A M(r) 34,000 proinflammatory outer membrane protein (oipA) of Helicobacter pylori

Abstract

The complete genome sequence revealed a family of 32 outer membrane proteins (OMPs) in Helicobacter pylori. We examined the effect of four OMPs (HP0638, HP0796, HP1501, and babA2) on the production of the proinflammatory cytokine, IL-8. Mutants of the four OMPs, as well as cagE and galE from H. pylori from the U.S. and Japan, were constructed by inserting a chloramphenicol-resistant cassette into the gene. Twenty-two pairs of parental and mutant H. pylori strains, as well as 160 clinical isolates (80 from Japanese and 80 from U.S.), were cocultured with gastric cancer cell lines. IL-8 production in the supernatant and adhesion was assayed by ELISA. HP0796, HP1501, babA2, and galE gene knockouts had no significant effect on IL-8 production. Knockout of the HP0638 gene in 81% of cag-positive strains reduced IL-8 production approximately 50%. The three cag-positive strains in which IL-8 levels were unchanged by HP0638 knockout had five or seven CT dinucleotide repeats in the 5' region, resulting in a frame shift and truncation. Strains with naturally inactive HP0638 gene were all from the U.S.; Japanese strains were always "on" and thus, on average, may be more virulent. Although cag-negative isolates produced a limited IL-8 response, cag-negative strains that contained a functional HP0638 gene produced more than 3-fold greater IL-8 than cag-negative nonfunctional HP0638 strains. We hypothesize that functional HP0638 gene may be an important virulence factor in relation to the risk of clinically significant outcomes of H. pylori infection. We denote HP0638 gene as outer inflammatory protein (oipA).

Figure 1

IL-8 production from three gastric cancer cell lines cocultured with clinical isolates of H. pylori from Japanese and U.S. patients. H. pylori was added to the cultured cells (bacterium-to-cell ratio of 100:1) for 24 h, and IL-8 in the supernatant was assayed by ELISA. The broken line indicates the IL-8 levels from each cancer cell without being cocultured with H. pylori (control). The ends of the bars indicates the 25th and 75th percentiles. The 50th percentile (median) is indicated with a line in the bar, and the 10th and 90th percentiles are indicated with error bars.

Figure 2

Relation between adherence of H. pylori (OD₄₉₀) to AGS cells and IL-8 production from AGS cells cocultured with H. pylori clinical isolates (A and B) and oipA knockout mutants (B). H. pylori was added to the cultured cells (bacterium-to-cell ratio of 100:1) for 24 h, and IL-8 in the supernatant was assayed by ELISA. H. pylori was added to the cultured cells (bacterium-to-cell ratio of 500:1) for 90 min, adherent H. pylori and cells were fixed at 4°C for 60 min, and adherence was assayed by ELISA using anti-H. pylori Ab (diluted 1:50) as a first Ab. The OD at 490 nm was used as the index of the number of H. pylori adhering to AGS cells. In B, the beginning of the array shows wild-type strains, and the end of the array shows oipA knockout mutants. The * indicates that the strains have nonfunctional oipA; therefore, the oipA knockout had no effect on IL-8 production.

Figure 3

IL-8 production from AGS cells cocultured with 16 cag-positive clinical isolates (A) and 6 cag-negative clinical isolates (B) from Japanese and U.S. patients and their oipA or cagE knockout mutants. H. pylori was added to the cultured cells (bacterium-to-cell ratio of 100:1) for 24 h, and IL-8 in the supernatant was assayed in triplicate by ELISA. Error bars indicate mean + SD. The broken line indicates the IL-8 levels from AGS cells without being cocultured with H. pylori (control).