THE PUTATIVE NEURAMINYLLACTOSE-BINDING HEMAGGLUTININ HPAA OF HELICOBACTER-PYLORI CCUG-17874 IS A LIPOPROTEIN

The ability of certain strains of Helicobacter pylori to cause sialic acid-sensitive agglutination of erythrocytes has been attributed to th e HpaA protein (D. G. Evans, T. K. Karjalainen, D. J. Evans, Jr., D. Y , Graham, and C.-H. Lee, J, Bacteriol. 175:674-683, 1993), the gene fo r which has been cloned and sequenced. On the basis of the hydropathy plot of HpaA and the presence of a potential lipoprotein signal sequen ce and modification site, and because of the similarities of these fea tures with those of the cell envelope lipoprotein Lpp20 of H. pylori, we examined the possibility that HpaA was also a lipoprotein. Posttran slational processing of the HpaA protein expressed by the cloned gene was sensitive to globomycin, an inhibitor of the lipoprotein-specific signal peptidase II. Antibodies raised to the putative sialic acid-bin ding region of HpaA failed to bind to the surface of H. pylori cells i n immunoelectron microscopy but instead were observed to have labeled the cytoplasm when thin sections were examined. This antibody recogniz ed a 29,000-M(r) protein in Western blots (immunoblots) of cell extrac ts of H. pylori and Escherichia call cells expressing the cloned hpaA gene. Determination of the sequence of hpaA from strain CCUG 17874 ind icated significant differences from that determined by Evans and cowor kers in the above-mentioned study, including extension of the gene int o the open reading frame 3 downstream of hpaA to produce a protein wit h an M(r) of 26,414. Localization of HpaA indicated that it was predom inantly located in the cytoplasmic fraction of the cell in both E. cal l and H. pylori. HpaA was not observed in the sarkosyl-insoluble outer membrane fraction. An isogenic mutant generated by insertional inacti vation of hpaA was unaffected in its ability to bind four different hu man cell lines as well as fixed sections of gastric tissue and had hem agglutination properties identical to those of the wild type. The data collectively suggest that HpaA is a nonessential lipoprotein internal to the H. pylori cell and that it is not involved in adhesion.