EVOGENE Seminar - Genus level variation in the Neisseria O-linked glycosylation system using mass spectrometry

Although broad-spectrum, protein glycosylation systems in bacteria are more and more common, investigation of these systems from a genus level perspective is currently limited. The broad spectrum O-linked protein glycosylation (pgl) system in pathogenic Neisseria species displays high glycan intra- and interstrain diversity with more than 30 glycoforms described to date. Variability in glycan structure and antigenicity are attributable to differences in content and expression status of glycan synthesis (pgl) genes. To further broaden our understanding of pgl gene evolution and the function of various pgl genes, we utilized global and targeted mass spectrometry (MS) of the type strains for a broad array of neisserial species species to define the breadth of glycan structure, microheterogeneity and macroheterogeneity, glycoproteomes, and site specificities. We then attempted to correlate glycoform phenotypes with pgl genotypes for all these strains. The analyses revealed that there is great intraspecies variation in pgl systems and several new and unique sugars were identified. Moreover, all strains exhibit a high level of glycan microheterogeneity - by varying the length of the protein-associated glycans generated by competing glycosyl transferases adding different sugars to the same position and/or by modifying the various sugars with minor modification such as acetylation and methylation. Finally, the combination of high resolution HCD combined with targeted ETD MS, extends our knowledge of the pan glycoproteome as multiple glycoproteins and glycosylation sites were identified in all strains.