Altered expression and modification of proteases from an avirulent mutant of Porphyromonas gingivalis W50 (W50/BE1)

Abstract

Proteases ofPorphyromonas gingivalisare considered to be important factors in the virulence of this organism. A non-pigmenting mutant ofP. gingivalisW50 (W50/BE1) has been shown to be less virulent in animal models and to produce significantly less Arg-specific protease activity than the parent strain. Three proteases are present in the culture supernatant ofP. gingivalisW50: RI, RIA and RIB. All three proteases are derived fromprpR1,which encodes a polypeptide of 1706 amino acids that is organized into distinct domains (pro, α, β and γ). The aim of the present investigation was to purify and characterize the Arg-specific proteases produced by the avirulent W50/BE1 strain. Significant differences were observed between the proteases ofP. gingivalisW50 and W50/BE1. The levels of RI present in the culture supernatant of W50/BE1 were lower than those present in W50, and RIA and RIB were absent. RI from W50/BE1 was composed of three polypeptide chains, unlike the enzyme from W50, which is a heterodimer. The remainder of the Arg-specific protease activity in W50/BE1 was derived from a second gene,prR2,and was present in two fractions, RIIAs/BE (soluble) and RIIAv/BE (vesicle-bound). This activity contained two peptide chains: a ∼ 54 kDa chain corresponding to the protease domain and a ∼ 26 kDa chain, derived from the propeptide domain of the PrRII precursor. No enzyme with large glycan additions, equivalent to RIB in the vesicle fraction of the wild-type W50, was present. These data indicate that the reduced level of extracellular protease activity in W50/BE1 reflects reduced synthesis and/or export ofprpR1enzymes, which is only partially compensated by synthesis ofprR2-derived enzymes, and that all of these proteases undergo altered post-translational modification compared to the parent strain.