Functional Characterization of Flagellin Glycosylation in Campylobacter jejuni 81-176

The major flagellin of Campylobacter jejuni strain 81-176, FlaA, has been shown to be glycosylated at 19 serine or threonine sites, and this glycosylation is required for flagellar filament formation. Some enzymatic components of the glycosylation machinery of C. jejuni 81-176 are localized to the poles of the cell in an FlhF-independent manner. Flagellin glycosylation could be detected in flagellar mutants at multiple levels of the regulatory hierarchy, indicating that glycosylation occurs independently of the flagellar regulon. Mutants were constructed in which each of the 19 serine or threonines that are glycosylated in FlaA was converted to an alanine. Eleven of the 19 mutants displayed no observable phenotype, but the remaining 8 mutants had two distinct phenotypes. Five mutants (mutations S417A, S436A, S440A, S457A, and T481A) were fully motile but defective in autoagglutination (AAG). Three other mutants (mutations S425A, S454A, and S460A) were reduced in motility and synthesized truncated flagellar filaments. The data implicate certain glycans in mediating filament-filament interactions resulting in AAG and other glycans appear to be critical for structural subunit-subunit interactions within the filament.Flagellins from many polarly flagellated bacteria are glycosylated (reviewed in reference 22). The best-characterized examples are the flagellins from Campylobacter spp. that are decorated with as many as 19 O-linked glycans that can contribute ∼10% to the weight of flagellin (38). The genes encoding the enzymes for biosynthesis of the glycans found on Campylobacter flagellins and the respective glycosyltransferases are located adjacent to the flagellin structural genes in one of the more hypervariable regions of the Campylobacter genome (3, 16, 28, 37). Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm) (23). In contrast, Campylobacter jejuni strain 81-176 contains only the pathway for synthesis of PseAc (9) and derivatives of PseAc that include an acetylated form (PseAcOAc), an acetamidino form (PseAm), and a form of PseAm with a glutamic acid moiety attached (PseAmOGln) (25, 34, 38). The flagellins of C. jejuni strain NCTC 11168 have recently been shown to be glycosylated with PseAc and LegAm, as well as two novel derivatives of PseAc, a di-O-methylglyceric acid and a related acetamidino form (24). Thus, although all of the flagellar glycans appear to be based on either PseAc and/or LegAm, there are variations among strains that contribute to serospecificity and reflect the heterogeneity of the flagellin glycosylation loci (23, 24).The function of the glycosyl modifications to flagellar structure and to the biology of campylobacters is not fully understood. Although most polarly flagellated bacteria appear to glycosylate flagellin, mutation of the genes involved in glycosylation does not generally result in loss of motility (22). However, flagella from C. jejuni, Campylobacter coli, and Helicobacter pylori, all members of the epsilon division of Proteobacteria, are unable to assemble a filament in the absence of a functional glycosylation system (7, 33). Also, changes in the glycans on campylobacter flagellins have been shown to affect autoagglutination (AAG) and microcolony formation on intestinal epithelial cells in vitro (5, 9). Thus, a mutant of C. jejuni 81-176 that was unable to synthesize PseAm assembled a flagellar filament, but the sites on the flagellin subunits that were normally glycosylated with PseAm were instead glycosylated with PseAc. This mutant was reduced in AAG, adherence, and invasion of INT407 cells and was also attenuated in a ferret diarrheal disease model (9). C. coli VC167 has both PseAc and LegAm pathways. Mutants that were defective in either pathway could still assemble flagellar filaments composed of subunits that were modified with the alternate sugar, but these mutants showed defects in AAG (7). A VC167 double mutant, defective in both PseAc and LegAm synthesis, was nonflagellated (7). Collectively, these data suggest that some glycosylation is required for either secretion of flagellin or for interactions between subunits within the filament.Flagellar biogenesis in C. jejuni is a complex process that is highly controlled by the alternate sigma factors σ²⁸ and σ⁵⁴, a two-component regulatory system composed of the sensor kinase FlgS and the σ⁵⁴-response regulator FlgR, and the flagellar export apparatus (15, 39). Both flgR and flgS genes undergo slip strand mismatch repair in C. jejuni strain 81-176, resulting in an on/off-phase variation of flagellar expression (13, 14). The major flagellin gene, flaA, and some other late flagellar genes are regulated by σ²⁸; the genes encoding the minor flagellin, flaB, and the hook and rod structures are regulated by σ⁵⁴. Here, we examine several aspects of glycosylation to flagellar function in C. jejuni 81-176. We demonstrate that some components of the flagellar glycosylation machinery are localized to the poles of the cell, but independently of the signal recognition particle-like flagellar protein, FlhF, and that flagellin glycosylation occurs independently of the flagellar regulon. We also show that the glycans on some amino acids appear to play a structural role in subunit interactions in the filament, while others affect interactions with adjacent filaments that result in AAG.     

Cell-surface alpha-glucan in Campylobacter jejuni 81-176

Campylobacter jejuni infection is a main source of severe gastroenteritis-related illnesses in humans and there is also evidence that it may be linked to neurological disorders. C. jejuni 81-176 is a virulent strain that has become the global model in the study of mechanisms and pathogenesis of C. jejuni infection. For this reason, we were engaged in studying the fine structures of cell-surface carbohydrate antigens of C. jejuni 81-176, namely, the capsule polysaccharide (CPS) and lipooligosaccharide (LOS). Serologically, C. jejuni 81-176 has been classified as belonging to serogroups HS23 and HS36, and indeed previous studies have shown that the LOS and CPS structures possess components similar to those expressed by serostrains HS23 and HS36. Here, we describe that in addition to the LOS and CPS, this strain also produced an independent cell-surface (1-->4)-alpha-glucan capsule.     

N-linked protein glycosylation is required for full competence in Campylobacter jejuni 81-176

The recent sequencing of the virulence plasmid of Campylobacter jejuni 81-176 revealed the presence of genes homologous to type IV secretion systems (TFSS) that have subsequently been found in Helicobacter pylori and Wolinella succinogenes. Mutational analyses of some of these genes have implicated their involvement in intestinal epithelial cell invasion and natural competence. In this report, we demonstrate that one of these type IV secretion homologs, Cjp3/VirB10, is a glycoprotein. Treatment with various glycosidases and binding to soybean agglutinin indicated that the structure of the glycan present on VirB10 contains a terminal GalNAc, consistent with previous reports of N-linked glycans in C. jejuni. Site-directed mutagenesis of five putative N-linked glycosylation sites indicated that VirB10 is glycosylated at two sites, N32 and N97. Mutants in the N-linked general protein glycosylation (pgl) system of C. jejuni are significantly reduced in natural transformation, which is likely due, in part, to lack of glycosylation of VirB10. The natural transformation defect in a virB10 mutant can be complemented in trans by using a plasmid expressing wild-type VirB10 or an N32A substitution but not by using a mutant expressing VirB10 with an N97A substitution. Taken together, these results suggest that glycosylation of VirB10 specifically at N97 is required for the function of the TFSS and for full competence in C. jejuni 81-176.     

Genetic analysis of lipooligosaccharide core biosynthesis in Campylobacter jejuni 81-176

We report isolation and characterization of Campylobacter jejuni 81-176 lgtF and galT lipooligosaccharide (LOS) core mutants. It has been suggested that the lgtF gene of C. jejuni encodes a two-domain glucosyltransferase that is responsible for the transfer of a β-1,4-glucose residue on heptosyltransferase I (Hep I) and for the transfer of a β-1,2-glucose residue on Hep II. A site-specific mutation in the lgtF gene of C. jejuni 81-176 resulted in expression of a truncated LOS, and complementation of the mutant in trans restored the core mobility to that of the wild type. Mass spectrometry and nuclear magnetic resonance of the truncated LOS confirmed the loss of two glucose residues, a β-1,4-glucose on Hep I and a β-1,2-glucose on Hep II. Mutation of another gene, galT, encoding a glycosyltransferase, which maps outside the region defined as the LOS biosynthetic locus in C. jejuni 81-176, resulted in loss of the β-(1,4)-galactose residue and all distal residues in the core. Both mutants invaded intestinal epithelial cells in vitro at levels comparable to the wild-type levels, in marked contrast to a deeper inner core waaC mutant. These studies have important implications for the role of LOS in the pathogenesis of Campylobacter-mediated infection.     

A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176

Campylobacter jejuni strain 81-176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K-digested whole-cell preparations. A site-specific insertional mutant in the kpsM gene results in loss of expression of a high-molecular-weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder-like glycan (apparent Mr 26-50 kDa). The kpsM mutant of 81-176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM-dependent HMW glycan and the kpsM-independent ladder-like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81-176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an alpha2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81-176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM-dependent capsule undergoes phase variation at a high frequency.     

Dynamic proteome changes in Campylobacter jejuni 81-176 after high pressure shock and subsequent recovery

Campylobacter jejuni is one of the most intriguing human foodborne bacterial pathogen. Its survival throughout the food processing chain and its pathogenesis mechanisms in humans remain enigmatic. Living in the animal guts and particularly in avian intestine as a commensal bacterium, this microorganism is frequently isolated from meat products. Ultra high pressure (HP) is a promising alternative to thermal technology for microbial safety of foodstuffs with less organoleptic and nutritional alterations. Its application could be extended to meat products potentially contaminated by C. jejuni. To evaluate the response of Campylobacter to this technological stress and subsequent recovery at a molecular level, a dynamic 2-DE-based proteomic approach has been implemented. After cultivation, C. jejuni cells were conditioned in a high-pressure chamber and transferred to fresh medium for recovery. The protein abundance dynamics at the proteome scale were analyzed by 2-DE during the cellular process of cell injury and recovery. Monitoring protein abundance through time unraveled the basic metabolisms involved in this cellular process. The significance of the proteome evolution modulated by HP and subsequent recovery is discussed in the context of a specific cellular response to stress and recovery of C. jejuni with 69 spots showing significant changes through time.     

Isolation and characterization of the flagellar hook of Campylobacter jejuni

Abstract A method for purification of the flagellar hook of Campylobacter jejuni is described. The hook was shown to be composed of a subunit protein, which has a molecular mass of 92,000 and an isoelectric point of pI 4.8. A monoclonal antibody and a polyvalent antiserum was raised against the purified flagellar hook of C. jejuni . Immuno-electronmicroscopy revealed that the epitope recognized by the monoclonal antibody is surface-located. However, this antibody reacted only with the hook of the immunization strain, but not with other strains or other flagellated bacteria. Thus, our data indicate that the immunodominant epitopes are located on the surface of the hook and that these epitopes are strain-specific.     

Polyisoprenol specificity in the Campylobacter jejuni N-linked glycosylation pathway

Campylobacter jejuni contains a general N-linked glycosylation pathway in which a heptasaccharide is sequentially assembled onto a polyisoprenyl diphosphate carrier and subsequently transferred to the asparagine side chain of an acceptor protein. The enzymes in the pathway function at a membrane interface and have in common amphiphilic membrane-bound polyisoprenyl-linked substrates. Herein, we examine the potential role of the polyisoprene component of the substrates by investigating the relative substrate efficiencies of polyisoprene-modified analogues in individual steps of the pathway. Chemically defined substrates for PglC, PglJ, and PglB are prepared via semisynthetic approaches. The substrates included polyisoprenols of varying length, double bond geometry, and degree of saturation for probing the role of the hydrophobic polyisoprene in substrate specificity. Kinetic analysis reveals that all three enzymes exhibit distinct preferences for the polyisoprenyl carrier whereby cis-double bond geometry and alpha-unsaturation of the native substrate are important features, while the precise polyisoprene length may be less critical. These findings suggest that the polyisoprenyl carrier plays a specific role in the function of these enzymes beyond a purely physical role as a membrane anchor. These studies underscore the potential of the C. jejuni N-linked glycosylation pathway as a system for investigating the biochemical and biophysical roles of polyisoprenyl carriers common to prokaryotic and eukaryotic glycosylation.     

Mutation of waaC, encoding heptosyltransferase I in Campylobacter jejuni 81-176, affects the structure of both lipooligosaccharide and capsular carbohydrate

Campylobacter jejuni 81-176 lipooligosaccharide (LOS) is composed of two covalently linked domains: lipid A, a hydrophobic anchor, and a nonrepeating core oligosaccharide, consisting of an inner and outer core region. We report the isolation and characterization of the deepest rough C. jejuni 81-176 mutant by insertional mutagenesis into the waaC gene, encoding heptosyltransferase I that catalyzes the transfer of the first L-glycero-D-manno-heptose residue to 3-deoxy-D-manno-octulosonic residue (Kdo)-lipid A. Tricine gel electrophoresis, followed by silver staining, showed that site-specific mutation in the waaC gene resulted in the expression of a severely truncated LOS compared to wild-type strain 81-176. Gas-liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy showed that the waaC LOS species lacked all sugars distal to Kdo-lipid A. Parallel structural studies of the capsular polysaccharides of the wild-type strain 81-176 and waaC mutant revealed loss of the 3-O-methyl group in the waaC mutant. Complementation of the C. jejuni mutant by insertion of the wild-type C. jejuni waaC gene into a chromosomal locus resulted in LOS and capsular structures identical to those expressed in the parent strain. We also report here the presence of O-methyl phosphoramidate in wild-type strain 81-176 capsular polysaccharide.     

Evidence for a system of general protein glycosylation in Campylobacter jejuni

A genetic locus from Campylobacter jejuni 81-176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5alpha containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site-specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site-specific mutation of each of the seven genes in 81-176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild-type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81-176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.     

Molecular characterization of a strain-specific sequence in Campylobacter jejuni

Campylobacter jejuni strains 81116 and NCTC 11392 were shown to contain a region of DNA that was not present in other strains. This region was cloned from the chromosome of strain 81116 and the nucleotide sequence determined. It was found to contain an insert of 742 bp which was flanked by direct repeats of 45 bp. Although the nature of this sequence is unknown at present, it is clear that it only presents in certain strains of Camp. jejuni and it may therefore be useful as an epidemiological tool.     

Polar flagellar biosynthesis and a regulator of flagellar number influence spatial parameters of cell division in Campylobacter jejuni

Spatial and numerical regulation of flagellar biosynthesis results in different flagellation patterns specific for each bacterial species. Campylobacter jejuni produces amphitrichous (bipolar) flagella to result in a single flagellum at both poles. These flagella confer swimming motility and a distinctive darting motility necessary for infection of humans to cause diarrheal disease and animals to promote commensalism. In addition to flagellation, symmetrical cell division is spatially regulated so that the divisome forms near the cellular midpoint. We have identified an unprecedented system for spatially regulating cell division in C. jejuni composed by FlhG, a regulator of flagellar number in polar flagellates, and components of amphitrichous flagella. Similar to its role in other polarly-flagellated bacteria, we found that FlhG regulates flagellar biosynthesis to limit poles of C. jejuni to one flagellum. Furthermore, we discovered that FlhG negatively influences the ability of FtsZ to initiate cell division. Through analysis of specific flagellar mutants, we discovered that components of the motor and switch complex of amphitrichous flagella are required with FlhG to specifically inhibit division at poles. Without FlhG or specific motor and switch complex proteins, cell division occurs more often at polar regions to form minicells. Our findings suggest a new understanding for the biological requirement of the amphitrichous flagellation pattern in bacteria that extend beyond motility, virulence, and colonization. We propose that amphitrichous bacteria such as Campylobacter species advantageously exploit placement of flagella at both poles to spatially regulate an FlhG-dependent mechanism to inhibit polar cell division, thereby encouraging symmetrical cell division to generate the greatest number of viable offspring. Furthermore, we found that other polarly-flagellated bacteria produce FlhG proteins that influence cell division, suggesting that FlhG and polar flagella may function together in a broad range of bacteria to spatially regulate division.     

Molecular characterization of a cryptic plasmid from Campylobacter jejuni

Campylobacter jejuni is a leading bacterial cause of human enterocolitis. Molecular genetic characterization of this pathogen has been hampered by the lack of genetic tools that are functional in this organism. Cloning vectors commonly used in other organisms usually do not replicate within C. jejuni. To develop a system for functional analysis of C. jejuni genes, a small plasmid (pCJ01) identified in a poultry isolate of C. jejuni was sequenced and characterized in this study. By using inverse PCR, the full sequence of pCJ01 was amplified and subsequently determined. Results indicate that pCJ01 is a circular molecule of 3212 bp, with a G + C content of 33.5%. A typical plasmid replication origin with iteron sequences is identified upstream of the DNA sequences encoding replication initiation proteins. Four open reading frames (ORFs) are present in pCJ01. ORF1 and ORF2 share high homology with the putative RepA and RepB proteins, respectively, of known C. coli plasmids. ORF3 and ORF4, of unknown function, do not exhibit homology with any sequences deposited in the GenBank database. Hydropathy analysis predicts that ORF3 and ORF4 contain multiple stretches of hydrophobic amino acids, suggesting that they may encode transmembrane proteins. Since pCJ01 is a small plasmid and can be readily prepared from C. jejuni, it may be modified for use in molecular characterization of C. jejuni virulence genes.     

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.     

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.