Expanding allelic diversity of Helicobacter pylori vacA

Abstract

The diversity of the gene encoding the vacuolating cytotoxin (vacA) of Helicobacter pylori was analyzed in 98 isolates obtained from different geographic locations. The studies focused on variation in the previously defined s and m regions of vacA, as determined by PCR and direct sequencing. Phylogenetic analysis revealed the existence of four distinct types of s-region alleles: aside from the previously described s1a, s1b, and s2 allelic types, a novel subtype, designated s1c, was found. Subtype s1c was observed exclusively in isolates from East Asia and appears to be the major s1 allele in that part of the world. Three different allelic forms (m1, m2a, and m2b) were detected in the m region. On the basis of sequence alignments, universal PCR primers that allow effective amplification of the s and m regions from H. pylori isolates from all over the world were defined. Amplimers were subsequently analyzed by reverse hybridization onto a line probe assay (LiPA) that allows the simultaneous and highly specific hybridization of the different vacA s- and m-region alleles and tests for the presence of the cytotoxin-associated gene (cagA). This PCR-LiPA method permits rapid analysis of the vacA and cagA status of H. pylori strains for clinical and epidemiological studies and will facilitate identification of any further variations.

FIG. 1

Phylogenetic tree of vacA s-region nucleotide sequences, including two reference sequences (U07145 and U29401). Clusters of subtypes s1a, s1b, s1c, and s2 are indicated. The sequence from PE9012, containing recombinant s1a-s1b sequences, is underlined. Letters in the names of the isolates indicate the country or location of origin: Australia (AU), Costa Rica (CR), China (CH), Hong Kong (HK), Japan (JA), The Netherlands (NL), New Zealand (NZ), Peru (PE), Portugal (PO), Thailand (TH), and the United States (US). A reference bar is shown for molecular distance.

FIG. 2

Alignment of partial nucleotide sequences of the vacA s region, showing differences between s2, s1a, s1b, and s1c subtypes. The recombinant s1a-s1b sequence from PE9012 is also shown. Each of the 100-bp sequences presented here starts at position 36 of the vacA open reading frame. To permit alignment between type s1 and s2 sequences, a dot indicates the absence of a nucleotide in the s1 variants. Identical nucleotides are indicated by a hyphen.

FIG. 3

Alignment of partial amino acid sequences of the vacA s region. Consistent differences between s1a, s1b, and s1c subtypes are indicated in boldface. To obtain proper alignment between type s1 and s2 sequences, a dot indicates the absence of an amino acid in s1 variants. Identical amino acids are indicated by a hyphen. The sequences U07145 and U29401 are also presented as references for s1a and s2, respectively.

FIG. 4

Phylogenetic tree of vacA m-region sequences, including four reference sequences (U05676, U05677, U07145, and U29401). Clusters of type m1 and subtype m2a and m2b are indicated. The origins of the isolates are defined in the legend to Fig. 1.

FIG. 5

Use of the reverse hybridization LiPA to determine the vacA and cagA genotypes of H. pylori strains whose sequences were known. The strains (genotypes) are as follows: AU10 (s1a, m1, cagA positive), PO12 (s1b, m1, cagA positive), HK44 (s1c, m1, cagA positive), PO24 (s2, m2a, cagA negative), and HK46 (s1c, m2b, cagA positive). The positions of the specific probes, as listed in Table 2, are indicated.