B-272. Broad Spectrum O-Linked Protein Glycosylation in the Human Pathogen Neisseria gonorrhoeae

Å. Vik, F. Aas, W. M. Egge-Jacobsen, J. Haug Anonsen, M. Koomey;
Univ. of Oslo, Oslo, NORWAY.

Glycoproteins have been found in both Gram-negative and Gram-positive bacterial species and both N-linked and O-linked glycans have been identified. Most prokaryotic glycosylation systems are dedicated to modification of single proteins or sets of related proteins that are surface localized (flagellin, pilin etc.). To date, two generalized protein glycosylation systems have been identified: the Mycobacterium tuberculosis O-mannosylation system for exported lipoproteins and the Campylobacter jejuni (Cje) N-linked system which targets at least 25 proteins found predominantly in the periplasm. Both of these pathways parallel related processes in eukaryotes. Here we describe the first generalized O-linked glycosylation system in a Gram-negative species. PilE, the pilin subunit of Neisseria gonorrhoeae (Ngo) type IV pili undergoes O-linked glycosylation. A set of Pgl (pilin glycosylation) proteins responsible for the synthesis, membrane translocation and transfer of the glycan to PilE have been identified. Genetic analyses and complementation show that this system is remarkably similar to that of Cje save for the use of a Ser-directed oligosaccharyltransferase (Otase) rather than an Asn-directed Otase. We now show that Ngo glycosylates multiple proteins and have gone on to unambiguously identify eight of these additional substrates. Like in Cje, targets of the Ngo glycosylation system are destined to function periplasmically. Interestingly, several of the glycosylated proteins in Ngo are functionally related and seem to be involved in electron transfer and maintenance of the periplasmic redox state. We have further shown that the sites of covalent modification were associated with regions of low complexity and features analogous to those influencing target selection in both eukaryotic mucin-type glycosylation and mycobacterial protein mannosylation systems. These findings highlight previously unappreciated similarities in both O- and N-linked systems in prokaryotes and in conserved features dictating O-linked occupancy sites in prokaryotic and mammalian proteins.