Porphyromonas gingivalis gingipain propeptides inhibit gingipains and bacterial growth

Objectives: This study aims to characterize the inhibitory potential of Kgp and RgpB propeptides against the mature cognate enzymes.

Methods: Mature Kgp was purified from a P. gingivalis Kgp adhesin-binding domain (ABM1) mutant which releases the Kgp catalytic domain (Kgp_catΔABM1) into the culture supernatant, allowing simpler purification. Purification of Kgp_catΔABM1 and RgpB from P. gingivalis strain HG66 was carried out at pH 5.3 to prevent enzyme degradation. Recombinant propeptides of Kgp and RgpB were produced in Escherichia coli and purified using nickel-affinity chromatography. The propeptides were incubated with the purified Kgp_catΔABM1 or RgpB with chromogenic or fluorescent substrates to monitor proteolytic activity. P. gingivalis growth assays were conducted in a protein-based medium.

Results: Both propeptides exhibited selectivity towards their cognate enzyme with 60-90% inhibition at an enzyme:inhibitor concentration of 1:10 with the rRgpB propeptide displaying stronger inhibitory activity. The rKgp and rRgpB propeptides displayed competitive inhibition kinetics with a K_i of 2.3 mM and 9 nM, respectively. Their specificity for the gingipains was demonstrated by their inability to inhibit papain, a closely related cysteine proteinase. Both propeptides at 100 mg/L caused a 50% reduction of P. gingivalis growth in the protein based medium.

Conclusions: Gingipain propeptides are capable of inhibiting mature gingipains; therefore the development of specific small peptide inhibitors based on cognate propeptide sequences may be a viable therapeutic strategy against pathogenic bacteria that rely on proteolytic activity for virulence.