Outer membrane vesicles of Tannerella forsythia: biogenesis, composition, and virulence.

Tannerella forsythia is the only 'red-complex' bacterium covered by an S-layer, which has been shown to affect virulence. Here, outer membrane vesicles (OMVs) enriched with putative glycoproteins are described as a new addition to the virulence repertoire of T. forsythia. Investigations of this bacterium are hampered by its fastidious growth requirements and the recently discovered mismatch of the available genome sequence (92A2 = ATCC BAA-2717) and the widely used T. forsythia strain (ATCC 43037). T. forsythia was grown anaerobically in serum-free medium and biogenesis of OMVs was analyzed by electron and atomic force microscopy. This revealed OMVs with a mean diameter of ~100 nm budding off from the outer membrane while retaining the S-layer. An LC-ESI-TOF/TOF proteomic analysis of OMVs from three independent biological replicates identified 175 proteins. Of these, 14 exhibited a C-terminal outer membrane translocation signal that directs them to the cell/vesicle surface, 61 and 53 were localized to the outer membrane and periplasm, respectively, 22 were predicted to be extracellular, and 39 to originate from the cytoplasm. Eighty proteins contained the Bacteroidales O-glycosylation motif, 18 of which were confirmed as glycoproteins. Release of pro-inflammatory mediators from the human monocytic cell line U937 and periodontal ligament fibroblasts upon stimulation with OMVs followed a concentration-dependent increase that was more pronounced in the presence of soluble CD14 in conditioned media. The inflammatory response was significantly higher than that caused by whole T. forsythia cells. Our study represents the first characterization of T. forsythia OMVs, their proteomic composition and immunogenic potential.

The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation.

Conserved C-terminal domains (CTD) have been shown to act as a signal for the translocation of certain proteins across the outer membrane of Bacteroidetes via a type IX secretion system (T9SS). The genome sequence of the periodontal pathogen Tannerella forsythia predicts the presence of the components for a T9SS in conjunction with a suite of CTD proteins. T. forsythia is covered with a two-dimensional crystalline surface (S-) layer composed of the glycosylated CTD proteins TfsA and TfsB. To investigate, if T9SS is functional in T. forsythia, T9SS-deficient mutants were generated by targeting either TF0955 (putative C-terminal signal peptidase) or TF2327 (PorK ortholog), and the mutants were analyzed with respect to secretion, assembly and glycosylation of the S-layer proteins as well as proteolytic processing of the CTD and biofilm formation. In either mutant, TfsA and TfsB were incapable of translocation, as evidenced by the absence of the S-layer in transmission electron microscopy of ultrathin-sectioned bacterial cells. Despite being entrapped within the periplasm, mass spectrometry analysis revealed that the S-layer proteins were modified with the complete, mature glycan found on the secreted proteins, indicating that protein translocation and glycosylation are two independent processes. Further, the T9SS mutants showed a denser biofilm with fewer voids compared with the wild-type. This study demonstrates the functionality of T9SS and the requirement of CTD for the outer membrane passage of extracellular proteins in T. forsythia, exemplified by the two S-layer proteins. In addition, T9SS protein translocation is decoupled from O-glycan attachment in T. forsythia.