Poster Presentation BacPath 13: Molecular Analysis of Bacterial Pathogens Conference 2015

Type IX secretion substrates are attached to the cell surface via a novel linker that is conjugated to the secreted proteins by a sortase-like mechanism (#139)

Paul D. Veith 1 , Dina Chen 1 , Michelle D. Glew 1 , Dhana G. Gorasia 1 , Eric C. Reynolds 1
  1. Oral Health Cooperative Research Centre, Melbourne Dental School, BIO21 Institute, University of Melbourne, Parkville, Victoria, Australia

Protein substrates (exoproteins) of the Type IX Secretion System (T9SS) in the Gram negative oral pathogen, Porphyromonas gingivalis have a conserved C-terminal signal that directs their secretion across the outer membrane. Once secreted, the signal is cleaved and the exoprotein is modified with an anchoring polymer that is related to anionic lipopolysaccharide (A-LPS) since it is recognised by the MAb-1B5 antibody that also recognises free A-LPS. The lipid A portion of A-LPS is embedded in the outer membrane, thereby providing an explanation for the attachment of exoproteins to the cell surface. While the structure of A-LPS has been published, the structure of the anchoring polymer has not yet been determined, nor has the site(s) of protein modification been elucidated. Examination of wbaP mutants that were unable to produce A-LPS revealed that the exoproteins were modified at their C-terminus with short peptides and amino acids present in the growth medium indicative of a transpeptidase or sortase-type reaction. To characterise the anchoring polymer, exoproteins partially purified from the wild type strain were first deglycosylated and then digested with either trypsin or proteinase K. MS analyses of the residual modification released by proteinase K cleavage or attached to peptide showed that the “linker” has a mass of 648 Da and is linked to the mature C-terminus of each exoprotein by a peptide bond. The 648 Da modification is novel and comprised of three major parts that are generated by fragmentation inside the mass spectrometer. The first part is 104 Da, the second 198 Da and third component is 346 Da and is hydrophobic. The potential structure of these components and their relationship to what is known about A-LPS biosynthesis will be discussed.