RESUMO
Helicobacter pylori CagA is translocated into gastric epithelial cells by a type IV secretion system and interacts with the Src homology 2 phosphatase, altering cell morphology. Multiple EPIYA motifs in CagA are associated with increased activity in cells and with gastric cancer. The aim of this work was to study the heterogeneity in activity in cells of multiple H. pylori single colonies isolated from a single patient and its association with polymorphism in cagA. The presence of cagA, cagE, cagT, and cag10 was studied with 318 H. pylori isolates from the antra and corpora of 18 patients. AGS gastric epithelial cells were infected with 75 isolates, and interleukin-8 (IL-8) secretion, cytoskeletal changes, CagA translocation, and tyrosine phosphorylation were measured. The cagA 3'-variable region was sequenced for 30 isolates to determine the number and types of EPIYA motifs. Isolates from an individual stomach were usually genetically related and had quantitatively similar phenotypic effects on cells (IL-8 induction and cytoskeletal changes). However, strains from different patients with similar CagA EPIYA motif patterns varied widely in these phenotypes. Among isolates with an EPIYA-ABC pattern, the phenotype was variable: IL-8 induction ranged from 200 to 1,200 pg/ml, and morphological changes occurred in 20 to 70% of cells. In several cases, cagA sequence diversity appeared to explain the lack of CagA activity, as isolates with an EPIYA-ACC pattern or a modified B motif had reduced cell activity. cag pathogenicity island-positive H. pylori isolates displayed a high level of heterogeneity in the capacity to induce IL-8 secretion and morphological changes; an absent or modified B motif was associated with low activity.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Epiteliais/microbiologia , Variação Genética , Helicobacter pylori/classificação , Helicobacter pylori/patogenicidade , Estômago/citologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Linhagem Celular , Criança , Células Epiteliais/imunologia , Feminino , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Helicobacter pylori/isolamento & purificação , Humanos , Interleucina-8/biossíntese , Masculino , México , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA , Estômago/imunologiaRESUMO
Genetic diversity of the human gastric pathogen Helicobacter pylori in an individual host has been observed; whether this diversity represents diversification of a founding strain or a mixed infection with distinct strain populations is not clear. To examine this issue, we analyzed multiple single-colony isolates from two to four separate stomach biopsies of eight adult and four pediatric patients from a high-incidence Mexican population. Eleven of the 12 patients contained isolates with identical random amplified polymorphic DNA, amplified fragment length polymorphism, and vacA allele molecular footprints, whereas a single adult patient had two distinct profiles. Comparative genomic hybridization using whole-genome microarrays (array CGH) revealed variation in 24 to 67 genes in isolates from patients with similar molecular footprints. The one patient with distinct profiles contained two strain populations differing at 113 gene loci, including the cag pathogenicity island virulence genes. The two strain populations in this single host had different spatial distributions in the stomach and exhibited very limited genetic exchange. The total genetic divergence and pairwise genetic divergence between isolates from adults and isolates from children were not statistically different. We also analyzed isolates obtained 15 and 90 days after experimental infection of humans and found no evidence of genetic divergence, indicating that transmission to a new host does not induce rapid genetic changes in the bacterial population in the human stomach. Our data suggest that humans are infected with a population of closely related strains that vary at a small number of gene loci, that this population of strains may already be present when an infection is acquired, and that even during superinfection genetic exchange among distinct strains is rare.