1  

Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori 1 

2 

Running title: Helicobacter pylori Lipopolysaccharide 3 

4 

5 

Hong Li1,2, Tingting Liao2, Aleksandra W. Debowski2,3,  Hong Tang1, Hans-Olof 6 

Nilsson4, Keith A. Stubbs3, Barry J. Marshall2, Mohammed Benghezal*2,5. 7 

8 

1West China Marshall Research Centre for Infectious Diseases, Centre of Infectious 9 

Diseases, West China Hospital of Sichuan University, Chengdu 610041, China 10 

2Helicobacter pylori Research Laboratory, School of Pathology & Laboratory 11 

Medicine, Marshall Centre for Infectious Disease Research and Training, The 12 

University of Western Australia, M504, L Block, QEII Medical Centre, Nedlands, WA 6009, 13 

Australia 14 

15 

University of Western Australia, Nedlands, Australia 16 

3School of Chemistry and Biochemistry, University of Western Australia, Crawley, 17 

Australia 18 

4Ondek Pty Ltd., School of Pathology & Laboratory Medicine, Marshall Centre for 19 

Infectious Disease Research and Training, University of Western Australia, Nedlands, 20 

Australia 21 

5SwissVitamin Institute, Route de la Corniche 1, CH-1066 Epalinges, Switzerland 22 

2  

*Corresponding author: Mohammed Benghezal, mohammed.benghezal@uwa.edu.au 1 

or mbenghezal@swissvitamin.ch 2 

3 

Abbreviations: Fuc, fucose; Glc, glucose; P, phosphate; GlcN, glucosamine; Gal, 4 

galactose; Kdo, 3-deoxy-D-manno-octulosonic acid; GlcNAc, N-acetylglucosamine; 5 

PEtN, phosphoethanolamine; ACP, acyl carrier protein; DD-Hep, 6 

D-glycero-D-mannoheptose; LD-Hep, L-glycero-D-mannoheptose; Und-PP, 7 

undecaprenyl pyrophosphate; ManNAc, N-acetylmannosamine; Neu5Ac, 8 

N-acetylneuraminic acid; Rib, ribofuranose; FutA, fucosyltransferase A; FutB, 9 

fucosyltransferase B; FutC, fucosyltransferase C. 10 

11 

Abstract 12 

This review covers the current knowledge and gaps in Helicobacter pylori 13 

lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram-negative 14 

bacterium which colonises the luminal surface of the human gastric epithelium. Both 15 

a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry 16 

of the Lewis antigen decorated O-antigen of H. pylori LPS promote immune escape 17 

and chronic infection. To date the complete structure of H. pylori LPS is not available 18 

and the proposed model is a linear arrangement composed of the inner core defined as 19 

the hexa-saccharide (Kdo-LD-Hep-LD-Hep-DD-Hep-Gal-Glc), the outer core 20 

composed of a conserved trisaccharide (-GlcNAc-Fuc-DD-Hep-) linked to the third 21 

heptose of the inner core, the glucan, the heptan and a variable O-antigen, generally 22 

3  

consisting of a poly-LacNAc decorated with Lewis antigens. Although the 1 

glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O-antigen 2 

chains have been identified and characterised, there are many gaps in regards to the 3 

biosynthesis of the core LPS. These limitations warrant additional mutagenesis and 4 

structural studies to obtain the complete LPS structure and corresponding biosynthetic 5 

pathway of this important gastric bacterium. 6 

7 

Introduction 8 

Lipopolysaccharide (LPS) is a large and variable complex glycolipid, and is an 9 

integral structural component of the outer membrane of Gram-negative bacteria. 10 

Localised in the outer leaflet of the outer membrane, LPS molecules maintain the 11 

barrier function of the outer membrane and mediate several interactions between the 12 

bacterium and its surrounding environment [1,2]. 13 

To date the proposed structure of Helicobacter pylori LPS (Figure 1), follows the 14 

same basic structure of other Gram-negative LPS molecules. It is composed of three 15 

domains: a hydrophobic domain termed lipid A (or endotoxin), which is embedded in 16 

the outer membrane; a relatively conserved non-repeating core oligosaccharide; and a 17 

variable outermost polysaccharide (or O-antigen). The three LPS domains differ in 18 

structure, and therefore confer different biological properties: the lipid A domain 19 

interacts with immune receptors and confers the LPS molecule with a range of 20 

immunological and potential endotoxic properties; the core oligosaccharide influences 21 

permeation properties of the outer membrane; and the O-antigen contributes to the 22 

4  

antigenicity and serospecificity of the molecule [3]. 1 

Helicobacter pylori is a spiral-shaped, microaerophilic Gram-negative bacterium 2 

commonly colonising humans with a prevalence up to 90% in certain countries. H. 3 

pylori is exceptionally well adapted to the human stomach mucosa, a niche where it 4 

can persist for decades in absence of antibiotic treatment [4-6]. The structural features 5 

of H. pylori LPS impart two major persistence mechanisms: 1) Decoration of the 6 

O-antigen with Lewis antigens promotes host mimicry to facilitate immune escape [7], 7 

and 2) the unique structure of its lipid A-core confers resistance to host cationic 8 

antimicrobial peptides [8]. Therefore, the study of the H. pylori LPS biosynthetic 9 

pathway represents an important step towards a better understanding of bacterial host 10 

adaptation and may bring eradication solutions through the identification of new 11 

therapeutic targets. 12 

Since the first report of the structure of H. pylori LPS purified from strain NCTC 13 

11637 [9], numerous genetic, biochemical and structural studies have been conducted 14 

to elucidate the LPS biosynthetic pathway and to further characterize the structure of 15 

the LPS of this bacterium. Of note, is that a difficulty in studying LPS biosynthesis in 16 

H. pylori lies in the fact that the genes known to be involved in LPS biosynthesis are 17 

scattered over the whole H. pylori genome instead of being organised in an operon, as 18 

is usually the case in other Gram-negative bacteria. In addition, attempts to produce 19 

knockout mutants of the gene HP0279 (encoding heptosyltransferase I, which 20 

transfers the first LD-Hep to Kdo) have been unsuccessful [10], suggesting that the 21 

minimal LPS structure in H. pylori might require one Hep moiety, in addition to lipid 22 

5  

A and Kdo. This is in contrast to Escherichia coli where the minimal LPS structure 1 

only requires the lipid A and the Kdo domains [11] for viability. 2 

To date most of H. pylori enzymes involved in the biosynthesis of lipid A and 3 

O-antigen have been identified and characterized. However, the enzymes responsible 4 

for the biosynthesis of the core region of LPS remain to be identified. Earlier studies 5 

of the LPS core region of H. pylori in strains 26695, SS1, and a serogroup O:3 isolate 6 

postulated the core region to be a branched structure [12-14]. However, recent 7 

reinvestigations have overturned this paradigm and have demonstrated a linear core 8 

structure in these strains [15-17]. Of particular interest is the identification of a new 9 

trisaccharide element GlcNAc-Fuc-DD-Hep in the proximal outer core, which has 10 

been shown to be conserved in all three strains [15-17]. 11 

This review summarises the current knowledge of enzymes and pathways involved in 12 

H. pylori LPS biosynthesis taking into account structural data obtained from the 13 

recent reinvestigation of the LPS structure of strain 26695 [17] (Figure 1). 14 

15 

Structure of Lipid A and its Biological Roles 16 

Lipid A is termed endodoxin. It is detected by host serum LPS-binding protein (LBP), 17 

dependent on the cofactors cluster of differentiation 14 (CD14) and myeloid 18 

differentiation factor 2 (MD2) and then recognized by the opsonic receptor CD14 to 19 

form a triple complex that subsequently activates Toll-like receptor 4 (TLR4) to 20 

trigger a signalling cascade, leading to the production of cytokines, clotting factors 21 

and the secretion of cationic antimicrobial peptides (CAMPs) and additional 22 

6  

stimulatory molecules [18]. CAMPs help to clear the invading pathogen by binding to 1 

negatively charged structural motifs (e.g. the phosphate groups on lipid A), leading to 2 

cell lysis and death [19]. However, if the immune response is overwhelmingly strong, 3 

sepsis and septic shock can occur, leading to multiple organ dysfunction syndrome 4 

and death [20].  5 

Previous reports have shown that TLR4 is also a receptor for H. pylori LPS [21-23]. 6 

Cullen et al. reported that in comparison to E. coli LPS, higher concentrations of H. 7 

pylori LPS are required for activation of TLR4，which is due to the constitutive 8 

modifications of H. pylori LPS by several enzymes [8]. When the modification 9 

enzymes are inactivated through mutation, H. pylori LPS displays a hexa-acylated, 10 

bis-phosphorylated lipid A, strongly activating TLR4 [8,24]. Cullen et al. also 11 

reported convincing data that H. pylori LPS did not activate TLR2 even at LPS 12 

concentrations as high as 10,000 ng/ml [8]. In contrast, other reports have suggested 13 

that H. pylori LPS recognition is mediated by TLR2 rather than TLR4 [25-28], as 14 

observed for Porphyromonas gingivalis LPS [29]. The discrepancy between these 15 

findings may be a consequence of contaminated LPS preparations with lipoproteins 16 

activating TLR2 [8,30]. Interestingly, a very recent study reported that TLR10 is a 17 

functional receptor involved in H. pylori LPS recognition [31]. 18 

Compared with other Enterobacteriaceae lipid A, H. pylori lipid A weakly activates 19 

TLR4 and displays a 1000-fold lower endotoxicity [3]. The molecular basis of this 20 

weak TLR4 elicitation is the modifications of lipid A through low level 21 

phosphorylation and an unusual acylation pattern and acyl chain length. In E. coli, 22 

7  

Neisseria meningitidis, and Pseudomonas aeruginosa, the lipid A structure contains 1 

six 14- or 12-carbon lipids, and carries two phosphate groups at positions 1- and 4'-. 2 

These characteristics are generally considered to be optimal for eliciting the maximal 3 

inflammatory response via TLR4 [32]. However, the major chemical species in H. 4 

pylori lipid A is a β-1,6-linked D-glucosamine disaccharide backbone acylated by four 5 

rather than six fatty acids with longer chain lengths (two 18-carbon and two 6 

16-carbon lipids), and lacks the usual 4'-phosphate group, and the 1-position 7 

phosphate group is replaced by a PEtN moiety (Figure 1). 8 

9 

Assembly of the Lipid A in H. pylori 10 

The biosynthesis of lipid A is well-conserved throughout Gram-negative bacteria and 11 

takes place on the cytoplasmic leaflet of the inner membrane through a nine-step 12 

biosynthetic pathway known as the Raetz pathway (8 Lpx enzymes and one Kdo 13 

glycosyltransferase are involved) (Figure 2) [11,33,34]. 14 

In H. pylori, homologues of seven Lpx proteins other than LpxM and the Kdo 15 

transferase have been identified in the Kyoto Encyclopedia of Genes and Genomes 16 

database (KEGG) [35]. The nine-step Kdo2-Lipid A biosynthesis in H. pylori is 17 

proposed to start from UDP-GlcNAc (Figure 2). The first step is catalysed by LpxA to 18 

add an acyl chain to UDP-GlcNAc, forming UDP-3-O-acyl-GlcNAc. The second step 19 

is catalysed by LpxC to remove the acetyl group from UDP-3-O-acyl-GlcNAc, 20 

producing UDP-3-O-acyl-GlcN. LpxD in the third step adds a second acyl chain to 21 

produce UDP-2,3-diacyl-GlcN. In the fourth step, part of the LpxD product is cleaved 22 

8  

at the pyrophosphate bond by LpxH, forming Lipid X (2,3-diacyl-GlcN-1P). In the 1 

fifth step, the Lipid X and UDP-2,3-diacyl-GlcN are condensed by LpxB to produce 2 

the characteristic β-1,6-linked GlcN disaccharide backbone of lipid A. The sixth step 3 

is catalysed by a specific kinase LpxK to produce Lipid IVA. In the seventh step, a 4 

bi-functional Kdo transferase (HP0957) (also known as WaaA or KdtA), is 5 

responsible for the transfer of two anionic 8-carbon Kdo sugars from the glycosyl 6 

donor CMP-Kdo to Lipid IVA, forming the Kdo2-IVA [33,36]. The final two steps are 7 

catalysed by two acyltransferases LpxL and LpxM, adding two secondary acyl chains 8 

to the distal GlcN unit of Kdo2-IVA to produce a hexa-acylated Kdo2-Lipid A. 9 

Based on KEGG H. pylori LPS biosynthesis and pentose phosphate pathways [35], 10 

the biosynthetic pathways for the generation of the two nucleotide activated sugars 11 

UDP-GlcNAc and CMP-Kdo are summarized (Figure S1 and S2, respectively). 12 

13 

Constitutive Modifications of the Lipid A in H. pylori 14 

Lipid A modifications in H. pylori are constitutive, which is unusual as most bacteria 15 

modify lipid A upon specific environmental cues [33]. The modifications to H. pylori 16 

lipid A result in low elicitation of the immune system, which is an important 17 

mechanism evolved by H. pylori to evade the innate immune response, and therefore 18 

enabling chronic infection. These modifications are likely the result of the long 19 

co-evolution of H. pylori with humans, at least since their joint exit from Africa about 20 

60,000 years ago [37]. 21 

H. pylori lipid A modification is a highly ordered and complex process that occurs via 22 

9  

a five-step enzymatic pathway (Figure 3). First, the 1-phosphate group of Kdo2-Lipid 1 

A is removed by LpxE, which is followed by the addition of a PEtN residue by EptA. 2 

Next a Kdo hydrolase removes the terminal Kdo sugar and subsequently, the 3 

4'-phosphate group is removed by a second phosphatase by LpxF. The final 4 

modification occurs in the outer membrane, where the two 3-O-linked acyl chains are 5 

removed by LpxR, thus resulting in a tetra-acylated lipid A. All the enzymes 6 

responsible for these steps have been identified and characterized [8,33,36,38-41]. It 7 

has been demonstrated that the HP0579-0580 (encoding the Kdo hydrolase) mutant 8 

has greater than a 180-fold increase in polymyxin B sensitivity as a consequence of 9 

the defect in downstream modifications to the lipid A 4'-phosphate group [33]. 10 

Mutants lacking lpxE, lpxF or lpxE/F had a 16-, 360- and 1020-fold increase in 11 

sensitivity to polymyxin B and a variety of other naturally occurring CAMPs, 12 

respectively, and 2-, 6- and 10-fold increase in activation of TLR4, respectively. 13 

Interestingly, the lpxE/F mutant loses its ability to colonize the murine stomach, 14 

demonstrating the central importance of dephosphorylation of the lipid A domain of H. 15 

pylori LPS [8]. 16 

17 

Structure and Biological Roles of the Core Oligosaccharide 18 

The core oligosaccharide, the central domain of LPS between lipid A and the 19 

O-antigen, can be further divided into two parts, the inner core and outer core (Figure 20 

1). Within a bacterial genus, the structure of the inner core tends to be well conserved, 21 

and the fact that core oligosaccharides from distantly related bacteria share inner core 22 

10  

structural features is a reflection of the importance of the LPS core in OM integrity 1 

[11]. The inner core typically contains two LD-Hep residues, designated as Hep I and 2 

Hep II. 3 

In E. coli, the inner Hep I is decorated with a PEtN residue by the kinase WaaP, and 4 

the Hep II is modified with another DD-Hep residue (Hep III) and phosphate (P) 5 

catalysed by WaaQ and WaaY, respectively (Figure 4). The WaaP enzyme has the 6 

most important role among these activities, as the modifications must proceed in the 7 

strict order of WaaP→WaaQ→WaaY [42]. The deletion of waaP resulted in a mutant 8 

that expressed LPS with a complete carbohydrate backbone and grew as the parent 9 

strain, however, it showed a markedly increased sensitivity to hydrophobic 10 

compounds and was avirulent like a deep rough stain [42]. It was subsequently 11 

demonstrated that truncation of LPS beyond the Hep II residue, through mutation of 12 

waaG, also led to an increase in sensitivity to hydrophobic compounds, and was 13 

found to have an indirect effect on inner core phosphorylation: the absence of an outer 14 

core resulted in inefficient phosphorylation by WaaP (40% of wild-type levels) [43]. 15 

Among Gram-negative bacteria, P. aeruginosa has the most phosphorylated inner 16 

core (Figure 4). Mutants lacking inner core Hep or phosphate groups have never been 17 

isolated or constructed, suggesting that Hep-linked phosphates are essential for P. 18 

aeruginosa viability [32]. The addition of negatively charged phosphoryl groups to 19 

the inner core enables adjacent LPS molecules to be cross linked by divalent cations, 20 

such as Ca2+, Mg2+, which stabilizes the outer membrane [44]. 21 

While the inner cores of E. coli and P. aeruginosa, are markedly negatively charged, 22 

11  

H. pylori has evolved modifications to reduce the negative charge on the inner core 1 

including: 1) Hep II is not decorated with phosphate moieties; and 2) the negatively 2 

charged outer Kdo is removed by Kdo hydrolase [33]. Only Hep I is decorated with 3 

PEtN, which is suggested to be transferred by HP1417 [3]. In addition, Hep III is a 4 

DD-Hep that is relatively rare among bacteria [45] and it is substituted with a 5 

branched disaccharide Gal-Glc [17] (Figure 4). 6 

The outer core of H. pylori shows substantial variability among strains. One of the 7 

unique features of the H. pylori outer core is the presence of the homopolymers. 8 

DD-heptan (consisting of DD-Hep) and α-1,6-glucan (consisting of Glc) were 9 

identified in strain 26695 [17]. DD-heptan was also found in other isolated clinical 10 

strains [46-48] (Figure 5, 6 and 7). Recently, another homopolymer β-1,2-riban 11 

(consisting of 3-5 β-ribofuranose) was identified in the outer core of strain SS1 [16] 12 

(Figure 5). Besides DD-heptan, α-1,6-glucan and β-1,2-riban, an α-1,4-glucan 13 

homopolymer was reported in LPS isolated from a Danish strain (D10) [48], NCTC 14 

strain 11637 and two clinical strains [49,50]. 15 

The outer core structure of H. pylori 26695 LPS was initially postulated to contain a 16 

side branch α-1,6-glucan substituted at the first DD-Hep of the heptan 17 

[12,13,45,51-54]. However, recent analysis of the 26695 LPS has revealed that the 18 

α-1,6-glucan is linked directly to a DD-Hep residue of a trisaccharide element 19 

composed of GlcNAc-Fuc-DD-Hep [17]. This trisaccharide element has also been 20 

identified in H. pylori strains SS1 and O:3 [15,16]. Interestingly, the reinvestigation of 21 

the 26695 LPS structure revealed that the heptan is directly linked to the glucan, 22 

12  

making the LPS almost completely linear in structure, with the exception of the 1 

Gal-Glc disaccharide on Hep III (Figure 1). 2 

The core oligosaccharide of H. pylori LPS contributes to the bacterium’s pathogenesis 3 

and colonisation. The inner core is involved in binding to laminin, which is an 4 

extracellular matrix glycoprotein in the basement membrane. This binding inhibits the 5 

recognition of laminin by the epithelial cell receptors (integrin), which damages 6 

gastric mucosal integrity [55-57]. The binding also stimulates pepsinogen secretion, 7 

leading to erosion of the mucus layer at the luminal surface [58,59]. In terms of the 8 

roles of the outer core, the α-1,6-glucan has been shown to be involved in initial 9 

colonisation of the host [51,60,61]. While the DD-heptan is not required for initial 10 

colonization [61], it is thought to provide increased length and flexibility to the LPS 11 

such that it covers the bacterial surface, and consequently interferes with the 12 

interaction of bacterial virulence factors with the epithelium of host cells. This is 13 

based on the observation that most clinical strains expressing heptan but lacking 14 

Lewis antigens have been isolated from asymptomatic hosts [61]. The presence of a 15 

novel D-galactan and an extended β-1,2-riban in the gerbil passaged strain of an 16 

mutant devoid of O-antigen (SS1HP0826::kan) supports this view [62]. 17 

18 

Assembly of the Inner and Outer Core LPS 19 

The carbohydrate Kdo is the link between the Lipid A and the inner core Hep residues, 20 

with Hep III being decorated with a Gal and Glc (Figure 1). Hep I is transferred to 21 

Kdo by Hep I transferase RfaC (HP0279) [3,33]. Hep II is then transferred to Hep I by 22 

13  

RfaF (HP1191) [33]. HP0479 was initially proposed to be the Hep III transferase [45], 1 

however mass spectrometric analysis later found that it was involved in the assembly 2 

of the outer core [17] thus, the enzyme responsible for the addition of the Hep III has 3 

yet to be identified. In addition the glycosyltransferase for attaching the Gal residue of 4 

the disaccharide branched on Hep III has also not been identified. The Glc residue of 5 

the branched disaccharide was found to be transferred by HP1416 [53,63]. The donor 6 

for Hep I and Hep II are ADP-LD-Hep, whereas for Hep III, the donor is 7 

ADP-DD-Hep. The biosynthetic pathway for the generation of ADP-LD-Hep and 8 

ADP-DD-Hep starts from Glc (Figure S3). It is noteworthy that all the genes involved 9 

in the synthesis of ADP-LD-Hep from D-sedo-heptulose-7P, are clustered together in 10 

the genome (HP0857 to HP0860). The disruption of the last gene, rfaD (HP0859) 11 

required for ADP-LD-Hep biosynthesis, was shown to result in a severe LPS 12 

truncation, decreased growth rate, greater susceptibility to novobiocin, reduced 13 

adhesion to AGS cells, and failed to induce the “hummingbird” phenotype in AGS 14 

cells, suggesting that there is a link between LPS and the type IV secretion system 15 

(T4SS) [64]. The viability of Hep-less rfaD mutant indicates that H. pylori can 16 

survive without any Hep residue in its LPS structure. This observation however is 17 

difficult to reconcile with the lethality of the mutation of HP0279 transferring Hep I to 18 

the inner core LPS [3]. Finally, UDP-Gal and UDP-Glc, the glycosyl donors for the 19 

Gal and Glc residues attached to Hep III respectively are also produced from Glc 20 

(Figure S4). 21 

The outer core begins with the conserved trisaccharide GlcNAc-Fuc-DD-Hep attached 22 

14  

after Hep III. The GTs required for the transfer of the first two carbohydrate motifs 1 

have not been identified yet, whereas the enzyme responsible for the transfer of 2 

DD-Hep was found to be HP0479, which was the first DD-Hep transferase to be 3 

identified in H. pylori [17,45]. Adjacent to the trisaccharide is an α-1,6 glucan 4 

followed by an α-1,3 heptan. The enzyme responsible for attachment of the glucan 5 

was found to be HP0159 [51,53,63], but the enzyme for transfer of the heptan is yet to 6 

be identified. The donor for Fuc is GDP-L-Fuc produced by the de novo pathway 7 

which has been characterized in detail [65] and a salvage pathway, recycling L-Fuc 8 

from the host [66]. The salvage pathway remains to be investigated as the Fuc kinase 9 

and GDP-Fuc pyrophosphorylase are yet to be identified (Figure S5). 10 

The carbohydrates of the core oligosaccharide are sequentially attached to the 11 

Kdo2-lipid A on the cytosolic leaflet of the inner membrane to form the lipid A-core. 12 

This molecule is then flipped across the inner membrane by the MsbA flippase on the 13 

periplasmic leaflet of the inner membrane for subsequent ligation to O-antigen in the 14 

periplasmic space [34] (Figure 8). 15 

16 

Structure and Biological Roles of the O-antigen 17 

The O-antigen of H. pylori strains usually comprises a Gal-GlcNAc backbone chain, 18 

which can be divided into two types on the basis of its linkage. The Type 1 chain is 19 

composed of Gal-(β-1,3)-GlcNAc, and gives rise to Lewis a (Lea), Lewis b (Leb), 20 

Lewis c (Lec), Lewis d (Led or H-1) and sialyl-Lea. The Type 2 chain is composed of 21 

Gal-(β-1,4)-GlcNAc (LacNAc), and gives rise to Lewis x (Lex), Lewis y (Ley) and 22 

15  

sialyl-Lex (Figure 9). The Type 2 Lex and Ley are overwhelmingly (80-90%) expressed 1 

in H. pylori strains from various geographical regions worldwide based on screens 2 

using anti-Le antibody probes [67]. It has been suggested that Type 1 Lea and Leb are 3 

predominantly expressed in Asian hosts [68]. Interestingly, H. pylori strains 4 

simultaneously expressing both Type 1 and Type 2 antigens have also been isolated 5 

from both Asian and Western populations [68,69]. These findings indicate a mosaic of 6 

expression of Type 1 and Type 2 antigens, occurring within the same strain or with 7 

one host carrying different stains.  8 

While typeable strains are identified using a panel of anti-Le antibodies some strains 9 

show no reaction. The lack of Lewis antigen reactivity might be due to false negatives 10 

of the serological assay used in these studies. For example, the clinical strains AF1 11 

and 007 were serologically non-typeable, whereas a mass spectrometric structural 12 

analysis showed that each strain had a high degree of fucosylation, producing a 13 

polymeric internal Lex chain terminating with Lex and Ley [46]. This suggests that 14 

expression of Lex and Ley may have been underestimated by serological analysis. 15 

Alternatively, certain strains are truly missing Lewis antigens [47,61] and carry other 16 

novel chains, including the trisaccharide repeating unit 17 

3-C-methyl-D-mannose-(α-1,3-L-rhamnose-(α1,3)-D-rhamnose (found in the Danish 18 

strains D1, D3 and D6 [46,70]). 19 

In the human stomach, the Lea and Leb antigens are mainly expressed on the apical 20 

surface of the superficial faveolar epithelium of the antrum and corpus, whereas Lex 21 

and Ley are mainly expressed in the glands of the antrum and corpus [71,72]. This 22 

16  

similarity in structure between Lewis antigens on stomach cells and H. pylori LPS 1 

O-antigen may represent a form of molecular mimicry or immune tolerance that 2 

enables H. pylori LPS antigen to be shielded from immune recognition because of the 3 

similarity to “self” antigens [73]. On the other hand, this similarity has been 4 

implicated in the pathogenesis of autoimmunity [74-77]. Specifically, antibodies 5 

against both Type 1 and Type 2 were found to react strongly with gastric mucin and 6 

anti-Lex targeted polymorphonuclear lymphocytes (PMN) as PMN express CD15 (Lex) 7 

on their surface, whereas the β-chain of the gastric proton pump (H+, K+-ATPase) is 8 

glycosylated with Ley and is therefore attacked by anti-Ley antibodies [76]. All these 9 

autoimmune reactions lead to H. pylori-related atrophic gastritis. With Lewis antigen 10 

mimicry camouflaging H. pylori from detection by the host, but inducing autoimmune 11 

gastric diseases, H. pylori has consequently been referred to as a “wolf in sheep’s 12 

clothing” [7]. 13 

Lex has been shown to contribute to the adhesion of H. pylori to gastric mucosa and 14 

the glycoprotein galectin-3 has been identified to be the receptor for Lex [78]. 15 

Consistent with its important role, the mouse-adapted strain SS1 has been found to 16 

predominantly express Lex with traces of Ley, and similar results have been reported 17 

for strains J99, 26695 and NCTC11637 [12-14,79]. It is noteworthy that the 18 

expression of O-antigen is subject to phenotypic variation at different pH values 19 

(Figure 10). When strain 26695 was grown in liquid medium at pH 7, the majority of 20 

GlcNAc residues on the O-chain were glycosylated with Fuc residues to form 21 

polymeric Lex, including chain termination by a Lex unit, whereas at pH 5, only some 22 

17  

GlcNAc residues were glycosylated with Fuc residues, while the majority were 1 

substituted with Gal and the chain was terminated by a Ley unit [79]. As the pH in the 2 

stomach varies from the very acidic pH 2 of the mucus layer to the almost neutral 3 

epithelial surface, H. pylori near the epithelial surface have optimal Lex expression, 4 

thus promoting adherence to the cell surface. Those H. pylori with reduced Lex 5 

expression can stay in the mucus layer as a free-swimming form and act as a reservoir 6 

for subsequent infection [67]. Furthermore, Lex and Ley can interact with the C-type 7 

lectin DC-SIGN on dendritic cells to block development of T helper cells (Th1), thus 8 

down-regulating the inflammatory response [80].The other C-type lectin that interacts 9 

with the O-chain is surfactant protein D (SP-D), which is found in the gastric mucosa 10 

at the luminal surface and within gastric pits of mucus-secreting cells [81]. SP-D is 11 

involved in antibody-independent pathogen recognition and clearance, and it has been 12 

shown that H. pylori colonisation is more robust in SP-D –/– mice [81]. 13 

Screening of the H. pylori SP-D escape variant (J178V) from the parent strain J178 14 

[82], showed that the phase-variable futA gene was upregulated in J178V, which 15 

resulted in a greater degree of fucosylation of the O-chain rather than the addition of 16 

Gal or Glc. With SP-D having a low affinity for Fuc relative to its affinity for Glc or 17 

Gal (Figure 11), the upregulation of the futA gene in J178V enables avoidance of 18 

SP-D mediated elimination. Thus, bacteria with more fucosylation have a selective 19 

advantage during colonisation [82]. 20 

21 

Assembly of the O-antigen and its Ligation to the Lipid A-Core 22 

18  

Three pathways are known for biosynthesis of the O-antigen: 1) the Wzy-dependent 1 

pathway; 2) the ABC-transporter-dependent pathway; and 3) the synthase-dependent 2 

pathway. The first two pathways are widespread in occurrence, but the 3 

synthase-dependent pathway is less common [11]. The three pathways have similar 4 

initiation reactions: the formation of an undecaprenyl pyrophosphate (und-PP) linked 5 

sugar, commonly catalysed by WecA, a GlcNAc-1P transferase, to form 6 

und-PP-GlcNAc. However, the three pathways differ with respect to O-antigen 7 

polymerisation and translocation. In the Wzy-dependent pathway, short individual 8 

und-PP-linked O-antigen units are assembled in the cytoplasm and then exported by 9 

the Wzx flippase to the periplasm, where they are polymerised by Wzy, which is 10 

regulated by Wzz, to modulate the final O-antigen chain length. In the 11 

ABC-transporter-dependent pathway, the entire O-antigen is assembled in the 12 

cytoplasm, prior to export by the ABC-transporter, which is formed by Wzm and Wzt. 13 

Wzm forms the membrane channel and Wzt provides the energy for transport. The 14 

third pathway relies on two enzymes WbbE and WbbF, to simultaneously extend and 15 

extrude the O-chain across the inner membrane [11]. 16 

Assembly of the O-antigen in H. pylori follows a novel Wzk-dependent pathway [83] 17 

(Figure 8) which differs from the ABC-transporter-dependent pathway in the 18 

translocation step. The ABC-transporter pathway requires both Wzm and Wzt for the 19 

translocation of the O-antigen, whereas, Wzk is the only protein required in H. pylori. 20 

Intriguingly, Wzk is not related to any previously identified O-antigen flippase, but is 21 

instead homologous to Campylobacter jejuni PglK, which is responsible for flipping 22 

19  

Und-PP-heptosaccharide for protein N-glycosylation. Wzk was demonstrated to 1 

restore the C. jejuni pglK mutant in flipping the Und-PP-heptosaccharide, suggesting 2 

an evolutionary connection between LPS biosynthesis and protein N-glycosylation 3 

[83]. 4 

The assembly of O-antigen in H. pylori starts with WecA transferring GlcNAc to the 5 

und-PP carrier to form und-PP-GlcNAc. Subsequently, the GlcNAc transferase, RfaJ 6 

(HP1105) [54], and Gal transferase (HP0826) [17,84] alternatively add GlcNAc and 7 

Gal to form the LacNAc backbone. FutA (HP0379) [85] then attaches a fucose 8 

molecule (through a α-1,3 linkage) to selected GlcNAc residues of the LacNAc 9 

backbone, to form Lex. The number of heptad repeats in the C-terminal region of 10 

FutA and FutB corresponds to the number of GlcNAc residues in the LacNAc 11 

backbone to be fucosylated, and so function as an enzymatic ruler [86]. FutC 12 

(HP0093, 0094) [87-89] transfers a Fuc residue (through a α-1,2 linkage) to the 13 

terminal Gal to form Ley. Although sialyl-Lex has been confirmed to be expressed in 14 

H. pylori strain P466 [12], the homologue for the required α-2,2 Neu5Ac transferase 15 

has not been identified in H. pylori. The glycosyl donor for Neu5Ac is CMP-Neu5Ac 16 

and HP0326 has been suggested to encode CMP-Neu5Ac synthetase [90] (Figure S6). 17 

However, homologues of other enzymes involved in the biosynthesis of 18 

CMP-Neu5Ac have not been identified in H. pylori (Figure S6). 19 

After assembly and translocation to the periplasm, the O-antigen is ligated with the 20 

lipid A-core to form the full-length LPS molecule. Of note, the GlcNAc residue after 21 

the heptan of the outer core has been inferred to be the anchor point for the O-antigen 22 

20  

[17], however no experimental data, either structural or genetic, is available to support 1 

this hypothesis. Thus the precise definition of the core and O-antigen of H. pylori LPS 2 

remains to be established experimentally. 3 

Although the machinery underlying the full-length LPS transport to the outer 4 

membrane remains to be characterized in H. pylori, the presence of homolog genes of 5 

the LPS transport pathway in its genome suggests that this bacterium forms LPS 6 

transport bridges as well [91,92]. 7 

In an attempt to summarise the current knowledge of LPS structure and biosynthesis, 8 

a model is presented here based on the literature data. In this LPS model the lipid A, 9 

the core (inner and outer) and the O-antigen are annotated along with the proteins 10 

involved in the biosynthesis of LPS in H. pylori, while the missing GTs are indicated 11 

with a question mark (Figure 12). 12 

13 

Future Directions for the Study of LPS in H. pylori 14 

Despite the characterization of the carbohydrate moieties in the structure of H. pylori 15 

LPS, all the GTs responsible for LPS assembly, including the important trisaccharide 16 

motif and the third heptosyltransferase of the core LPS, have yet to be identified 17 

(Figure 12). A difficulty to studying LPS biosynthesis in H. pylori lies in the fact that 18 

so far the genes found to be involved in LPS biosynthesis are scattered over the whole 19 

H. pylori genome instead of being organised in an operon as it is usually the case in 20 

other Gram-negative bacteria. Thus, through the use of molecular biology, 21 

carbohydrate chemistry and structural analysis of LPS from wild-type and the 22 

21  

associated LPS mutants should be conducted to determine the overall structure and 1 

associated biosynthetic pathways in H. pylori. At the time of writing this review, the 2 

genomes of 48 sequenced H. pylori strains have been annotated in Carbohydrate 3 

Active Enzyme database (CAZy) [93] , and more than 20 ORFs (approximately 1–2% 4 

of the whole genome) from each strain have been annotated as GTs (experimentally 5 

confirmed or putative). The ORFs have been further classified into 12 GT families, 6 

among which several putative GTs have not been previously studied (summarised in 7 

Table 1). Finally, purification of the core LPS accumulating in the waaL mutant [83] 8 

and its structural analysis will precisely identify the anchor point of the O-antigen in 9 

H. pylori to validate the current LPS model (Figure 12) or to redefine the overall 10 

structure of H. pylori LPS. Although H. pylori LPS is a key determinant in 11 

establishing the colonisation and persistence, its LPS structure and corresponding 12 

biosynthetic pathway remain to be fully characterised. Establishing the complete LPS 13 

structure of this important human gastric pathogen is crucial to a better understanding 14 

of H. pylori pathogenesis such as resistance to CAMPs and immune escape, and may 15 

bring new therapeutic solutions targeting the LPS or the corresponding enzymes 16 

involved in its biosynthesis. 17 

 18 

Acknowledgements and disclosures 19 

This work was supported by an Early Career Research Fellowship from the National 20 

Health and Medical Research Council (NHMRC) (APP1073250), ECR Fellowship 21 

Support Grant from the University of Western Australia, and an Ada Bartholomew 22 

22  

Medical Research Trust Grant to A.W.D. An ARC Future Fellowship (FT100100291) 1 

supported K.A.S. A grant from West China Hospital, Sichuan University to L.H. and 2 

H.T. entitled “1.3.5 project for disciplines of excellence, West China Hospital, 3 

Sichuan University”. 4 

Competing interest 5 

The funders had no role in the decision to publish the review, or its preparation. 6 

Hans-Olof Nilsson is an Employee of Ondek Pty Ltd. Mohammed Benghezal is a 7 

former Employee of Ondek Pty Ltd. Hong Li, Tingting Liao, Aleksandra W. 8 

Debowski, Keith A. Stubbs and Barry J. Marshall received research funds as well as 9 

reimbursement of expenses for attending a symposium, congress, national or 10 

international meeting. 11 

12 

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83. Hug I, Couturier MR, Rooker MM, Taylor DE, Stein M, et al. (2010) Helicobacter pylori 7 

Lipopolysaccharide Is Synthesized via a Novel Pathway with an Evolutionary Connection to 8 

Protein N-Glycosylation. PLoS Pathogens 6: e1000819. 9 

84. Endo T, Koizumi S, Tabata K, Ozaki A (2000) Cloning and expression of beta 10 

1,4-galactosyltransferase gene from Helicobacter pylori. Glycobiology 10: 809-813. 11 

85. Rasko DA, Wang G, Palcic MM, Taylor DE (2000) Cloning and characterization of the alpha(1,3/4) 12 

fucosyltransferase of Helicobacter pylori. Journal of Biological Chemistry 275: 4988-4994. 13 

86. Nilsson C, Skoglund A, Moran AP, Annuk H, Engstrand L, et al. (2006) An enzymatic ruler 14 

modulates Lewis antigen glycosylation of Helicobacter pylori LPS during persistent infection. 15 

Proceedings of the National Academy of Sciences of the United States of America 103: 16 

2863-2868. 17 

87. Wang G, Boulton PG, Chan NWC, Palcic MM, Taylor DE (1999) Novel Helicobacter pylori alpha 18 

1,2-fucosyltransferase, a key enzyme in the synthesis of Lewis antigens. Microbiology 145: 19 

3245-3253. 20 

88. Stein DB, Lin Y-N, Lin C-H (2008) Characterization of Helicobacter pylori alpha 21 

1,2-Fucosyltransferase for Enzymatic Synthesis of Tumor-Associated Antigens. Advanced 22 

Synthesis & Catalysis 350: 2313-2321. 23 

89. Drouillard S, Driguez H, Samain E (2006) Large-scale synthesis of H-antigen oligosaccharides by 24 

expressing Helicobacter pylori alpha 1,2-fucosyltransferase in metabolically engineered 25 

Escherichia coli cells. Angewandte Chemie-International Edition 45: 1778-1780. 26 

90. Berg DE, Hoffman PS, Appelmelk BJ, Kusters JG (1997) The Helicobacter pylori genome 27 

sequence: genetic factors for long life in the gastric mucosa. Trends in Microbiology 5: 28 

468-474. 29 

91. Liechti G, Goldberg JB (2012) Outer membrane biogenesis in Escherichia coli, Neisseria 30 

meningitidis, and Helicobacter pylori: paradigm deviations in H. pylori. Front Cell Infect 31 

Microbiol 2: 29. 32 

92. Okuda S, Freinkman E, Kahne D (2012) Cytoplasmic ATP hydrolysis powers transport of 33 

lipopolysaccharide across the periplasm in E. coli. Science 338: 1214-1217. 34 

93. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, et al. (2009) The 35 

30  

Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics. 1 

Nucleic Acids Research 37: D233-D238. 2 

94. Whitfield C, Trent MS (2014) Biosynthesis and Export of Bacterial Lipopolysaccharides. Annu Rev 3 

Biochem: 99-128. 4 

95. Altman E, Chandan V, Li J, Vinogradov E (2011) A reinvestigation of the lipopolysaccharide 5 

structure of Helicobacter pylori strain Sydney (SS1). FEBS Journal 278: 3484-3493. 6 

7 

8 

31  

Figure Legends 1 

Figure 1. Proposed LPS Structure of H. pylori Reference Strain 26695 2 

The proposed complete model is based on the reinvestigation of H. pylori LPS 3 

structure [15-17]. The three domains of LPS are indicated: lipid A, core 4 

oligosaccharide (further divided into inner core and outer core) and O-antigen. 5 

6 

Figure 2. Proposed Kdo2-lipid A Biosynthetic Pathway in H. pylori 7 

Based on a recent review [94] and KEGG H. pylori LPS biosynthesis pathway [35] , 8 

the H. pylori Kdo2-lipid A is proposed to be produced via a nine-step enzymatic 9 

pathway (eight Lpx enzymes and one Kdo glycosyltransferases). Step 1: the addition 10 

of a acyl chain to UDP-GlcNAc by LpxA (HP1375); Step 2; the removal of the acetyl 11 

group from UDP-3-O-acyl-GlcNAc by LpxC (HP1052); Step 3, the addition of a 12 

second acyl chain by LpxD (HP0196); Step 4: the pyrophosphate bond is cleaved by 13 

LpxH, producing lipid X; Step 5: the condensation of lipid X and 14 

UDP-2,3-diacyl-GlcN by LpxB (HP0867) to produce the characteristic 15 

tetra-acyl-disaccharide-1-phosphate; Step 6: the phosphorylation at the 4'- position by 16 

kinase LpxK (HP0328) to produce lipid IVA; Step 7：the addition of two Kdo by the 17 

bi-functional KdtA (HP0957) to form Kdo2-IVA; Step 8: the addition of secondary 18 

acyl chain to the 2'- position by LpxL (HP0280); Step 9: the addition of the other 19 

secondary acyl chain to the 3'- position by LpxM (homologue in H. pylori 20 

unidentified). 21 

22 

32  

Figure 3. The Constitutive Lipid A Modification Pathway in H. pylori 1 

H. pylori produces a highly modified lipid A species via a five step enzymatic 2 

pathway [8]. Step 1: the 1-phosphate group of Kdo2-lipidA is cleaved by LpxE 3 

(HP0021); Step 2: the addition of PEtN by EptA (HP0022) at the 1-position; Step 3: 4 

KdoH1 (HP0579) and KdoH2 (HP0580) act in concert to remove the terminal Kdo 5 

sugar; Step 4: the 4'-phosphate group is removed by LpxF (HP1580), leaving an 6 

unmodified hydroxyl group; Step 5: the removal of the 3-O-linked acyl chains by 7 

LpxR (HP0694) to produce a tetra-acylated lipid A. The first four modification 8 

reactions occur at the periplasmic leaflet of the inner membrane. The final step occurs 9 

in the outer membrane. For simplicity, the core oligosaccharide and O-antigen are not 10 

shown. 11 

12 

Figure 4. LPS Inner Core Structure in E. coli, P. aeruginosa and H. pylori 13 

In E. coli, the inner Hep I is decorated with PEtN by kinase WaaP; Hep II is added 14 

with a Hep III and modified with a phosphate (P) residue by WaaQ and WaaY, 15 

respectively. P. aeruginosa has the most phosphorylated inner core among 16 

Gram-negative bacteria: Hep I is modified with three phosphate residues and a PEtN, 17 

Hep II is modified with three phosphate residues and a carbamoyl (Cm) residue. In 18 

contrast, in H. pylori the inner core lacks phosphate residues, the Kdo II is removed 19 

by Kdo hydrolase and Hep I is decorated with PEtN (HP1417 is predicted to be the 20 

PEtN transferase). For simplicity, the outer core and O-antigen are not indicated. 21 

22 

33  

Figure 5. LPS Outer Core Structures in H. pylori strain 26695, SS1 and 1 

serogroup O:3 2 

H. pylori strain 26695 (A) outer core was based on references [12,13,45,51-54]; SS1 3 

(A) is based on references [12,13,45,54]; O:3 (A) is based on references [12,14]; O:3 4 

(B) is based on reference [45]; 26695 (B), SS1 (B) and O:3 (C) are based on the 5 

reinvestigation of these strains [15,17,95]. (m=4, n=3 in 26695B); (n=3-5 in SS1(B)); 6 

(m=5-6 and n=2-3 in O:3(A)); m and n in other structures were not indicated in the 7 

papers. For simplicity, lipid A, the inner core and the O-antigen are not indicated. 8 

9 

Figure 6. LPS Outer Core Structures in Other H. pylori strains 10 

H. pylori strain J99 outer core is based on reference [12]; strains 11 

11637(O:1)/P466/H428/H507/UA915/UA948/UA955/J223/CA2/CA4/CA5/CA6/F-512 

8C/F-15A/R-58A/F-58C/GU2/R-7A/H607 are based on [12-14,46,69] and n=2-3; 13 

strain PJ1 is based on reference [45]; PJ2 is based on references [60,61], O-antigen is 14 

devoid and glucan: n=3-9; strains O:6 and MO19 are based on references [12,14] and 15 

m=5, n (heptan)=2, and n (glucan)=2-3. For simplicity, lipid A, the inner core and the 16 

O-antigen are not indicated. 17 

18 

Figure 7. LPS Outer Core Structures in Various Clinical Strains of H. pylori 19 

The structures of H. pylori strains 1C2, 12C2, 62C, 7A, 75A and 77C are based on 20 

reference [47]. * indicate that these heptan homopolymers are capped with an 21 

incomplete Lewis antigen (Fuc, GlcNAc), n=4-5. The Danish strains D2, D4, D4 are 22 

34  

based on reference [48], all these strains lack a typical Lewis antigen. For simplicity, 1 

the inner core and lipid A are not indicated. 2 

3 

Figure 8. Assembly of the O-antigen and its Ligation with Lipid A-core in H. 4 

pylori 5 

Assembly of H. pylori O-antigen and its ligation with Lipid A-core requires three 6 

inner membrane bound enzymes: WecA (HP1581), Wzk (HP1206) and WaaL 7 

(HP1039) [83]. WecA initiates the assembly of O-antigen by transferring GlcNAc to 8 

the und-PP carrier to form und-PP-GlcNAc. Subsequently, cytoplasmic GTs including 9 

HP1105, HP0826, FutA and FutC add corresponding sugars to form the O-antigen, 10 

which is flipped to the periplasm by the O-antigen flippase Wzk. Also assembled in 11 

the cytoplasm, the Lipid A-core is flipped by flippase MsbA (HP1082) to the 12 

periplasm, where the flipped O-antigen is ligated with Lipid A-core by the O-antigen 13 

ligase WaaL, forming the full-length H. pylori LPS. 14 

15 

Figure 9. Structures of Lewis antigens in H. pylori 16 

H. pylori Lewis antigen comprises a Gal-GlcNAc backbone chain, which can be 17 

divided into two types on the basis of its linkage. The Type 1 chain is composed of 18 

Gal-(β-1,3)-GlcNAc, and gives rise to Lea, Leb, Lec (also referred as Type 1 19 

precursor), Led (also referred as H-1) and sialyl-Lea. The Type 2 chain is composed of 20 

Gal-(β-1,4)-GlcNAc, and gives rise to Lex, Ley and sialyl-Lex. Linkage and 21 

corresponding GTs are indicated. 22 

35  

1 

Figure 10. O-antigen Structural Variations at Different pH 5 and pH 7 2 

At pH 5, most of the O-chain is substituted with Gal, only partially with Lex and is 3 

terminated with Ley. At pH 7, most of the O-chain is fucosylated to form polymeric 4 

Lex. 5 

6 

Figure 11. O-antigen Structural Variations Between the Parent Strain J178 and 7 

the SP-D escape variant (J178V) 8 

The parent strain J178 has a terminal H-1 unit and contains Lex, and most of the 9 

O-chain is substituted with Gal or Glc. However, the O-chain of the variant J178V has 10 

a greater level of fucosylation, only partial decoration with Gal or Glc, and is 11 

terminated with Ley. 12 

13 

Figure 12. Enzymes Involved in LPS biosynthesis in H. pylori 14 

Based on the literature data, a model for the H. pylori LPS structure and biosynthesis 15 

is summarised. The three domains of LPS are indicated: lipid A, core oligosaccharide 16 

(further divided into inner core and outer core) and O-antigen. The known enzymes 17 

involved in the LPS biosynthesis pathway are indicated by protein names, whereas the 18 

missing GTs are indicated by a question mark. The n (heptan)=4, and n (glucan)=3 19 

[17]. 20 

21 

Table 1 Glycosyltranferases in H. pylori from the CAZy database Family ORF Function Mechanism Structure

fold Clan

26695 G27 GT-2 HP0102 HPG27_94 Not previously studied inverting GT-A I GT-4 HP0421 HPG27_952 cholesterol α-glucosyltransferase (CGT) retaining GT-B IV GT-8 HP0159 HPG27_146 α-1,6-glucosyltransferase (RfaJ) retaining GT-A III GT-8 HP1105 HPG27_1046 β-1,3-N-acetyl-glucosaminyltransferase retaining GT-A IIIGT-8 HP1416 HPG27_ 1339 a-(1,2/4)-glucosyltransferase (RfaJ) retaining GT-A IIIGT-8 HP0208 HPG27_190 α-1,2-glycosyltransferase (RfaJ-2) retaining GT-A IIIGT-8 HP1578 HPG27_1515 Not previously studied retaining GT-A IIIGT-8 C694_01040* ※ Not previously studied retaining GT-A IIIGT-8 C694_01045* ※ Not previously studied retaining GT-A IIIGT-9 HP0479 HPG27_437 α-1,2-DD-heptosyltransferase inverting GT-B II GT-9 HP0279 HPG27_258 LPS heptosyltransferase-I (RfaC) inverting GT-B II GT-9 HP1191 HPG27_1136 LPS heptosyltransferase-II (RfaF) inverting GT-B II GT-9 HP1283 HPG27_1235 Not previously studied inverting GT-B II GT-9 HP1284 HPG27_1236 Not previously studied inverting GT-B II GT-9 ※ HPG27_1229 Not previously studied inverting GT-B II GT-9 ※ HPG27_1230 Not previously studied inverting GT-B II GT-10 HP0379 HPG27_613 α-1,3/4-L-fucosyltransferase (FutA) inverting GT-B II Glycosyltranferases in H. pylori from the CAZy database, continued Family ORF Function Mechanism Structure

fold Clan

26695 G27 GT-10 HP0651 HPG27_1018 α-1,3/4-L-fucosyltransferase (FutB) inverting GT-B II GT-11 HP0093* HPG27_86 α-1,2-L-fucosyltransferase (FutC) inverting GT-B orphan GT-11 HP0094* HPG27_86 α-1,2-L-fucosyltransferase (FutC) inverting GT-B orphan GT-19 HP0867 HPG27_821 lipid A disaccharide synthase (LpxB) inverting GT-B II GT-25 HP0826 HPG27_785 β-1,4-galactosyltransferase inverting GT-B □

GT-25 C694_03200* HPG27_579 Not previously studied inverting GT-B □GT-25 C694_03205* HPG27_580 Not previously studied inverting GT-B □GT-25 HP0805 HPG27_761 Not previously studied inverting GT-B □GT-28 HP1155 HPG27_1099 Peptidoglycan (MurG) inverting GT-B II GT-30 HP0957 HPG27_905 Kdo transferase (KdtA or WaaA) inverting GT-B II GT-51 HP0597 HPG27_557 Peptidoglycan PBP-1A inverting # orphan GT-82 HP0217 ※ Putative β-1,4-GalNAc transferase inverting GT-A I

※homologue was not found in this strain; *fragment; □unassigned in CAZy; PBP-1A, penicillin-binding protein 1A; ＃GT-51 adopts a unique bacteriophage-lysozyme-like fold.   

  

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori

Hong Li1,2, Tingting Liao2, Aleksandra W. Debowski2,3, Hong Tang1, Hans-Olof

Nilsson4, Keith A. Stubbs3, Barry J. Marshall2, Mohammed Benghezal*2,5.

Supplementary Information

Figure S1. The Biosynthetic Pathway for UDP-GlcNAc in H. pylori

Based on KEGG H. pylori LPS biosynthesis pathway [1], the biosynthesis of

UDP-GlcNAc is proposed to involve six steps starting from Glc. Step 1: the

phosphorylation of Glc by kinase Glk (HP1103); Step 2: the conversion of Glc-6P to

frucose-6P by isomerase Pgi (HP1166); Step 3: the conversion to GlcN-6P by

aminotransferase HP1532; Step 4: the conversion of GlcN-6P to GlcN-1P by mutase

GlmM (HP0075); Step 5 and 6 are catalysed by a bi-functional GlmU (GlcNAc-1P

uridyltransferase/GlcN-1P acetyltransferase, HP0683).

Figure S2. The Biosynthetic Pathway for CMP-Kdo in H. pylori

Based on KEGG H. pylori LPS biosynthesis and pentose phosphate pathways [1], the

biosynthesis of CMP-Kdo is proposed to involve eight steps starting from Glc. Step 1:

the phosphorylation of Glc by kinase Glk (HP1103); Step 2: the conversion of Glc-6P

to frucose-6P by isomerase Pgi (HP1166); Step 3: the conversion of frucose-6P to

xylulose-5P by transketolase (HP1088); Step 4: the conversion to ribulose-5P by

epimerase Rpe (HP1386); Step 5, the conversion to arabinose-5P by isomerase

(HP1429); Step 6, the conversion to Kdo-8P by KdsA (HP0003); Step 7, the

conversion of Kdo-8P to Kdo by phosphatase (HP1570); Step 8, the production of

CMP-Kdo by synthetase KdsB (HP0230).

Figure S3. The Biosynthetic Pathway for ADP-LD-Hep and ADP-DD-Hep in

H. pylori

Based on KEGG H. pylori LPS biosynthesis and pentose phosphate pathways [1], the

biosynthesis of ADP-DD-Hep is proposed to involve eight steps starting from Glc.

Step 1: the phosphorylation of Glc by kinase Glk (HP1103); Step 2: the conversion of

Glc-6P to frucose-6P by isomerase Pgi (HP1166); Step 3: the conversion of

frucose-6P to erythrose-4P or xylulose-5P by transketolase (HP1088); Step 4: the

conversion of erythrose-4P or xylulose-5P to sedo-heptulose-7P by transaldolase

(HP1495) or transketolase (HP1088), respectively; Step 5: the conversion to

DD-Hep-7P by isomerase GmhA (HP0857); Step 6: the conversion to DD-Hep-1,7PP

by the bi-functional RfaE (kinase/adenosyltransferase, HP0858); Step 7: the

conversion to DD-Hep-1P by phosphatase GmhB (HP0860); Step 8: the production of

ADP-DD-Hep by the bi-functional RfaE (kinase/ adenosyltransferase, HP0858). For

the production of ADP-LD-Hep, it is converted from ADP-DD-Hep by epimerase RfaD

(HP0859).

Figure S4. The Biosynthetic Pathway for UDP-Glc and UDP-Gal in H. pylori

Based on KEGG H. pylori Gal metabolism pathway [1], the biosynthesis of UDP-Glc

is proposed to involve three steps. Step 1: the phosphorylation of Glc by kinase Glk

(HP1103); Step 2: the conversion of Glc-6P to Glc-1P by mutase AlgC (HP1275);

Step 3: the production of UDP-Glc by pyrophosphorylase GalU (HP0646). For the

production of UDP-Gal, it is converted from UDP-Glc by epimease GalE (HP0360).

Figure S5. The Biosynthetic Pathway for GDP-Fuc in H. pylori

Based on KEGG H. pylori fructose and mannose metabolism pathway [1] and on

reference [2], the de novo biosynthesis of GDP-Fuc involves seven steps starting from

Glc. Step 1: the phosphorylation of Glc by kinase Glk (HP1103); Step 2: the

conversion of Glc-6P to frucose-6P by isomerase Pgi (HP1166); Step 3: the

conversion of frucose-6P to mannose-6P by the isomerase function of HP0043; Step 4:

the conversion of mannose-6P to mannose-1P by mutase AlgC (HP1275); Step 5: the

production of GDP-mannose by the pyrophosphorylase function of HP0043; Step 6

and 7: the conversion of GDP-mannose to GDP-Fuc by a dehydratase (HP0044) and

GDP-Fuc synthase NolK (HP0045). The salvage pathway for the production of

GDP-Fuc is based on reference [3]. The L-Fuc is released from host glycoconjugates

by α-L-fucosidase (FUCA2). The conversion of L-Fuc to GDP-Fuc requires Fuc

kinase (homologue unidentified in H. pylori) and GDP-Fuc pyrophosphorylase

(homologue unidentified in H. pylori).

Figure S6. The Biosynthetic Pathway for CMP-Neu5Ac in H. pylori

Based on KEGG H. pylori amino sugar and nucleotide sugar metabolism pathway [1]

and on reference [4], the biosynthesis of CMP-Neu5Ac involves five steps starting

from UDP-GlcNAc. Step 1: the conversion of UDP-GlcNAc to ManNAc by an

epimerase (homologue unidentified in H. pylori); Step 2: the conversion of ManNAc

to ManNAc-6P by a kinase (homologue unidentified in H. pylori ); Step 3: the

conversion of ManNAc-6P to Neu5Ac-9P by a synthase (homologue unidentified in

H. pylori); Step 4: the conversion of Neu5Ac-9P to Neu5Ac by a phosphatase

(homologue unidentified in H. pylori); Step 5: the production of CMP-Neu5Ac by a

synthetase (HP0326).

References

1. Kanehisa MKM (2000) KEGG: Kyoto Encyclopedia of Genes and Genomes.

Nucleic Acids Res. 28: 27-30.

2. Wu B, Zhang Y, Wang PG (2001) Identification and characterization of

GDP-d-mannose 4,6-dehydratase and GDP-l-fucose snthetase in a GDP-l-fucose

biosynthetic gene cluster from Helicobacter pylori. Biochem. Biophys. Res.

Commun. 285: 364-371.

3. Liu TW, Ho CW, Huang HH, Chang SM, Popat SD, et al. (2009) Role for

alpha-L-fucosidase in the control of Helicobacter pylori-infected gastric cancer

cells. Proc. Natl.Acad. Sci. U S A 106: 14581-14586.

4. Yarema KJ, Goon S, Bertozzi CR (2001) Metabolic selection of glycosylation

defects in human cells. Nat. Biotechnol. 19: 553-558.