Two Different Families of hopQ Alleles in Helicobacter pylori

Helicobacter pylori is a gram-negative bacterium that persistently colonizes the stomachs of more than half of the world's human population. Colonization of the stomach by H. pylori consistently induces gastric inflammation, known as superficial chronic gastritis, and is a risk factor for development of peptic ulcer disease and gastric malignancies (16, 20). H. pylori exhibits a very high level of intraspecies genetic diversity (11, 31, 55). When H. pylori strains isolated from unrelated humans are compared, each isolate is genetically unique, with a level of relatedness among most orthologous sequences ranging from about 90 to 99% nucleotide identity (1, 4, 21, 24, 42, 49). This diversity is the consequence of numerous point mutations, combined with a very high rate of intraspecies genetic recombination (11, 24, 49, 55). In addition, certain genes are present in some H. pylori strains but absent from others (3, 6, 13, 45). Insight into genetic variation among H. pylori strains has been gained by comparing the complete genome sequences of two different H. pylori strains (26695 and J99) (6, 51). Overall, the nucleotide sequences of these two genomes are about 94% identical. However, 206 different genes have been identified that are present in only one strain but not the other (117 genes present only in strain 26695 and 89 present only in strain J99) (6). The majority of H. pylori-infected persons remain asymptomatic for decades, but a subset develop peptic ulcer disease, distal gastric adenocarcinoma, or gastric lymphomas (16, 20). Genetic variation among H. pylori strains could be a factor that helps to explain these diverse clinical outcomes of H. pylori infection. Thus far, two candidate markers for distinguishing virulent H. pylori strains (i.e., those associated with peptic ulceration or gastric carcinoma) from less virulent strains have been studied extensively: presence or absence of the cag pathogenicity island and polymorphisms in vacA alleles. The cag pathogenicity island is a ∼40-kb DNA segment that encodes a highly antigenic protein (CagA) and a type IV secretion pathway utilized for translocating CagA into host cells (2, 7, 14, 15, 35, 46, 47, 52). Upon entry into eukaryotic cells, CagA undergoes phosphorylation of tyrosine residues and induces cytoskeletal alterations in cells (7, 15, 35, 46, 47). The cag pathogenicity island also encodes factors that activate proinflammatory signal transduction pathways in epithelial cells and that probably contribute to an enhanced inflammatory response in the gastric mucosa (2, 14, 52). The vacA gene encodes a secreted “vacuolating toxin” (9, 34, 39). Effects of VacA on cells include the formation of intracellular vacuoles, formation of anion-selective channels in the plasma membrane, induction of apoptosis, and alterations in the permeability of epithelial monolayers. In contrast to the cag island, which is present in some H. pylori strains but absent from others, all H. pylori strains contain a vacA allele. Two families of vacA alleles (designated type s1 and type s2) can be differentiated by analysis of the 5′ end of vacA (8). Type s1 and type s2-VacA protoxins undergo cleavage of amino-terminal signal sequences at two different sites, and as a result, secreted type s2-VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment that is absent from secreted type s1 toxins (8, 30, 32). The presence of this segment renders type s2-VacA toxins defective in vacuolating toxin activity. Type s1-vacA alleles are found predominantly in H. pylori strains that contain the cag pathogenicity island, and type s2-vacA alleles are found predominantly in strains that lack the cag pathogenicity island (8, 22, 44, 54). In many different studies, infection with cag+/s1-vacA strains has been associated with a higher risk of peptic ulceration than has infection with cag-negative/s2-vacA strains (8, 18, 22, 25, 29, 43, 44, 48, 53, 54). A role for the cag pathogenicity island and VacA in the pathogenesis of peptic ulcer disease also has been demonstrated in animal models (36, 50). In several studies, strains classified as cag+/s1-vacA have been associated with an increased risk of distal gastric carcinoma (12, 33). Notably, associations between cag+/s1-vacA strains and severe clinical illness have not been detected as readily in Asian populations as in Western populations (38). In addition to the cag pathogenicity island and vacA, several other candidate markers for distinguishing virulent H. pylori strains from less virulent strains have been proposed. These include iceA, babA2, and oipA (22, 41, 54, 56). In an effort to identify new markers of virulence, a recent study used whole-genome DNA microarray methodology to analyze gene content in 15 different H. pylori strains (45). The cag pathogenicity island was present in 10 strains and was absent or present in an incomplete form in 5 strains. Ten genes located elsewhere in the H. pylori genome were reported to be “coinherited” with the cag island (i.e., present more frequently in cag+ strains than in cag-negative strains). This group included six genes predicted to encode proteins of unknown function (hypothetical proteins), two genes predicted to encode type II DNA methyltransferases, and two genes predicted to encode outer membrane proteins (babA and omp27). It was suggested that these genes coinherited with the cag island might contribute to the virulence of cag+ strains (45). In the present study, we sought to examine in further detail the diversity that is reported to exist in the omp27 locus. H. pylori omp27, corresponding to HP1177 in the genome of H. pylori 26695 and JHP1103 in the genome of H. pylori J99 (6, 51), belongs to the hop family of paralogous genes and has recently been renamed hopQ (5). We report here that hopQ alleles can be classified into two highly divergent families and report the use of PCR-based methodology for classifying H. pylori strains based on hopQ genotypes. We report that type I hopQ alleles are found significantly more commonly in cag+/s1-vacA strains from patients with peptic ulcer disease than in cag-negative/s2-vacA strains from patients without ulcer disease. These data confirm and extend the microarray results reported by Salama et al. (45) and suggest that variation in hopQ genotypes among H. pylori strains may be a factor that influences the clinical outcome of H. pylori infections.