Abstract
The gastric pathogen Helicobacter pylori can express the histo blood group antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y, whereas a small population express the type I carbohydrates, Lewis A and B. The expression of Lewis A and Lewis X, as in the case of H. pylori strain UA948, requires the addition of fucose in alpha1,4 and alpha1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been identified in each of the genomes sequenced. The characteristic adenosine and cytosine tracts in the amino terminus and repeated regions in the carboxyl terminus are present in the DNA encoding the two UA948fucT genes, but these genes also contain differences when compared with previously identified H. pylori fucTs. The UA948fucTa gene encodes an approximately 52-kDa protein containing 475 amino acids, whereas UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type II carbohydrate acceptors, by UA948FucTa, does not appear to be affected by fucosylation at other sites on the carbohydrate acceptor, but the rate of fucose transfer is affected by terminal fucosylation of type I acceptors. Through mutational analysis we demonstrate that only FucTa is active in this H. pylori isolate and that inactivation of this enzyme eliminates expression of all Lewis antigens.
Highlights
§ Supported by A Doctoral Research Award from the Medical Research Council of Canada and an Alberta Heritage Foundation for Medical Research Studentship and Incentive Award
It has been suggested that the expression of Lewis antigens by H. pylori may be the cause for an autoimmune response leading to chronic type B gastritis and gastric as well as duodenal ulcers (4), but the role of Lewis antigens in the disease process has recently been questioned by two separate groups (5, 6)
In this study we demonstrate that a single H. pylori FucT enzyme from UA948 contains both ␣(1,3) and ␣(1,4) FucT activity responsible for the production of both Lewis A (Lea) and Lewis X (Lex) in the LPS O-side chain, while the other copy of the fucT gene does not encode a functional FucT enzyme
Summary
Bacterial Strains and Media—H. pylori strains were cultured by standard methods described by Taylor et al (31). DNA Manipulation Techniques—Standard DNA manipulation techniques including the isolation, transformation, and restriction enzyme digestion analysis of plasmid DNA were detailed by Sambrook et al (33) Both strands of the appropriate PCR fragments were sequenced using the Thermosequenase sequencing kit according to the manufacturer’s instructions. Analysis of LPS by Acrylamide Gel Electrophoresis and Immunoblot— Whole cell extracts of the H. pylori strains were treated with proteinase K, processed, and subjected to electrophoresis as described previously (1). These gels were stained with either zinc imidazole, according to the method of Hardy et al (38), or transferred to nitrocellulose membrane (Micron Separations Inc., Westboro, MA; pore size, 0.22 m) according to the method described by Towbin et al (39). Blots were developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer’s specifications, and images were visualized on BioMax BM film (Eastman Kodak Co., Rochester, NY)
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