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A link between the assembly of flagella and lipooligosaccharide of the Gram-negative bacterium Campylobacter jejuni.

ABSTRACT: Campylobacter jejuni is the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Two major surface features, the outer membrane lipooligosaccharide and flagella, are highly variable and are often targets for modification. Presumably, these modifications provide a competitive advantage to the bacterium. In this work, we identify a gene encoding a phosphoethanolamine (pEtN) transferase (Cj0256) that serves a dual role in modifying not only the lipooligosaccharide lipid anchor lipid A with pEtN, but also the flagellar rod protein FlgG. Generation of a mutant in C. jejuni 81-176 by interruption of cj0256 resulted in the absence of pEtN modifications on lipid A as well as FlgG. The cj0256 mutant showed a 20-fold increase in sensitivity to the cationic antimicrobial peptide, polymyxin B, as well as a decrease in motility. Transmission EM of the cj0256 mutant revealed a population (approximately 95%) lacking flagella, indicating that, without pEtN modification of FlgG, flagella production is hindered. Most intriguing, this research identifies a pEtN transferase showing preference for two periplasmic substrates linking membrane biogenesis and flagellar assembly. Cj0256 is a member of a large family of mostly uncharacterized proteins that may play a larger role in the decoration of bacterial surface structures.

SUBMITTER: Cullen TW

PROVIDER: S-EPMC2841920 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain-Barre syndrome.

Project description:Molecular mimicry between microbial and self-components is postulated as the mechanism that accounts for the antigen and tissue specificity of immune responses in postinfectious autoimmune diseases. Little direct evidence exists, and research in this area has focused principally on T cell-mediated, antipeptide responses, rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome, the most frequent cause of acute neuromuscular paralysis, occurs 1-2 wk after various infections, in particular, Campylobacter jejuni enteritis. Carbohydrate mimicry [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-] between the bacterial lipooligosaccharide and human GM1 ganglioside is seen as having relevance to the pathogenesis of Guillain-Barré syndrome, and conclusive evidence is reported here. On sensitization with C. jejuni lipooligosaccharide, rabbits developed anti-GM1 IgG antibody and flaccid limb weakness. Paralyzed rabbits had pathological changes in their peripheral nerves identical with those present in Guillain-Barré syndrome. Immunization of mice with the lipooligosaccharide generated a mAb that reacted with GM1 and bound to human peripheral nerves. The mAb and anti-GM1 IgG from patients with Guillain-Barré syndrome did not induce paralysis but blocked muscle action potentials in a muscle-spinal cord coculture, indicating that anti-GM1 antibody can cause muscle weakness. These findings show that carbohydrate mimicry is an important cause of autoimmune neuropathy.

| S-EPMC509213 | biostudies-literature

A PAS domain-containing regulator controls flagella-flagella interactions in Campylobacter jejuni.

Project description:The bipolar flagella of the foodborne bacterial pathogen Campylobacter jejuni confer motility, which is essential for virulence. The flagella of C. jejuni are post-translationally modified, but how this process is controlled is not well understood. In this work, we have identified a novel PAS-domain containing regulatory system, which modulates flagella-flagella interactions in C. jejuni. Inactivation of the cj1387c gene, encoding a YheO-like PAS6 domain linked to a helix-turn-helix domain, resulted in the generation of a tightly associated "cell-train" morphotype, where up to four cells were connected by their flagella. The morphotype was fully motile, resistant to vortexing, accompanied by increased autoagglutination, and was not observed in aflagellated cells. The Δcj1387c mutant displayed increased expression of the adjacent Cj1388 protein, which comprises of a single endoribonuclease L-PSP domain. Comparative genomics showed that cj1387c (yheO) orthologs in bacterial genomes are commonly linked to an adjacent cj1388 ortholog, with some bacteria, including C. jejuni, containing another cj1388-like gene (cj0327). Inactivation of the cj1388 and cj0327 genes resulted in decreased autoagglutination in Tween-20-supplemented media. The Δcj1388 and Δcj0327 mutants were also attenuated in a Galleria larvae-based infection model. Finally, substituting the sole cysteine in Cj1388 for serine prevented Cj1388 dimerization in non-reducing conditions, and resulted in decreased autoagglutination in the presence of Tween-20. We hypothesize that Cj1388 and Cj0327 modulate post-translational modification of the flagella through yet unidentified mechanisms, and propose naming Cj1387 the Campylobacter Flagella Interaction Regulator CfiR, and the Cj1388 and Cj0327 protein as CfiP and CfiQ, respectively.

| S-EPMC4519771 | biostudies-literature

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

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| S-EPMC2258656 | biostudies-literature

Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species.

Project description:Campylobacter jejuni subsp. jejuni and Campylobacter coli are leading causes of gastroenteritis, with virulence linked to cell surface carbohydrate diversity. Although the associated gene clusters are well studied for C. jejuni subsp. jejuni, C. coli has been largely neglected. Here we provide comparative analysis of the lipooligosaccharide (LOS) and capsular polysaccharide (CPS) gene clusters, using genome and cluster sequence data for 36 C. coli strains, 67 C. jejuni subsp. jejuni strains and ten additional Campylobacter species. Similar to C. jejuni subsp. jejuni, C. coli showed high LOS/CPS gene diversity, with each cluster delineated into eight gene content classes. This diversity was predominantly due to extensive gene gain/loss, with the lateral transfer of genes likely occurring both within and between species and also between the LOS and CPS. Additional mechanisms responsible for LOS/CPS diversity included phase-variable homopolymeric repeats, gene duplication/inactivation, and possibly host environment selection pressure. Analyses also showed that (i) strains of C. coli and Campylobacter upsaliensis possessed genes homologous to the sialic acid genes implicated in the neurological disorder Guillain-Barré syndrome (GBS), and (ii) C. coli LOS classes were differentiated between bovine and poultry hosts, potentially aiding post infection source tracking.

| S-EPMC3622452 | biostudies-literature

Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources.

Project description:Campylobacter jejuni strains exhibit significant variation in the genetic content of the lipooligosaccharide (LOS) biosynthesis loci with concomitant differences in LOS structure. The C. jejuni LOS loci have been grouped into six classes based on gene content and organization. Utilizing PCR amplifications of genes from these loci, we were able to classify a majority (80%) of the LOS biosynthesis loci from 123 strains of C. jejuni that included 39 of the Penner serotype reference strains. We found that a particular LOS class was not always associated with a specific Penner serotype, and 14 of 16 Guillain-Barré syndrome-associated isolates tested in this study shared the same LOS class. The remaining isolates that could not be classified were often distinguishable from each other based on the results of gene-specific PCR and the lengths of their LOS biosynthesis loci as determined by long (XL) PCR. Sequence analysis of two of these unique XL PCR products demonstrated two new LOS classes. These results support the hypothesis that the LOS locus is a hot spot for genetic exchange and rearrangements. Analysis of the LOS biosynthesis genes by PCR assays can be used for typing C. jejuni and offers the advantage of inferring potential LOS structures.

| S-EPMC1151924 | biostudies-literature

Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni.

Project description:Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM(2) ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative beta-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.

| S-EPMC97763 | biostudies-literature

Lipooligosaccharide of Campylobacter jejuni: similarity with multiple types of mammalian glycans beyond gangliosides.

Project description:Campylobacter jejuni is well known for synthesizing ganglioside mimics within the glycan component of its lipooligosaccharide (LOS), which have been implicated in triggering Guillain-Barré syndrome. We now confirm that this pathogen is capable of synthesizing a much broader spectrum of host glycolipid/glycoprotein mimics within its LOS. P blood group and paragloboside (lacto-N-neotetraose) antigen mimicry is exhibited by RM1221, a strain isolated from a poultry source. RM1503, a gastroenteritis-associated strain, expresses lacto-N-biose and sialyl-Lewis c units, the latter known as the pancreatic tumor-associated antigen, DU-PAN-2 (or LSTa). C. jejuni GC149, a Guillain-Barré syndrome-associated strain, expresses an unusual sialic acid-containing hybrid oligosaccharide with similarity to both ganglio and Pk antigens and can, through phase variation of its LOS biosynthesis genes, display GT1a or GD3 ganglioside mimics. We show that the sialyltransferase CstII and the galactosyltransferase CgtD are involved in the synthesis of multiple mimic types, with LOS structural diversity achieved through evolving allelic substrate specificity.

| S-EPMC3069439 | biostudies-literature

Campylobacter jejuni translocation across intestinal epithelial cells is facilitated by ganglioside-like lipooligosaccharide structures.

Project description:Translocation across intestinal epithelial cells is an established pathogenic feature of the zoonotic bacterial species Campylobacter jejuni. The number of C. jejuni virulence factors known to be involved in translocation is limited. In the present study, we investigated whether sialylation of C. jejuni lipooligosaccharide (LOS) structures, generating human nerve ganglioside mimics, is important for intestinal epithelial translocation. We here show that C. jejuni isolates expressing ganglioside-like LOS bound in larger numbers to the Caco-2 intestinal epithelial cells than C. jejuni isolates lacking such structures. Next, we found that ganglioside-like LOS facilitated endocytosis of bacteria into Caco-2 cells, as visualized by quantitative microscopy using the early and late endosomal markers early endosome-associated protein 1 (EEA1), Rab5, and lysosome-associated membrane protein 1 (LAMP-1). This increased endocytosis was associated with larger numbers of surviving and translocating bacteria. Next, we found that two different intestinal epithelial cell lines (Caco-2 and T84) responded with an elevated secretion of the T-cell attractant CXCL10 to infection by ganglioside-like LOS-expressing C. jejuni isolates. We conclude that C. jejuni translocation across Caco-2 cells is facilitated by ganglioside-like LOS, which is of clinical relevance since C. jejuni ganglioside-like LOS-expressing isolates are linked with severe gastroenteritis and bloody stools in C. jejuni-infected patients.

| S-EPMC3418735 | biostudies-literature

Phase variation of Campylobacter jejuni 81-176 lipooligosaccharide affects ganglioside mimicry and invasiveness in vitro.

Project description:The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.

| S-EPMC127662 | biostudies-literature

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