Proteomics reveals multiple phenotypes associated with n-linked glycosylation in Campylobacter jejuni

Abstract

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 DIASWATH-MS. Deletion of the PGLB oligosaccharyltransferase (PGLB) resulted in a significant change in abundance of 185 proteins, 137 of which were restored to their wild-type levels by reintroduction of PGLB (PGLBPGLB). Deletion of PGLB was associated with significantly reduced abundances of PGL targets and increased stressrelated 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). PglB chemotaxis to some substrates (Asp, glutamate, succinate and -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.