title: Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.
abstract: The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.
authors: Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors: Dhana G
authors: DG
authors: Gorasia
authors: Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors: Paul D
authors: PD
authors: Veith
authors: Melbourne Advanced Microscopy Facility and Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.
authors: Eric G
authors: EG
authors: Hanssen
authors: Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors: Michelle D
authors: MD
authors: Glew
authors: Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors: Keiko
authors: K
authors: Sato
authors: Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors: Hideharu
authors: H
authors: Yukitake
authors: Division of Microbiology and Oral Infection, Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors: Koji
authors: K
authors: Nakayama
authors: Oral Health CRC, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Victoria, Australia.
authors: Eric C
authors: EC
authors: Reynolds
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism / Q000648:ultrastructure*; D058947:Bacterial Secretion Systems / Q000378:metabolism / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D016966:Porphyromonas gingivalis / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems