Dennis Linton

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.

Helicobacter pullorum sp. nov.-genotype and phenotype of a new species isolated from poultry and from human patients with gastroenteritis.

Campylobacter-like organisms were isolated from the liver, duodenum and caecum of broiler and layer chickens, and from humans with gastroenteritis. They formed a unique DNA homology group and a polyphasic taxonomic analysis was made of 16 strains. Analysis of the nucleotide sequence of the 16S rRNA gene from seven of the strains identified them as belonging to a single species, within the genus Helicobacter. This conclusion was supported by the studies of relative DNA homology and of total protein electrophoretic patterns. The new species could be biochemically differentiated from other helicobacters and its ultrastructure in the electron microscope was typical of the genus except that the flagellum was not sheathed. We propose the name Helicobacter pullorum sp. nov. for this group. Like H. fennelliae or H. cinaedi it represents another non-gastric urease-negative Helicobacter species colonizing the lower bowel. Its isolation from the livers of chickens with vibrionic hepatitis is significant. We describe a species-specific PCR assay for H. pullorum sp. nov. which will facilitate its identification and further studies of its epidemiology.

Phase variation of a β-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo‐oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain–Barré and Miller–Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1‐like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside‐mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal β‐1,3‐linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a β‐1,3 galactosyltransferase responsible for converting GM2‐like LOS structures to GM1‐like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside‐mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1‐like LOS structure as well as the ganglioside GM2‐like LOS structure proposed in the literature.

Functional analysis of the Campylobacter jejuni N‐linked protein glycosylation pathway

We describe in this report the characterization of the recently discovered N‐linked glycosylation locus of the human bacterial pathogen Campylobacter jejuni, the first such system found in a species from the domain Bacteria. We exploited the ability of this locus to function in Escherichia coli to demonstrate through mutational and structural analyses that variant glycan structures can be transferred onto protein indicating the relaxed specificity of the putative oligosaccharyltransferase PglB. Structural data derived from these variant glycans allowed us to infer the role of five individual glycosyltransferases in the biosynthesis of the N‐linked heptasaccharide. Furthermore, we show that C. jejuni‐ and E. coli‐derived pathways can interact in the biosynthesis of N‐linked glycoproteins. In particular, the E. coli encoded WecA protein, a UDP‐GlcNAc: undecaprenylphosphate GlcNAc‐1‐phosphate transferase involved in glycolipid biosynthesis, provides for an alternative N‐linked heptasaccharide biosynthetic pathway bypassing the requirement for the C. jejuni‐derived glycosyltransferase PglC. This is the first experimental evidence that biosynthesis of the N‐linked glycan occurs on a lipid‐linked precursor prior to transfer onto protein. These findings provide a framework for understanding the process of N‐linked protein glycosylation in Bacteria and for devising strategies to exploit this system for glycoengineering.

Campylobacter - a tale of two protein glycosylation systems

Post-translational glycosylation is a universal modification of proteins in eukarya, archaea and bacteria. Two recent publications describe the first confirmed report of a bacterial N-linked glycosylation pathway in the human gastrointestinal pathogen Campylobacter jejuni. In addition, an O-linked glycosylation pathway has been identified and characterized in C. jejuni and the related species Campylobacter coli. Both pathways have similarity to the respective N- and O-linked glycosylation processes in eukaryotes. In bacteria, homologues of the genes in both pathways are found in other organisms, the complex glycans linked to the glycoproteins share common biosynthetic precursors and these modifications could play similar biological roles. Thus, Campylobacter provides a unique model system for the elucidation and exploitation of glycoprotein biosynthesis.

Adaptation of Campylobacter jejuni NCTC11168 to High-Level Colonization of the Avian Gastrointestinal Tract

ABSTRACT The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148:473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5, flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.

A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni

Flagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maf family represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.

Identification of N-Acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni.

It was demonstrated recently that there is a system of general protein glycosylation in the human enteropathogen Campylobacter jejuni. To char‐ acterize such glycoproteins, we identified a lectin, Soybean agglutinin (SBA), which binds to multiple C. jejuni proteins on Western blots. Binding of lectin SBA was disrupted by mutagenesis of genes within the previously identified protein glycosylation locus. This lectin was used to purify putative glycoproteins selectively and, after sodium dodecyl sulphate‐ polyacrylamide gel electrophoresis (SDS–PAGE), Coomassie‐stained bands were cut from the gels. The bands were digested with trypsin, and peptides were identified by mass spectrometry and database searching. A 28 kDa band was identified as PEB3, a previously characterized immunogenic cell surface protein. Bands of 32 and 34 kDa were both identified as a putative periplasmic protein encoded by the C. jejuni NCTC 11168 coding sequence Cj1670c. We have named this putative glycoprotein CgpA. We constructed insertional knockout mutants of both the peb3 and cgpA genes, and surface protein extracts from mutant and wild‐type strains were analysed by one‐ and two‐dimensional polyacrylamide gel electrophoresis (PAGE). In this way, we were able to identify the PEB3 protein as a 28 kDa SBA‐reactive and immunoreactive glycoprotein. The cgpA gene encoded SBA‐reactive and immunoreactive proteins of 32 and 34 kDa. By using specific exoglycosidases, we demonstrated that the SBA binding property of acid‐glycine extractable C. jejuni glycoproteins, including PEB3 and CgpA, is a result of the presence of α‐linked N‐acetylgalactosamine residues. These data confirm the existence, and extend the boundaries, of the previously identified protein glycosylation locus of C. jejuni. Furthermore, we have identified two such glycoproteins, the first non‐flagellin campylobacter glycoproteins to be identified, and demonstrated that their glycan components contain α‐linked N‐acetylgalactosamine residues.

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide.

N‐acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo‐oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain–Barré syndrome (GBS) and Miller–Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB‐deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild‐type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography–mass spectrometry and fast atom bombardment–mass spectrometry analysis of LOS from wild‐type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N‐acetyl‐d‐mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non‐motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.

Characterization of N-Linked Protein Glycosylation in Helicobacter pullorum

The first bacterial N-linked glycosylation system was discovered in Campylobacter jejuni, and the key enzyme involved in the coupling of glycan to asparagine residues within the acceptor sequon of the glycoprotein is the oligosaccharyltransferase PglB. Emerging genome sequence data have revealed that pglB orthologues are present in a subset of species from the Deltaproteobacteria and Epsilonproteobacteria, including three Helicobacter species: H. pullorum, H. canadensis, and H. winghamensis. In contrast to C. jejuni, in which a single pglB gene is located within a larger gene cluster encoding the enzymes required for the biosynthesis of the N-linked glycan, these Helicobacter species contain two unrelated pglB genes (pglB1 and pglB2), neither of which is located within a larger locus involved in protein glycosylation. In complementation experiments, the H. pullorum PglB1 protein, but not PglB2, was able to transfer C. jejuni N-linked glycan onto an acceptor protein in Escherichia coli. Analysis of the characterized C. jejuni N-glycosylation system with an in vitro oligosaccharyltransferase assay followed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry demonstrated the utility of this approach, and when applied to H. pullorum, PglB1-dependent N glycosylation with a linear pentasaccharide was observed. This reaction required an acidic residue at the -2 position of the N-glycosylation sequon, as for C. jejuni. Attempted insertional knockout mutagenesis of the H. pullorum pglB2 gene was unsuccessful, suggesting that it is essential. These first data on N-linked glycosylation in a second bacterial species demonstrate the similarities to, and fundamental differences from, the well-studied C. jejuni system.