Abstract
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.
Highlights
Chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth is a major public health problem
These proteins contain a C-terminal signal that allows their secretion through the T9SS and it is cleaved by the protein PG0026 at the cell surface
We identify a mechanism by which gingipains and other proteins attach to the cell surface of this bacterium
Summary
An inflammatory disease of the supporting tissues of the teeth is a major public health problem. It has been demonstrated that the CTD- containing proteins are secreted and attached to the cell surface via the type IX secretion system (T9SS) [9,10,11,12]. These CTD proteins have been found to be extensively modified and migrate as diffuse bands on SDS-PAGE with molecular weights at least 20 kDa higher than that predicted from their sequence [12, 13]. The A-LPS modification is thought to be the anchor that tethers CTD proteins to the cell surface [18], the mechanism of attachment is unknown
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