Identification of a d-glycero-d-manno-Heptosyltransferase Gene from Helicobacter pylori

Lipopolysaccharide (LPS) is a major surface antigen of gram-negative bacteria and is essential for the physical integrity and function of the bacterial outer membrane. The basic structure of Helicobacter pylori LPS is similar to those of other bacterial species and consists of three distinct regions: an O-chain polysaccharide composed of repeating units, a core oligosaccharide, and a hydrophobic lipid A moiety (40). H. pylori LPS may play several roles in pathogenesis and has been implicated in causing abnormal acid secretion and in inducing apoptosis of epithelial cells and gastritis in mice (22, 36-39, 42). More recently, the focus has been on the ability of many strains of H. pylori to express O-polysaccharide chains that mimic Lewis blood group antigens (33). These antigens are naturally expressed on the surface of epithelial cells of several human tissues, including the gastric mucosa (30). A majority of the core regions of LPSs from different gram-negative bacteria contain l-glycero-d-manno-heptopyranose (ld-heptose) (Fig. ​(Fig.1).1). One of the unique features of H. pylori LPS is the abundance of d-glycero-d-manno-heptose (dd-heptose) (Fig. ​(Fig.1)1) residues in its core region (33). The occurrence of dd-heptose in bacterial LPS is relatively uncommon. A limited number of bacterial species are known to contain dd-heptose residues in their core region, including Proteus mirabilis R110/1959, Yersinia enterocolitica, several strains of Yersinia pestis, Aeromonas hydrophila, and Rhodocyclus spp. (18). In Klebsiella pneumoniae strain 01/R20, the outer core region of LPS contains an α-1,2-linked dd-heptoglycan (48). Other bacteria, such as Haemophilus ducreyi (18), Vibrio parahaemolyticus (18), Vibrio salmonicida (18), Coxiella burnetii (18), Proteus penneri (54), Actinobacillus pleuropneumoniae (45), and Mannheimia hemolytica (4), have one or two internal dd-heptose residues in their core regions. Additionally, the glycan chain of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 was reported to contain dd-heptose residues (57). To date, only the dd-heptosyltransferase gene of H. ducreyi has been cloned and characterized (12, 15, 53). FIG. 1. Chemical structures of l-glycero-d-manno-heptopyranose and d-glycero-d-manno-heptopyranose. In H. pylori LPS, dd-heptose residues form an integral component of the core oligosaccharide as well as the linking region that connects the outer core oligosaccharide to the O chain. In some H. pylori isolates the linking region is composed of a long dd-heptoglycan polymer, while in other strains a single dd-heptose residue links the O chain to the core (33). Recently, nontypeable strains of H. pylori expressing dd-heptoglycan but lacking traditional Lewis antigens have been identified (2, 20, 44). The presence of the outer core region consisting of multiple dd-heptose residues and the dd-heptoglycan domain along with the Lewis blood group antigen-containing O-chain polysaccharide distinguishes H. pylori LPS from those of other gram-negative bacteria (33). To continue our work directed towards elucidating the genetic basis of LPS biosynthesis in H. pylori, we have cloned and characterized the key outer core biosynthetic enzyme encoded by the HP0479 gene through insertional mutagenesis studies and structural analysis of the resulting LPS.