Proteomics Reveals Multiple Phenotypes Associated with N-linked Glycosylation in Campylobacter jejuni*

N-linked protein glycosylation (Pgl) in Campylobacter jejuni is required for chicken colonization and human virulence, yet its biological role remains unknown. pgl gene deletion resulted in a significant rearrangement of the C. jejuni proteome that leads to alterations in crucial phenotypes including stress response, nutrient uptake, electron transport and chemotaxis, and is essential for full activity of the Nap nitrate reductase. N-glycosylation therefore contributes to multiple “virulence” phenotypes in C. jejuni. Graphical Abstract Highlights Protein N-glycosylation is essential for nitrate reductase (Nap) activity in C. jejuni. Removal of N-glycosylation results in a metabolic switch from Asp to Pro uptake. N-glycosylation is required for optimal chemotaxis towards several substrates. Loss of N-glycosylation reduces survival following temperature and osmotic shock. 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.