Paul D. Veith

Prevotella intermedia , a Gram‐negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O ‐glycosylation system. The O ‐glycoproteome has been characterized in several species, including Tannerella forsythia , Porphyromonas gingivalis , and Flavobacterium johnsoniae . In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O ‐glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia . We observed an electron‐dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron‐dense or translucent. LC‐MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O ‐glycosylation sites within 224 glycoproteins. Interestingly, the O ‐glycosylation motif exhibited a broader range than reported in other species, with O ‐glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O ‐glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision‐induced dissociation and high‐energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O ‐glycan sequence was confirmed to be dHex‐dHex‐HexNAc (HPO 3 ‐C 6 H 12 O 5 )‐dHex‐Hex‐HexA‐Hex(dHex). Bioinformatic analyses predicted the localization of O ‐glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.