Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium,Campylobacter jejuni

Mass spectrometry investigations of partially purified Campylobacter jejuni protein PEB3 showed it to be partially modified with an Asn-linked glycan with a mass of 1406 Da and composed of one hexose, five N-acetylhexosamines and a species of mass 228 Da, consistent with a trideoxydiacetamidohexose. By means of soybean lectin affinity chromatography, a mixture of glycoproteins was obtained from a glycine extract, and two-dimensional gel proteomics analysis led to the identification of at least 22 glycoproteins, predominantly annotated as periplasmic proteins. Glycopeptides were prepared from the glycoprotein mixture by Pronase digestion and gel filtration. The structure of the glycan was determined by using nano-NMR techniques to be GalNAc-alpha1,4-GalNAc-alpha1,4-[Glcbeta1,3-]GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-beta1,N-Asn-Xaa, where Bac is bacillosamine, 2,4-diacetamido-2,4,6-trideoxyglucopyranose. Protein glycosylation was abolished when the pglB gene was mutated, providing further evidence that the enzyme encoded by this gene is responsible for formation of the glycopeptide N-linkage. Comparison of the pgl locus with that of Neisseria meningitidis suggested that most of the homologous genes are probably involved in the biosynthesis of bacillosamine.     

Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway

Campylobacter jejuni has a general N-linked glycosylation pathway, encoded by the pgl gene cluster. In C. jejuni, a heptasaccharide is transferred from an undecaprenyl pyrophosphate donor [GalNAc-alpha1,4-GalNAc-alpha1,4-(Glcbeta1,3)-GalNAc-alpha1,4-GalNAc-alpha1,4-GalNAc-alpha1,3-Bac-alpha1-PP-undecaprenyl, where Bac is bacillosamine (2,4-diacetamido-2,4,6-trideoxyglucose)] to the asparagine side chain of target proteins at the Asn-X-Ser/Thr motif. In this study, we have cloned, overexpressed in Escherichia coli, and purified PglC, the glycosyl-1-phosphate transferase responsible for the first step in the biosynthesis of the undecaprenyl-linked heptasaccharide donor. In addition, we report the first synthetic route to uridine 5'-diphosphobacillosamine. Using the uridine 5'-diphosphobacillosamine and undecaprenyl phosphate, we demonstrate the ability of PglC to produce undecaprenyl pyrophosphate bacillosamine using radiolabeled HPLC and mass spectral analysis. In addition, we revealed that PglC does not accept uridine 5'-diphospho-N-acetylglucosamine or uridine 5'-diphospho-N-acetylgalactosamine as substrates but will accept uridine 5'-diphospho-6-hydroxybacillosamine, an analogue of bacillosamine that retains the C-6 hydroxyl functionality from the biosynthetic precursor. The in vitro characterization of PglC as a bacillosamine 1-phosphoryl transferase provides direct evidence for the early steps in the C. jejuni N-linked glycosylation pathway, and the coupling of PglC with the latter glycosyltransferases (PglA, PglJ, PglH, and PglI) allows for the "one-pot" chemoenzymatic synthesis of the undecaprenyl pyrophosphate heptasaccharide donor.     

Biosynthesis of the N-linked glycan in Campylobacter jejuni and addition onto protein through block transfer

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.     

Characterization of the structurally diverse N-linked glycans of Campylobacter species

The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In this study, we have demonstrated that only Campylobacter species closely related to C. jejuni produce glycoproteins that interact with both a C. jejuni N-linked-glycan-specific antiserum and a lectin known to bind to the C. jejuni N-linked glycan. In order to further investigate the structure of Campylobacter N-linked glycans, we employed an in vitro peptide glycosylation assay combined with mass spectrometry to demonstrate that Campylobacter species produce a range of structurally distinct N-linked glycans with variations in the number of sugar residues (penta-, hexa-, and heptasaccharides), the presence of branching sugars, and monosaccharide content. These data considerably expand our knowledge of bacterial N-linked glycan structure and provide a framework for investigating the role of glycosyltransferases and sugar biosynthesis enzymes in glycoprotein biosynthesis with practical implications for synthetic biology and glycoengineering.     

Knockout mutagenesis of the kpsE gene of Campylobacter jejuni 81116 and its involvement in bacterium-host interactions

Campylobacter jejuni is a common cause of bacterial enteritis. The surface capsular polysaccharides are important for this bacterium to survive in the environment, but little is known about their involvement in bacterium-host interactions. This study showed that the C. jejuni capsular polysaccharides play an important role in adherence to and invasion of human embryonic epithelial cells. However, no significant role of capsular polysaccharides was shown in colonization of the chicken gut.     

Detection of a novel campylobacter cytotoxin

The culture filtrates from 10 Campylobacter species were screened for the presence of cytotoxins on a variety of selected tissue culture cell lines. Some Campylobacter jejuni strains showed no effects on tissue culture cell lines compared with other C. jejuni strains, especially C. jejuni 81116, which consistently produced a cytotoxin that was lethal to tissue culture cells. It was observed that CHO cells were the most sensitive cell line in detecting campylobacter cytotoxins. Samples containing the culture filtrate of C. jejuni 81116 prepared at various growth stages were used to determine the subcellular location of the cytotoxin. This C. jejuni 81116 cytotoxin appears to be a heat-stable toxin that is secreted from the cell during stationary phase; cytotoxin activity can be abolished with proteolytic enzymes.     

The structure of the lipid anchor of Campylobacter jejuni polysaccharide

Campylobacter jejuni is a leading cause of gastroenteritis in humans. Campylobacter jejuni produces extracellular polysaccharides that have been characterized structurally and shown to be independent of lipopolysaccharides. Furthermore, it has been suggested that these C. jejuni polysaccharides are capsular in nature, although their lipid anchor has not been identified. In this report, the occurrence of a lipid-linked capsular-like polysaccharide in C. jejuni is conclusively shown, and the lipid anchor identified as dipalmitoyl-glycerophosphate.     

Bacteriophage F336 recognizes the capsular phosphoramidate modification of Campylobacter jejuni NCTC11168

Bacteriophages infecting the food-borne human pathogen Campylobacter jejuni could potentially be exploited to reduce bacterial counts in poultry prior to slaughter. This bacterium colonizes the intestinal tract of poultry in high numbers, and contaminated poultry meat is regarded as the major source of human campylobacteriosis. In this study, we used phage F336 belonging to the Myoviridae family to select a C. jejuni NCTC11168 phage-resistant strain, called 11168R, with the aim of investigating the mechanisms of phage resistance. We found that phage F336 has reduced adsorption to 11168R, thus indicating that the receptor is altered. While proteinase K-treated C. jejuni cells did not affect adsorption, periodate treatment resulted in reduced adsorption, suggesting that the phage binds to a carbohydrate moiety. Using high-resolution magic angle spinning nuclear magnetic resonance (NMR) spectroscopy, we found that 11168R lacks an O-methyl phosphoramidate (MeOPN) moiety attached to the GalfNAc on the capsular polysaccharide (CPS), which was further confirmed by mass spectroscopy. Sequence analysis of 11168R showed that the potentially hypervariable gene cj1421, which encodes the GalfNAc MeOPN transferase, contains a tract of 10 Gs, resulting in a nonfunctional gene product. However, when 11168R reverted back to phage sensitive, cj1421 contained 9 Gs, and the GalfNAc MeOPN was regained in this strain. In summary, we have identified the phase-variable MeOPN moiety, a common component of the diverse capsular polysaccharides of C. jejuni, as a novel receptor of phages infecting this bacterium.     

Molecular origin of two polysaccharides of Campylobacter jejuni 81116

The nature of the polysaccharide molecules of the human enteric pathogen Campylobacter jejuni has been the subject of debate. Previously, C. jejuni 81116 was shown to contain two different polysaccharides, one acidic (polysaccharide A) and the other neutral (polysaccharide B), occurring in a 3 : 1 ratio, respectively. The aim of this study was to determine the molecular origin of these polysaccharides. Using a combination of centrifugation, gel permeation chromatography, chemical assays, and (1)H-NMR analysis, polysaccharide B was shown to be derived from lipopolysaccharide and polysaccharide A from capsular polysaccharide. Thus, C. jejuni 81116 produces both lipopolysaccharide-like molecules and capsular polysaccharide.     

The HS:1 serostrain of Campylobacter jejuni has a complex teichoic acid-like capsular polysaccharide with nonstoichiometric fructofuranose branches and O-methyl phosphoramidate groups

Recently, the CPS biosynthetic loci for several strains of Campylobacter jejuni were sequenced and revealed evidence for multiple mechanisms of structural variation. In this study, the CPS structure for the HS:1 serostrain of C. jejuni was determined using mass spectrometry and NMR at 600 MHz equipped with an ultra-sensitive cryogenically cooled probe. Analysis of CPS purified using a mild enzymatic method revealed a teichoic acid-like [-4)-alpha-d-Galp-(1-2)-(R)-Gro-(1-P](n), repeating unit, where Gro is glycerol. Two branches at C-2 and C-3 of galactose were identified as beta-d-fructofuranoses substituted at C-3 with CH(3)OP(O)(NH(2))(OR) groups. Structural heterogeneity was due to nonstoichiometric glycosylation at C-3 of galactose and variable phosphoramidate groups. Identical structural features were found for cell-bound CPS on intact cells using proton homonuclear and (31)P heteronuclear two-dimensional HR-MAS NMR at 500 MHz. In contrast, spectroscopic data acquired for hot water/phenol purified CPS was complicated by the hydrolysis and subsequent loss of labile groups during extraction. Collectively, the results of this study established the importance of using sensitive isolation techniques and HR-MAS NMR to examine CPS structures in vivo when labile groups are present. This study uncovered how incorporation of variable O-methyl phosphoramidate groups on nonstoichiometric fructose branches is used in C. jejuni HS:1 as a strategy to produce a highly complex polysaccharide from its small CPS biosynthetic locus and a limited number of sugars.     

Influence of growth conditions on diverse polysaccharide production by Campylobacter jejuni

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide. The present study was undertaken to determine the forms of polysaccharide-related compounds (PRCs) produced by C. jejuni and the culture conditions influencing their production. Expression of polysaccharides by C. jejuni was influenced by culture medium composition and growth phase. In addition to the production of lipooligosaccharide and capsular polysaccharide, a previously undescribed polysaccharide, not related to capsular polysaccharide, was shown to occur in C. jejuni in batch liquid and chemostat cultures. Thus, a variety of PRCs are produced by C. jejuni, and this should be considered when growing the bacterium in vitro for pathogenesis studies.     

The Campylobacter jejuni glycome

Microbial cell surface glycans in the form of glycolipids and glycoproteins frequently play important roles in cell-cell interaction and host immune responses. Given the likely importance of these surface structures in the survival and pathogenesis of Campylobacter jejuni, a concerted effort has been made to characterise these determinants genetically and structurally since the genome was sequenced in 2000. We review the considerable progress made in characterising the Campylobacter glycome including the lipooligosaccharide (LOS), the capsule and O- and N-linked protein glycosylation systems, and speculate on the roles played by glycan surface structures in the life-cycle of C. jejuni.     

Campylobacter sugars sticking out

The amazing repertoire of glycoconjugates that are found in Campylobacter jejuni includes lipooligosaccharides mimicking human glycolipids, capsular polysaccharides with complex and unusual sugars, and proteins that are post-translationally modified with either O- or N-linked glycans. Thus, the glycome of this important food-borne pathogen is an excellent toolbox for glycobiologists to understand the fundamentals of these pathways and their role in host-microbe interactions, develop new techniques for glycobiology and exploit these pathways for novel diagnostics and therapeutics. The exciting surge in recent research activities will be summarized in this review.     

Neisseria gonorrhoeae O-linked pilin glycosylation: functional analyses define both the biosynthetic pathway and glycan structure

Neisseria gonorrhoeae expresses an O-linked protein glycosylation pathway that targets PilE, the major pilin subunit protein of the Type IV pilus colonization factor. Efforts to define glycan structure and thus the functions of pilin glycosylation (Pgl) components at the molecular level have been hindered by the lack of sensitive methodologies. Here, we utilized a 'top-down' mass spectrometric approach to characterize glycan status using intact pilin protein from isogenic mutants. These structural data enabled us to directly infer the function of six components required for pilin glycosylation and to define the glycan repertoire of strain N400. Additionally, we found that the N. gonorrhoeae pilin glycan is O-acetylated, and identified an enzyme essential for this unique modification. We also identified the N. gonorrhoeae pilin oligosaccharyltransferase using bioinformatics and confirmed its role in pilin glycosylation by directed mutagenesis. Finally, we examined the effects of expressing the PglA glycosyltransferase from the Campylobacter jejuni N-linked glycosylation system that adds N-acetylgalactosamine onto undecaprenylpyrophosphate-linked bacillosamine. The results indicate that the C. jejuni and N. gonorrhoeae pathways can interact in the synthesis of O-linked di- and trisaccharides, and therefore provide the first experimental evidence that biosynthesis of the N. gonorrhoeae pilin glycan involves a lipid-linked oligosaccharide precursor. Together, these findings underpin more detailed studies of pilin glycosylation biology in both N. gonorrhoeae and N. meningitidis, and demonstrate how components of bacterial O- and N-linked pathways can be combined in novel glycoengineering strategies.     

Desulfovibrio desulfuricans PglB homolog possesses oligosaccharyltransferase activity with relaxed glycan specificity and distinct protein acceptor sequence requirements

Oligosaccharyltransferases (OTases) are responsible for the transfer of carbohydrates from lipid carriers to acceptor proteins and are present in all domains of life. In bacteria, the most studied member of this family is PglB from Campylobacter jejuni (PglB(Cj)). This enzyme is functional in Escherichia coli and, contrary to its eukaryotic counterparts, has the ability to transfer a variety of oligo- and polysaccharides to protein carriers in vivo. Phylogenetic analysis revealed that in the delta proteobacteria Desulfovibrio sp., the PglB homolog is more closely related to eukaryotic and archaeal OTases than to its Campylobacter counterparts. Genetic analysis revealed the presence of a putative operon that might encode all enzymes required for N-glycosylation in Desulfovibrio desulfuricans. D. desulfuricans PglB (PglB(Dd)) was cloned and successfully expressed in E. coli, and its activity was confirmed by transferring the C. jejuni heptasaccharide onto the model protein acceptor AcrA. In contrast to PglB(Cj), which adds two glycan chains to AcrA, a single oligosaccharide was attached to the protein by PglB(Dd). Site-directed mutagenesis of the five putative N-X-S/T glycosylation sites in AcrA and mass spectrometry analysis showed that PglB(Dd) does not recognize the "conventional bacterial glycosylation sequon" consisting of the sequence D/E-X(1)-N-X(2)-S/T (where X(1) and X(2) are any amino acid except proline), and instead used a different site for the attachment of the oligosaccharide than PglB(Cj.). Furthermore, PglB(Dd) exhibited relaxed glycan specificity, being able to transfer mono- and polysaccharides to AcrA. Our analysis constitutes the first characterization of an OTase from delta-proteobacteria involved in N-linked protein glycosylation.     

Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction.

A previously developed polymerase chain reaction (PCR) amplification of a target region in the flaA Campylobacter flagellin gene was evaluated and adapted for use with environmental water samples. The ability to detect Campylobacter jejuni or Campylobacter coli in seeded water samples was tested with various filters after concentration and freeze-thaw lysis of the bacterial cells. A nonradioactive probe for the amplified flagellin gene fragment detected as little as 1 to 10 fg of genomic DNA and as few as 10 to 100 viable C. jejuni cells per 100 ml of water filtered onto Fluoropore (Millipore Corp.) filters. No amplification was obtained with cellulose acetate filters, most likely because of binding of the DNA to the filter. Concentration and lysis of target cells on Fluoropore and Durapore (Millipore Corp.) filters allowed PCR to be performed in the same reaction tube without removing the filters. This methodology was then adapted for use with environmental water samples. The water supply to a broiler chicken production farm was suspected as the source of C. jejuni known to be endemic in grow-out flocks at the farm, despite the inability to culture the organisms by standard methods. The filtration-PCR method detected Campylobacter DNA in more than half of the farm water samples examined. Amplified campylobacter DNA was not detected in small volumes of regional surface water samples collected on a single occasion in February. The filtration-PCR amplification method provided a basis for detection of C. jejuni and C. coli in environmental waters with a high degree of specificity and sensitivity.     

Prevalence and survival of Campylobacter jejuni in unpasteurized milk.

Campylobacter jejuni was isolated from 1 to 108 (0.9%) milk samples obtained from the bulk tanks of nine grade A dairy farms and from 50 of 78 (64%) cows producing grade A milk. Survival of eight Campylobacter strains in unpasteurized milk (4 degrees C) varied greatly: the most tolerant strain showed a less than 2-log10 decrease in viable cells after 14 days, and the most sensitive strain showed a greater than 6-log10 decrease after 7 days. One strain was still recoverable 21 days after the inoculation of milk. Inactivation of the different strains corresponded with an increase in milk aerobic plate count and a decrease in milk pH; however, no absolute correlation could be made between the rates of change of these parameters and the rates of campylobacter inactivation. When held at 4 degrees C, C. jejuni was most stable in brucella broth, died most rapidly in unpasteurized milk, and was inactivated at an intermediate rate in sterile milk. Our results indicate the presence and possible persistence of C. jejuni in raw grade A milk and reaffirm the need for pasteurization of milk.     

Characterization of the Campylobacter jejuni heptosyltransferase II gene, waaF, provides genetic evidence that extracellular polysaccharide is lipid A core independent

Campylobacter jejuni produces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides in C. jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. In Escherichia coli and Salmonella enterica serovar Typhimurium the waaF gene encodes a heptosyltransferase, which catalyzes the transfer of the second L-glycero-D-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation of waaF results in a truncated core oligosaccharide. In this report we confirm experimentally that C. jejuni gene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. The Campylobacter waaF gene complements an S. enterica serovar Typhimurium waaF mutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF in C. jejuni, waaF mutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM(1), asialo-GM(1), and GM(2) ganglioside epitopes. The mutation of waaF did not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide by C. jejuni. The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-beta(1,3) N-acetylgalactosamine. In comparison, the waaF mutant possessed a truncated molecule consistent with that observed with waaF mutants in other bacterial species.     

Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.     

Isolation of Campylobacter jejuni from retail mushrooms

Campylobacter jejuni was isolated from 3 (1.5%) of 200 retail, polyvinyl chloride film-wrapped, fresh mushrooms. These results indicate that fresh mushrooms may indeed be a source of C. jejuni and support previously reported epidemiological data (Seattle-King County Department of Public Health, Surveillance of the Flow of Salmonella and Campylobacter in a Community, 1984) which revealed an an elevated relative risk of developing campylobacter enteritis in individuals who consume mushrooms.