Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism.
The type IX secretion system (T9SS) of Porphyromonas gingivalis secretes proteins possessing a conserved C-terminal domain (CTD) to the cell surface. The C-terminal signal is essential for these proteins to translocate across the outer membrane via the T9SS. On the surface the CTD of these proteins is cleaved prior to extensive glycosylation. It is believed that the modification on these CTD proteins is anionic lipopolysaccharide (A-LPS), which enables the attachment of CTD proteins to the cell surface. However, the exact site of modification and the mechanism of attachment of CTD proteins to the cell surface are unknown. In this study we characterized two wbaP (PG1964) mutants that did not synthesise A-LPS and accumulated CTD proteins in the clarified culture fluid (CCF). The CTDs of the CTD proteins in the CCF were cleaved suggesting normal secretion, however, the CTD proteins were not glycosylated. Mass spectrometric analysis of CTD proteins purified from the CCF of the wbaP mutants revealed the presence of various peptide/amino acid modifications from the growth medium at the C-terminus of the mature CTD proteins. This suggested that modification occurs at the C-terminus of T9SS substrates in the wild type P. gingivalis. This was confirmed by analysis of CTD proteins from wild type, where a 648 Da linker was identified to be attached at the C-terminus of mature CTD proteins. Importantly, treatment with proteinase K released the 648 Da linker from the CTD proteins demonstrating a peptide bond between the C-terminus and the modification. Together, this is suggestive of a mechanism similar to sortase A for the cleavage and modification/attachment of CTD proteins in P. gingivalis. PG0026 has been recognized as the CTD signal peptidase and is now proposed to be the sortase-like protein in P. gingivalis. To our knowledge, this is the first biochemical evidence suggesting a sortase-like mechanism in Gram-negative bacteria.
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Dhana G
DG
Gorasia
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Paul D
PD
Veith
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Dina
D
Chen
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Christine A
CA
Seers
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Helen A
HA
Mitchell
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Yu-Yen
YY
Chen
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Michelle D
MD
Glew
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Stuart G
SG
Dashper
Oral Health Cooperative Research Centre, Melbourne Dental School, and The Bio21 Institute, The University of Melbourne, Victoria, Australia.
Eric C
EC
Reynolds
D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058947:Bacterial Secretion Systems / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K; D017353:Gene Deletion; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D016966:Porphyromonas gingivalis / Q000201:enzymology / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D058947:Bacterial Secretion Systems; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; D019286:Endopeptidase K; D003546:Cysteine Endopeptidases