Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

Biochimie. 2019 Nov;166:161-172. doi: 10.1016/j.biochi.2019.06.010. Epub 2019 Jun 15.

Abstract

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Keywords: Inhibition; Limited proteolysis; Periodontal disease; Posttranslational modification; Prodomain; Secretion; Sortase.

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism
  • Enzyme Precursors / metabolism*
  • Gingipain Cysteine Endopeptidases / chemistry*
  • Gingipain Cysteine Endopeptidases / metabolism*
  • Porphyromonas gingivalis / enzymology*
  • Porphyromonas gingivalis / metabolism*
  • Protein Processing, Post-Translational
  • Secretory Pathway

Substances

  • Bacterial Proteins
  • Enzyme Precursors
  • Gingipain Cysteine Endopeptidases