Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin.

Flagellins from three strains of Campylobacter jejuni and one strain of Campylobacter coli were shown to be extensively modified by glycosyl residues, imparting an approximate 6000-Da shift from the molecular mass of the protein predicted from the DNA sequence. Tryptic peptides from C. jejuni 81-176 flagellin were subjected to capillary liquid chromatography-electrospray mass spectrometry with a high/low orifice stepping to identify peptide segments of aberrant masses together with their corresponding glycosyl appendages. These modified peptides were further characterized by tandem mass spectrometry and preparative high performance liquid chromatography followed by nano-NMR spectroscopy to identify the nature and precise site of glycosylation. These analyses have shown that there are 19 modified Ser/Thr residues in C. jejuni 81-176 flagellin. The predominant modification found on C. jejuni flagellin was O-linked 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (pseudaminic acid, Pse5Ac7Ac) with additional heterogeneity conferred by substitution of the acetamido groups with acetamidino and hydroxyproprionyl groups. In C. jejuni 81-176, the gene Cj1316c, encoding a protein of unknown function, was shown to be involved in the biosynthesis and/or the addition of the acetamidino group on Pse5Ac7Ac. Glycosylation is not random, since 19 of the total 107 Ser/Thr residues are modified, and all but one of these are restricted to the central, surface-exposed domain of flagellin when folded in the filament. The mechanism of attachment appears unrelated to a consensus peptide sequence but is rather based on surface accessibility of Ser/Thr residues in the folded protein.     

Pseudaminic acid, the major modification on Campylobacter flagellin, is synthesized via the Cj1293 gene

Flagellins from Campylobacter jejuni 81-176 and Campylobacter coli VC167 are heavily glycosylated. The major modifications on both flagellins are pseudaminic acid (Pse5Ac7Ac), a nine carbon sugar that is similar to sialic acid, and an acetamidino-substituted analogue of pseudaminic acid (PseAm). Previous data have indicated that PseAm is synthesized via Pse5Ac7Ac in C. jejuni 81-176, but that the two sugars are synthesized using independent pathways in C. coli VC167. The Cj1293 gene of C. jejuni encodes a putative UDP-GlcNAc C6-dehydratase/C4-reductase that is similar to a protein required for glycosylation of Caulobacter crescentus flagellin. The Cj1293 gene is expressed either under the control of a sigma 54 promoter that overlaps the coding region of Cj1292 or as a polycistronic message under the control of a sigma 70 promoter upstream of Cj1292. A mutant in gene Cj1293 in C. jejuni 81-176 was non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. This mutant was complemented in trans with the homologous C. jejuni gene, as well as the Helicobacter pylori homologue, HP0840, which has been shown to encode a protein with UDP-GlcNAc C6-dehydratase/C4-reductase activity. Mutation of Cj1293 in C. coli VC167 resulted in a fully motile strain that synthesized a flagella filament composed of flagellin in which Pse5Ac7Ac was replaced by PseAm. The filament from the C. coli Cj1293 mutant displayed increased solubility in SDS compared with the wild-type filament. A double mutant in C. coli VC167, defective in both Cj1293 and ptmD, encoding part of the independent PseAm pathway, was also non-motile and non-flagellated and accumulated unglycosylated flagellin intracellularly. Collectively, the data indicate that Cj1293 is essential for Pse5Ac7Ac biosynthesis from UDP-GlcNAc, and that glycosylation is required for flagella biogenesis in campylobacters.     

Novel glycosylation sites localized in Campylobacter jejuni flagellin FlaA by liquid chromatography electron capture dissociation tandem mass spectrometry

Glycosylation of flagellin in Campylobacter jejuni is essential for motility and virulence. It is well-known that flagellin from C. jejuni 81-176 is glycosylated by pseudaminic acid and its acetamidino derivative, and that Campylobactor coli VC167 flagellin is glycosylated by legionaminic acid and its derivatives. Recently, it was shown, by use of a metabolomics approach, that C. jejuni 11168 is glycosylated by dimethyl glyceric acid derivatives of pseudaminic acid, but the sites of glycosylation were not confirmed. Here, we apply an online liquid chromatography electron capture dissociation (ECD) tandem mass spectrometry approach to localize sites of glycosylation in flagellin from C. jejuni 11168. Flagellin A is glycosylated by a dimethyl glyceric acid derivative of pseudaminic acid at Ser181, Ser207 and either Thr464 or Thr 465; and by a dimethyl glyceric acid derivative of acetamidino pseudaminic acid at Ser181 and Ser207. For comparison, on-line liquid chromatography collision-induced dissociation of the tryptic digests was performed, but it was not possible to assign sites of glycosylation by that method.     

Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.     

Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence

Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc to flagellin. Mutation of Cj1316c or pseA had been shown to result in loss of the acetamidino form of pseudaminic acid (PseAm). Mutation of a second gene also resulted in loss of PseAm, as well as a minor modification that appears to be PseAm extended with N-acetyl-glutamic acid. Previously described mutants in C. jejuni 81-176 and Campylobacter coli VC167 that produced flagella lacking PseAm or PseAc failed to autoagglutinate. This suggests that interactions between modifications on adjacent flagella filaments are required for autoagglutination. Mutants (81-176) defective in autoagglutination showed a modest reduction in adherence and invasion of INT407 cells. However, there was a qualitative difference in binding patterns to INT407 cells using GFP-labelled 81-176 and mutants lacking PseAm. A mutant lacking PseAm was attenuated in the ferret diarrhoeal disease model.     

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.     

Structural heterogeneity of carbohydrate modifications affects serospecificity of Campylobacter flagellins

Flagellin from Campylobacter coli VC167 is post-translationally modified at > or = 16 amino acid residues with pseudaminic acid and three related derivatives. The predominant modification was 5,7-diacetamido-3,5,7,9 - tetradeoxy - l - glycero - l - manno - nonulosonic acid (pseudaminic acid, Pse5Ac7Ac), a modification that has been described previously on flagellin from Campylobacter jejuni 81-176. VC167 lacked two modi-fications present in 81-176 and instead had two unique modifications of masses 431 and 432 Da. Flagellins from both C. jejuni 81-176 and C. coli VC167 were also modified with an acetamidino form of pseudaminic acid (PseAm), but tandem mass spectrometry indicated that the structure of PseAm differed in the two strains. Synthesis of PseAm in C. coli VC167 requires a minimum of six ptm genes. In contrast, PseAm is synthesized in C. jejuni 81-176 via an alternative pathway using the product of the pseA gene. Mutation of the ptm genes in C. coli VC167 can be detected by changes in apparent Mr of flagellin in SDS-PAGE gels, changes in isoelectric focusing (IEF) patterns and loss of immunoreactivity with antiserum LAH2. These changes corresponded to loss of both 315 Da and 431 Da modifications from flagellin. Complementation of the VC167 ptm mutants with the 81-176 pseA gene in trans resulted in flagellins containing both 315 and 431 Da modifications, but these flagellins remained unreactive in LAH2 antibody, suggesting that the unique form of PseAm encoded by the ptm genes contributes to the serospecificity of the flagellar filament.     

Structural,genetic and functional characterization of the flagellin glycosylation process in Helicobacter pylori

Mass spectrometry analyses of the complex polar flagella from Helicobacter pylori demonstrated that both FlaA and FlaB proteins are post-translationally modified with pseudaminic acid (Pse5Ac7Ac, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno -n o n-ulosonic acid). Unlike Campylobacter, flagellar glycosylation in Helicobacter displays little heterogeneity in isoform or glycoform distribution, although all glycosylation sites are located in the central core region of the protein monomer in a manner similar to that found in Campylobacter. Bioinformatic analysis revealed five genes (HP0840, HP0178, HP0326A, HP0326B, HP0114) homologous to other prokaryote genes previously reported to be involved in motility, flagellar glycosylation or polysaccharide biosynthesis. Insertional mutagenesis of four of these homologues in Helicobacter (HP0178, HP0326A, HP0326B, HP0114) resulted in a non-motile phenotype, no structural flagella filament and only minor amounts of flagellin protein detectable by Western immunoblot. However, mRNA levels for the flagellin structural genes remained unaffected by each mutation. In view of the combined bioinformatic and structural evidence indicating a role for these gene products in glycan biosynthesis, subsequent investigations focused on the functional characterization of the respective gene products. A novel approach was devised to identify biosynthetic sugar nucleotide precursors from intracellular metabolic pools of parent and isogenic mutants using capillary electrophoresis-electrospray mass spectrometry (CE-ESMS) and precursor ion scanning. HP0326A, HP0326B and the HP0178 gene products are directly involved in the biosynthesis of the nucleotide-activated form of Pse, CMP-Pse. Mass spectral analyses of the cytosolic extract from the HP0326A and HP0326B isogenic mutants revealed the accumulation of a mono- and a diacetamido trideoxyhexose UDP sugar nucleotide precursor.     

Rapid construction of Campylobacter jejuni deletion mutants

AIMS: To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. METHODS AND RESULTS: We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. CONCLUSIONS: This method can be used for rapid construction of Camp. jejuni deletion mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this method should facilitate functional characterization of various Camp. jejuni genes.     

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.     

Heme utilization in Campylobacter jejuni

A putative iron- and Fur-regulated hemin uptake gene cluster, composed of the transport genes chuABCD and a putative heme oxygenase gene (Cj1613c), has been identified in Campylobacter jejuni NCTC 11168. Mutation of chuA or Cj1613c leads to an inability to grow in the presence of hemin or hemoglobin as a sole source of iron. Mutation of chuB, -C, or -D only partially attenuates growth where hemin is the sole iron source, suggesting that an additional inner membrane (IM) ABC (ATP-binding cassette) transport system(s) for heme is present in C. jejuni. Genotyping experiments revealed that Cj1613c is highly conserved in 32 clinical isolates. One strain did not possess chuC, though it was still capable of using hemin/hemoglobin as a sole iron source, supporting the hypothesis that additional IM transport genes are present. In two other strains, sequence variations within the gene cluster were apparent and may account for an observed negative heme utilization phenotype. Analysis of promoter activity within the Cj1613c-chuA intergenic spacer region revealed chuABCD and Cj1613c are expressed from separate iron-repressed promoters and that this region also specifically binds purified recombinant Fur(Cj) in gel retardation studies. Absorbance spectroscopy of purified recombinant His(6)-Cj1613c revealed a 1:1 heme:His(6)-Cj1613c binding ratio. The complex was oxidatively degraded in the presence of ascorbic acid as the electron donor, indicating that the Cj1613c gene product functions as a heme oxygenase. In conclusion, we confirm the involvement of Cj1613c and ChuABCD in heme/hemoglobin utilization in C. jejuni.     

Cj1121c, a novel UDP-4-keto-6-deoxy-GlcNAc C-4 aminotransferase essential for protein glycosylation and virulence in Campylobacter jejuni

Campylobacter jejuni produces glycoproteins that are essential for virulence. These glycoproteins carry diacetamidobacillosamine (DAB), a sugar that is not found in humans. Hence, the enzymes responsible for DAB synthesis represent potential therapeutic targets. We describe the biochemical characterization of Cj1121c, a putative aminotransferase encoded by the general protein glycosylation locus, to assess its role in DAB biosynthesis. By using overexpressed and affinity-purified enzyme, we demonstrate that Cj1121c has pyridoxal phosphate- and glutamate-dependent UDP-4-keto-6-deoxy-GlcNAc C-4 transaminase activity and produces UDP-4-amino-4,6-dideoxy-GlcNAc. This is consistent with a role in DAB biosynthesis and distinguishes Cj1121c from Cj1294, a homologous UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose C-4 aminotransferase that we characterized previously. We show that Cj1121c can also use this 4-keto-arabino sugar indirectly as a substrate, that Cj1121c and Cj1294 are active simultaneously in C. jejuni, and that the activity of Cj1121c is preponderant under standard growth conditions. Kinetic data indicate that Cj1121c has a slightly higher catalytic efficiency than Cj1294 with regard to the 4-keto-arabino substrate. By site-directed mutagenesis, we show that residues Glu-158 and Leu-131 are not essential for catalysis or for substrate specificity contrary to expectations. We further demonstrate that a cj1121c knock-out mutant is impaired for flagella-mediated motility, for invasion of intestinal epithelial cells, and for persistence in the chicken intestine, clearly demonstrating that Cj1121c is essential for host colonization and virulence. Finally, we show that cj1121c is necessary for protein glycosylation by lectin Western blotting. Collectively, these results validate Cj1121c as a promising drug target and provide the means to assay for inhibitors.     

Cj0011c, a periplasmic single- and double-stranded DNA-binding protein, contributes to natural transformation in Campylobacter jejuni

Campylobacter jejuni is an important bacterial pathogen causing gastroenteritis in humans. C. jejuni is capable of natural transformation, which is considered a major mechanism mediating horizontal gene transfer and generating genetic diversity. Despite recent efforts to elucidate the transformation mechanisms of C. jejuni, the process of DNA binding and uptake in this organism is still not well understood. In this study, we report a previously unrecognized DNA-binding protein (Cj0011c) in C. jejuni that contributes to natural transformation. Cj0011c is a small protein (79 amino acids) with a partial sequence homology to the C-terminal region of ComEA in Bacillus subtilis. Cj0011c bound to both single- and double-stranded DNA. The DNA-binding activity of Cj0011c was demonstrated with a variety of DNAs prepared from C. jejuni or Escherichia coli, suggesting that the DNA binding of Cj0011c is not sequence dependent. Deletion of the cj0011c gene from C. jejuni resulted in 10- to 50-fold reductions in the natural transformation frequency. Different from the B. subtilis ComEA, which is an integral membrane protein, Cj0011c is localized in the periplasmic space of C. jejuni. These results indicate that Cj0011c functions as a periplasmic DNA receptor contributing to the natural transformation of C. jejuni.     

Identification of a Campylobacter jejuni-secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter ( yplA ). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242 , and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.     

Cellular response of Campylobacter jejuni to trisodium phosphate

The highly alkaline compound trisodium phosphate (TSP) is used as an intervention to reduce the load of Campylobacter on poultry meat in U.S. poultry slaughter plants. The aim of the present study was to investigate the cellular responses of Campylobacter jejuni NCTC11168 when exposed to sublethal concentrations of TSP. Preexposure of C. jejuni to TSP resulted in a significant increase in heat sensitivity, suggesting that a combined heat and TSP treatment may increase reduction of C. jejuni. A microarray analysis identified a limited number of genes that were differently expressed after sublethal TSP exposure; however, the response was mainly associated with ion transport processes. C. jejuni NCTC11168 nhaA1 (Cj1655c) and nhaA2 (Cj1654c), which encode orthologues to the Escherichia coli NhaA cation/proton antiporter, were able to partially restore TSP, alkaline, and sodium resistance phenotypes to an E. coli cation/proton antiporter mutant. In addition, inhibition of resistance-nodulation-cell division (RND) multidrug efflux pumps by the inhibitor PaβN (Phe-Arg β-naphthylamide dihydrochloride) decreased tolerance to sublethal TSP. Therefore, we propose that NhaA1/NhaA2 cation/proton antiporters and RND multidrug efflux pumps function in tolerance to sublethal TSP exposure in C. jejuni.     

Evaluation of genetic markers and molecular typing methods for prediction of sources of Campylobacter jejuni and C. coli infections

Campylobacter jejuni and C. coli isolates from poultry, cattle, and humans were studied using pulsed-field gel electrophoresis (PFGE) and PCR of candidate livestock-associated marker genes. Human isolates showed 5.7 and 61% overlap with cattle and poultry isolates, respectively, by use of PFGE. No unambiguous association was found between marker genes (the Cj1321 and Cj1324 genes) and livestock-associated isolates.