Figure 7. PorU (PG0026), an extracellular component of T9SS in P. gingivalis, is required for proRgpB processing.

(A) P. gingivalis strains ΔK/ΔRAB, W83 and ΔPorU at early stationary phase were adjusted to OD₆₀₀=1.5 and centrifuged for 10 min at 5,000 × g. Bacteria were resuspended in RPMI media complemented with 5 mM DTT and 50 μM of gingipain-specific inhibitors KYT-1 and KYT-36. After 15 min, proRgpB6His (0.4 mg/ml) was added and incubated at room temperature. At various time, aliquots were removed to follow the maturation of the enzyme by Western blotting with the pAb anti-RgpB. The final products obtained after 96 hours of incubation were also analysed by Western blotting with mAb anti-6His and pAb anti-RgpBCTD domain. Mature RgpB bearing a C-terminal HisTag (lane M) was used as a control. (B) Comparison of RgpB-WT (expressed in the W83 strain) and RgpBC473A (expressed in the ΔRgpA strain) maturation by Western blot analysis with pAb anti-RgpB (WC, whole culture; WCE, whole cell extract; M, media; OMV, outer-membrane vesicles)