The redefinition of Helicobacter pylori lipopolysaccharide O-antigen and core-oligosaccharide domains

Abstract

Helicobacter pylori lipopolysaccharide promotes chronic gastric colonisation through O-antigen host mimicry and resistance to mucosal antimicrobial peptides mediated primarily by modifications of the lipid A. The structural organisation of the core and O-antigen domains of H. pylori lipopolysaccharide remains unclear, as the O-antigen attachment site has still to be identified experimentally. Here, structural investigations of lipopolysaccharides purified from two wild-type strains and the O-antigen ligase mutant revealed that the H. pylori core-oligosaccharide domain is a short conserved hexasaccharide (Glc-Gal-DD-Hep-LD-Hep-LD-Hep-KDO) decorated with the O-antigen domain encompassing a conserved trisaccharide (-DD-Hep-Fuc-GlcNAc-) and variable glucan, heptan and Lewis antigens. Furthermore, the putative heptosyltransferase HP1284 was found to be required for the transfer of the third heptose residue to the core-oligosaccharide. Interestingly, mutation of HP1284 did not affect the ligation of the O-antigen and resulted in the attachment of the O-antigen onto an incomplete core-oligosaccharide missing the third heptose and the adjoining Glc-Gal residues. Mutants deficient in either HP1284 or O-antigen ligase displayed a moderate increase in susceptibility to polymyxin B but were unable to colonise the mouse gastric mucosa. Finally, mapping mutagenesis and colonisation data of previous studies onto the redefined organisation of H. pylori lipopolysaccharide revealed that only the conserved motifs were essential for colonisation. In conclusion, H. pylori lipopolysaccharide is missing the canonical inner and outer core organisation. Instead it displays a short core and a longer O-antigen encompassing residues previously assigned as the outer core domain. The redefinition of H. pylori lipopolysaccharide domains warrants future studies to dissect the role of each domain in host-pathogen interactions. Also enzymes involved in the assembly of the conserved core structure, such as HP1284, could be attractive targets for the design of new therapeutic agents for managing persistent H. pylori infection causing peptic ulcers and gastric cancer.

TIFA Signaling in Gastric Epithelial Cells Initiates thecagType 4 Secretion System-Dependent Innate Immune Response toHelicobacter pyloriInfection

“…Furthermore, the crystal structure of H. pylori HldD revealed that both the substrate binding site and the overall structure were highly similar to those of the HldD of E. coli , suggesting a conserved enzymatic function ( 36 ). It has also been shown that deletion of several enzymes involved in assembly of the highly conserved LPS core in H. pylori results in mutants that are unable to colonize mice, typifying the essential nature of most PAMPs ( 37 , 38 ). Furthermore, despite the fact that waaC was the first LPS biosynthesis gene identified in H. pylori , attempts to generate deletion mutants proved unsuccessful ( 39 ).…”

“…ADP- l , d -heptose and ADP- d , d -heptose are incorporated into the highly conserved core structure of H. pylori LPS, which consists of a hexa-saccharide (glucose—galactose- d , d -heptose [Hep III]— l , d -heptose [Hep II]— l , d -heptose [Hep I]—KDO). The variable O-antigen is directly attached to the Hep III molecule ( 37 ). The LPS molecule is assembled in the bacterial cytosol, transported across the periplasm, and flipped across the outer bacterial membrane.…”

“…Several groups have recently provided insight into the LPS structure of various commonly studied H. pylori strains ( 37 , 50 – 53 ). Li et al proposed a redefinition of the H. pylori LPS structure to contain lipid A, a short, highly conserved core hexa-saccharide domain composed of glucose-galactose- d , d- heptose- l , d -heptose- l , d -heptose-2-KDO (keto-3-deoxyoctulosonic acid) and the direct attachment of the variable O-antigen to the d , d- heptose molecule ( 37 ). This is in contrast to the LPS structures of other Gram-negative bacteria, which contain an inner core resembling the hexa-saccharide domain of H. pylori and an outer core to which the O-antigen is attached.…”

“…This is in contrast to the LPS structures of other Gram-negative bacteria, which contain an inner core resembling the hexa-saccharide domain of H. pylori and an outer core to which the O-antigen is attached. Studies have shown that deletion of enzymes involved in the assembly of the highly conserved LPS core in H. pylori results in bacteria that are unable to colonize mice ( 14 , 37 , 38 , 45 ). Our own data suggest that hldE and gmhA , at least in the G27 strain background, appear to be essential in H. pylori and that deletion of hldD and gmhB results in highly filamentous and slow-growing bacteria.…”

Lipopolysaccharides of Gram-Negative Bacteria: Biosynthesis and Structural Aspects

“…The assembly of O-antigen polysaccharides has so far been described to take place in four different ways, viz., (i) the synthasedependent pathway present in Salmonella enterica O:54 in which a D-ManpNAc homopolymer is formed having alternating β-(1 3)and β-(1 4)-linkages (27), (ii) the ATP-binding cassette (ABC)transporter-dependent pathway present e.g. in some E. coli in which the chain elongation takes place in the cytoplasm on a primer-adaptor trisaccharide and is terminated through the addition of a non-reducing terminal modification (28), (iii) the Wzk-dependent pathway present in H. pylori in which the Lewis-antigen containing O-antigen (29) assembled in the cytoplasm is transferred to the periplasm by a flippase that is a homologue of N-glycosylation proteins in other bacteria (30,31), and (iv) the Wzx/Wzy-dependent pathway in which an oligosaccharide is built on the cytoplasmic side of the IM and then flipped by Wzx to the periplasm where it is polymerized by the action of Wzy (32); this pathway is present in the majority of E. coli serogroups. All four pathways of formation of O-antigen polysaccharides lead to sufficiently long polymers such that a repetitive structure can be identified, which is referred to as the repeating unit (RU) of the O-antigen.…”