Influence of Protein Glycosylation on Campylobacter fetus Physiology

Duma, Justin; Nothaft, Harald; Weaver, Danielle; Fodor, Christopher; Beadle, Bernadette; Linton, Dennis; Benoit, Stéphane L.; Scott, Nichollas E.; Maier, Robert J.; Szymanski, Christine M.

doi:10.3389/fmicb.2020.01191

Cited by 9 publications

(9 citation statements)

References 74 publications

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“…Functional analysis of the PglI proteins was carried out by reconstituting the C. jejuni N-glycosylation system in the E. coli three-plasmid system by complementing the pglI gene mutation on plasmid pACYC184 pgl /Δ pglI (as described in ref ). Plasmid pACYC184 pgl (expressing all components of the N-glycosylation pathway for biosynthesis of the full-length Cj -N-glycan/heptasaccharide) and pACYC184 pgl /Δ pglI complemented with pglI from Cj- 81116 were used as positive controls.…”

Section: Resultscontrasting

confidence: 72%

“…The pglI expression plasmid from Cj strain 81116 has been described previously . GEMS strain pglIs were PCR amplified (in triplicate) from chromosomal DNA (cDNA) from strains 1J2, 1J5, 1H2, and 1D7 with oligonucleotides (pglI-81116- Bam HI-F) and (81116-pglI-XhoI-R_short).…”

Section: Experimental Methodsmentioning

confidence: 99%

“…The previously described E. coli three-plasmid system was employed to follow PglI activity. Here, the N-glycan acceptor protein CmeA-His 6 is constitutively expressed from plasmid pHI18b and Cj - pgl genes are constitutively expressed from either plasmid pACYC184 pgl (positive control) or plasmid pACYC184 pgl / pglI::kan . The individual pglI alleles are constitutively expressed from pCE111-28 derivatives.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure

et al. 2021

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Campylobacter jejuni is a significant cause of human gastroenteritis worldwide, and all strains express an N-glycan that is added to at least 80 different proteins. We characterized 98 C. jejuni isolates from infants from 7 low- and middle-income countries and identified 4 isolates unreactive with our N-glycan-specific antiserum that was raised against the C. jejuni heptasaccharide composed of GalNAc-GalNAc-GalNAc(Glc)-GalNAc-GalNAc-diNAcBac. Mass spectrometric analyses indicated these isolates express a hexasaccharide lacking the glucose branch. Although all 4 strains encode the PglI glucosyltransferase (GlcTF), one aspartate in the DXDD motif was missing, an alteration also present in ∼4% of all available PglI sequences. Deleting this residue from an active PglI resulted in a nonfunctional GlcTF when the protein glycosylation system was reconstituted in E. coli, while replacement with Glu/Ala was not deleterious. Molecular modeling proposed a mechanism for how the DXDD residues and the structure/length beyond the motif influence activity. Mouse vaccination with an E. coli strain expressing the full-length heptasaccharide produced N-glycan-specific antibodies and a corresponding reduction in Campylobacter colonization and weight loss following challenge. However, the antibodies did not recognize the hexasaccharide and were unable to opsonize C. jejuni isolates lacking glucose, suggesting this should be considered when designing N-glycan-based vaccines to prevent campylobacteriosis.

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Section: Resultscontrasting

confidence: 72%

Section: Experimental Methodsmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

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Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure

et al. 2021

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“…Within the highly conserved N -linked general protein glycosylation pathway (Pgl), more than 80 proteins have been identified so far in C. jejuni which are characterized by a typical heptasaccharide motif (GalNAc-α1,4-GalNAc-α1,4-(Glc-β1,3-)GalNAc-α1,4-GalNAc-α1,4-GalNAc-α1,3-diNAcBac-β1; with diNAcBac as 2,4-diacetamido-2,4,6-trideoxyglucopyranose, Gal as galactose, NAc as N-acetylated carbohydrate, Glc as glucose) attached to asparagine residues (Cain et al 2019 ; Duma et al 2020 ). The consensus sequence of the glycosylation sites for efficient modifications by bacterial oligosaccharyltransferases is D/E—Y—N—X—S/T, with Y and X being variable amino acids except proline (Kowarik et al 2006 ).…”

Section: Adhesion: the First Step For Attacking Host Cellsmentioning

confidence: 99%

Campylobacter jejuni: targeting host cells, adhesion, invasion, and survival

Kemper

Hensel

2023

Appl Microbiol Biotechnol

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Campylobacter jejuni, causing strong enteritis, is an unusual bacterium with numerous peculiarities. Chemotactically controlled motility in viscous milieu allows targeted navigation to intestinal mucus and colonization. By phase variation, quorum sensing, extensive O-and N-glycosylation and use of the flagellum as type-3-secretion system C. jejuni adapts effectively to environmental conditions. C. jejuni utilizes proteases to open cell–cell junctions and subsequently transmigrates paracellularly. Fibronectin at the basolateral side of polarized epithelial cells serves as binding site for adhesins CadF and FlpA, leading to intracellular signaling, which again triggers membrane ruffling and reduced host cell migration by focal adhesion. Cell contacts of C. jejuni results in its secretion of invasion antigens, which induce membrane ruffling by paxillin-independent pathway. In addition to fibronectin-binding proteins, other adhesins with other target structures and lectins and their corresponding sugar structures are involved in host–pathogen interaction. Invasion into the intestinal epithelial cell depends on host cell structures. Fibronectin, clathrin, and dynein influence cytoskeletal restructuring, endocytosis, and vesicular transport, through different mechanisms. C. jejuni can persist over a 72-h period in the cell. Campylobacter-containing vacuoles, avoid fusion with lysosomes and enter the perinuclear space via dynein, inducing signaling pathways. Secretion of cytolethal distending toxin directs the cell into programmed cell death, including the pyroptotic release of proinflammatory substances from the destroyed cell compartments. The immune system reacts with an inflammatory cascade by participation of numerous immune cells. The development of autoantibodies, directed not only against lipooligosaccharides, but also against endogenous gangliosides, triggers autoimmune diseases. Lesions of the epithelium result in loss of electrolytes, water, and blood, leading to diarrhea, which flushes out mucus containing C. jejuni. Together with the response of the immune system, this limits infection time. Based on the structural interactions between host cell and bacterium, the numerous virulence mechanisms, signaling, and effects that characterize the infection process of C. jejuni, a wide variety of targets for attenuation of the pathogen can be characterized. The review summarizes strategies of C. jejuni for host–pathogen interaction and should stimulate innovative research towards improved definition of targets for future drug development. Key points • Bacterial adhesion of Campylobacter to host cells and invasion into host cells are strictly coordinated processes, which can serve as targets to prevent infection. • Reaction and signalling of host cell depend on the cell type. • Campylobacter virulence factors can be used as targets for development of antivirulence drug compounds.

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“…The discovery of a general protein glycosylation (Pgl) system in C. jejuni , and related organisms − also confirmed that bacteria are capable of modifying a diverse array of proteins with N -glycans. The biochemistry of N -glycosylation in C.…”

Section: Introductionmentioning

confidence: 99%

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation

2021

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Campylobacter jejuni is a bacterial pathogen encoding a unique N-linked glycosylation (pgl) system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited pglB-positive (wild-type; WT) and -negative (ΔpglB) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in ΔpglB relative to WT and restored by pglB reintroduction (ΔpglB::pglB). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and ΔpglB::pglB C. jejuni. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified C. jejuni glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.

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Influence of Protein Glycosylation on Campylobacter fetus Physiology

Cited by 9 publications

References 74 publications

Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure

Detecting Glucose Fluctuations in the Campylobacter jejuni N-Glycan Structure

Campylobacter jejuni: targeting host cells, adhesion, invasion, and survival

Exploiting pglB Oligosaccharyltransferase-Positive and -Negative Campylobacter jejuni and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation

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