C-terminal glycosylation of type IX secretion system cargo proteins in with both short and long secretion signals.

is a Gram-negative bacterium that is associated with periodontitis and acute necrotizing ulcerative gingivitis. utilizes the type IX secretion system (T9SS) to secrete and anchor virulence factors to the cell surface, presumably via C-terminal glycosylation. The identity of the linking sugar and the sites of modification on the cargo are unknown. Here, we employed hidden Markov models to predict cargo proteins in and conducted LC-MS/MS analyses of partially deglycosylated fractions to characterize the C-terminal glycosylation. A total of 80 cargo proteins were predicted based on the presence of a T9SS C-terminal domain (CTD) signal, and these were divided into 48 short CTDs and 32 long CTDs. Cleavage sites for five short and four long CTDs were experimentally determined, and glycosylation was observed at the mature C-terminus of six cargo. Two glycans were identified of delta masses 419.198 and 433.185 Da, corresponding to novel C-terminal amide linkages to N-alanyl dHex-HexNAc and N-alanyl (Me-dHex)-HexNAc, respectively. This indicated that both short and long CTDs supported cleavage and glycosylation. AlphaFold multimer modelling predicted that both kinds of CTDs could bind to the PorV shuttle protein in the same manner, with the conserved CTD motifs interacting with the same sites in PorV.