title: The S-layer proteins of Tannerella forsythia are secreted via a type IX secretion system that is decoupled from protein O-glycosylation.
abstract: 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.
authors.affiliation: Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Vienna, Austria.
authors.forename: M B
authors.initials: MB
authors.lastname: Tomek
authors.forename: L
authors.initials: L
authors.lastname: Neumann
authors.forename: I
authors.initials: I
authors.lastname: Nimeth
authors.forename: A
authors.initials: A
authors.lastname: Koerdt
authors.forename: P
authors.initials: P
authors.lastname: Andesner
authors.forename: P
authors.initials: P
authors.lastname: Messner
authors.forename: L
authors.initials: L
authors.lastname: Mach
authors.forename: J S
authors.initials: JS
authors.lastname: Potempa
authors.forename: C
authors.initials: C
authors.lastname: Schäffer
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000502:physiology*; D041963:Bacteroidetes / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018441:Biofilms / Q000254:growth & development; D055786:Gene Knockout Techniques; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D008562:Membrane Glycoproteins; C055764:S-layer proteins
keywords: C-terminal domain; C-terminal signal peptidase; S-layer glycosylation; T9SS; protein secretion