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Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway.

ABSTRACT: The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC, and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer of [³H]GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates.

SUBMITTER: Morrison JP

PROVIDER: S-EPMC2981441 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway.

Morrison James P JP Troutman Jerry M JM Imperiali Barbara B

Bioorganic & medicinal chemistry 20101029 23

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of ...[more]

PMID: 21036619

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Project description:Campylobacter jejuni is a major gastrointestinal pathogen generally acquired via consumption of poorly prepared poultry. N-linked protein glycosylation encoded by the pgl gene cluster targets >80 membrane proteins and is required for both nonsymptomatic chicken colonization and full human virulence. Despite this, the biological functions of N-glycosylation remain unknown. We examined the effects of pgl gene deletion on the C. jejuni proteome using label-based liquid chromatography/tandem mass spectrometry (LC-MS/MS) and validation using data independent acquisition (DIA-SWATH-MS). We quantified 1359 proteins corresponding to ∼84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIA-SWATH-MS. Deletion of the pglB oligosaccharyltransferase (ΔpglB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pglB (Δaaz.batpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including ClpB, GroEL, GroES, GrpE and DnaK. pglB mutants demonstrated reduced survival following temperature (4 °C and 46 °C) and osmotic (150 mm NaCl) shock and altered biofilm phenotypes compared with wild-type C. jejuni Targeted metabolomics established that pgl negative C. jejuni switched from aspartate (Asp) to proline (Pro) uptake and accumulated intracellular succinate related to proteome changes including elevated PutP/PutA (proline transport and utilization), and reduced DctA/DcuB (aspartate import and succinate export, respectively). ΔpglB chemotaxis to some substrates (Asp, glutamate, succinate and α-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tlp) proteins. Glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen. We demonstrate for the first time that N-glycosylation is required for a specific enzyme activity (Nap nitrate reductase) that is associated with reduced abundance of the NapAB glycoproteins. These data indicate a multifactorial role for N-glycosylation in C. jejuni physiology.

Dataset Information

Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway.

Publications

Development of a multicomponent kinetic assay of the early enzymes in the Campylobacter jejuni N-linked glycosylation pathway.

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