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

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 similar to 84% of the C. jejuni NCTC 11168 genome, and 1080 of these were validated by DIASWATH-MS. Deletion of the pgIB oligosaccharyltransferase (Delta pgIB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of pgIB (Delta pgIB::Delta pgIB)Deletion of pgIB was associated with significantly reduced abundances of pgl targets and increased stress-related proteins, including CIpB, GroEL, GroES, GrpE and DnaK. pgIB mutants demonstrated reduced survival following temperature (4 degrees C and 46 degrees 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). Delta pgIB chemotaxis to some substrates (Asp, glutamate, succinate and alpha-ketoglutarate) was reduced and associated with altered abundance of transducer-like (Tip) 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.