Fig 1.
Previously proposed and the redefined LPS structure in H. pylori.
The previously proposed LPS structure in strain 26695 wild-type (A), the redefined LPS structures of the G27 wild-type (B), G27ΔHP1284 (C) and G27ΔwaaL (D). The nomination of different domains of the LPS is annotated.
Fig 2.
MS analysis of H. pylori Wild-type G27 LPS.
Wild-type G27 LPS samples were subjected to (A): methanolysis; (B): mild HF hydrolysis, and (C): mild periodate oxidation, respectively. MALDI-TOF MS spectra were recorded after permethylation. The MS peaks corresponding to sodiated glycans are coloured red, and annotated with m/z values and glycan structures. Note that for the spectrum after mild HF hydrolysis, the most intense isotopic peaks are annotated. Other blank signals are mainly due to (A): an addition of a sodium atom; and (C): incomplete reduction. The MS data indicate the fundamental architecture of wild-type G27 LPS is the same as strain 26695, containing LacNAc, heptan, glucan, Trio, the phosphorylated Glc-Gal-tri-Hep-KDO structure and lipid A.
Fig 3.
Effects of HP1284 and waaL mutation on H. pylori LPS.
LPS samples from H. pylori wild-type and mutants were analysed by SDS-PAGE and silver stain. (A): Low resolution SDS-PAGE. Lane 1–3: G27 wild-type, HP1284 deletion and HP1284 complementation in strain G27; (B): High resolution Tricine-SDS-PAGE. Lane 1–4: wild-type, HP1284 deletion, HP1284 complementation and waaL deletion in strain G27; Lane 5–6: wild-type and HP1284 insertion mutant in strain 26695; Lane 7–8: wild-type and HP1284 deletion mutant in strain X47.
Fig 4.
MS Analysis of LPS from G27 HP1284 and waaL Deletion Mutants.
The LPS samples were methanolysed, permethylated and analysed by MS. MALDI-TOF spectra of LPS from G27 HP1284 and waaL deletion mutants are shown in (A) and (B), respectively. The MS peaks corresponding to sodiated heptan-glucan structures are coloured red and annotated with m/z values. Most blank peaks are due to contamination and the addition of a sodium atom. The MS data indicate the core-oligosaccharide of G27 LPS is a hexa-saccharide with a sequence of Glc-Gal-Hep-Hep-Hep-KDO. The deletion of HP1284 leads to an incompletely synthesized core, which does not affect its O-antigen.
Table 1.
Polymyxin B Minimal Inhibitory Concentration (MIC) of H. pylori G27, X47 and 26695 Wild-type Strains and LPS Mutants.
Table 2.
Viable Counts of H. pylori X47ΔHP1284 and X47ΔwaaL mutants Recovered from Mice at 2 or 8 Weeks post Challenge*.
Fig 5.
Proposed Model for the LPS Biosynthetic Pathways in H. pylori.
H. pylori LPS biosynthesis follows a novel Wzk-dependent pathway [36]. The assembly of the very long O-antigen occurs in the cytoplasm and is initiated by WecA [36] transferring the GlcNAc residue of the Trio onto the UndPP carrier. Successive glycosyltransferases are recruited to complete the synthesis of the whole O-antigen encompassing the conserved Trio and variable glucan, heptan and Lewis antigens. After assembly in the cytoplasm, the UndPP-linked O-antigen is translocated by flippase Wzk to the periplasm [36]. Also assembled in the cytoplasm, the de novo synthesized core-lipid A is flipped by flippase MsbA to the periplasm [28]. After constitutive modifications through dephosphorylation and deacylation [9], the modified core-lipid A is ligated with O-antigen by the ligase WaaL [36], and the Hep III residue is the attachment site.
Fig 6.
The conserved Trio and the short core of H. pylori LPS are important for colonisation.
Currently known glycosyltransferases are assigned to the redefined complete structure of H. pylori LPS. The lipid A, the core-oligosaccharide domain and the Trio are proposed to be conserved and are important for host colonisation, whereas the Lewis antigen, heptan and glucan are variable and non-essential for colonisation.