title: PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.
abstract: Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Heath
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Christine A
authors.initials: CA
authors.lastname: Seers
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Veith
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Butler
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Nor A
authors.initials: NA
authors.lastname: Nor Muhammad
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Yu-Yen
authors.initials: YY
authors.lastname: Chen
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Nada
authors.initials: N
authors.lastname: Slakeski
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Peng
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Lianyi
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Stuart G
authors.initials: SG
authors.lastname: Dashper
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Cross
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cleal
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Caroline
authors.initials: C
authors.lastname: Moore
authors.affiliation: Oral Health Cooperative Research Centre, Melbourne Dental School, Bio21 Institute, The University of Melbourne, Melbourne, Australia.
authors.forename: Eric C
authors.initials: EC
authors.lastname: Reynolds
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D058635:Lipid-Linked Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010641:Phenotype; D016966:Porphyromonas gingivalis / Q000235:genetics / Q000378:metabolism*; D000072417:Protein Domains; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D058635:Lipid-Linked Proteins; D008070:Lipopolysaccharides; D010447:Peptide Hydrolases