A_R. (B) DNA sequence alignment. (C) A chimera of EPIYA-D_L/EPIYA-A_R. (D) DNA sequence alignment. Black: EPIYA motif. (E) General scheme of site-specific recombination. Modified from Fig. 11-4 in . (F–I) Estimation of possible spacer region within EPIYA motif. (J) Consensus sequence around the EPIYA motif.

Figure 4. Rearrangement by illegitimate recombination between two short similar sequences.

(A) Deletion of an EPIYA motif. (B) DNA sequence alignment. (C) Duplication of the entire EPIYA region. (D) DNA sequence alignment. Sequences presumed to be involved in recombination are boxed. Black: EPIYA motif. Gray: CM sequence.

Figure 5. Amino acids at sites with positive selection for change on the cagA phylogenetic tree.

The tree is based on nucleotide sequences of the entire gene. Each codon number refers to those in strain 26695. (A) 82nd codon. (B) 634th codon. (C) 910th codon. (D) 911th codon. (E) 989th codon. (F) 1111th codon. (G) 1160th codon.