Poster Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

Proteomics reveals multiple phenotypes associated with N-linked glycosylation in Campylobacter jejuni (#449)

Joel Cain 1 2 , Ashleigh Dale 1 2 , Paula Niewold 2 3 , William Klare 1 2 , Lok Man 1 2 , Melanie White 2 3 , Nichollas Scott 1 4 , Stuart Cordwell 1 2 3 5
  1. School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  3. Discipline of Pathology, University of Sydney, Sydney, NSW, Australia
  4. Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Victoria, Australia
  5. Sydney Mass Spectrometry, University of Sydney, Sydney, NSW, Australia


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 non-symptomatic chicken colonization and full human intestinal epithelial cell virulence. Despite this, the biological functions of N-glycosylation remain unknown.


Here 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 analysis (DIA-SWATH-MS). Targeted metabolomics was used to assess the impact  of loss of N-glycosylation on intracellular levels of key respiratory metabolites. Other conventional microbial assays, tube-based chemotactic responses, biofilm formation, cell morphology and susceptibility to temperature and osmotic stress, were performed to establish how protein-level changes translated into macro-level phenotypes critical to cell function and pathogenicity. 

Key findings:

We quantified 1359 proteins corresponding to ~84% of the predicted C. jejuni NCTC 11168 genome. 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 (ΔpglB::ΔpglB). Deletion of pglB was associated with significantly reduced abundances of known pgl targets and increased levels of stress-related proteins. Commensurately, pglB mutants demonstrated reduced survival following temperature (4°C and 46°C) and osmotic (150 mM NaCl) shock, and showed altered biofilm phenotypes compared to wild-type C. jejuni. Targeted metabolomics established that glycosylation negative C. jejuni were depleted of all respiration-associated proteins that allow the use of alternative electron acceptors under low oxygen conditions, which correlated with protein-level changes to known small-molecule transporters. These data indicate a multi-factorial role for N-glycosylation in C. jejuni physiology.