Activities of the Porphyromonas gingivalis PrtP proteinase determined by construction of prtP-deficient mutants and expression of the gene in Bacteroides species

PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene e ncoding this proteinase, prtP, was cloned into the Escherichia coli-Bactero ides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cel ls, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was det ected by fibrinogen zymography in cells or spent growth medium of several B acteroides species harboring the recombinant plasmids. The proteinase was r ecovered from Bacteroides fragilis ATCC 25285(pFD340-prtP) cells by 3-[(3-c holamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS) extraction and characterized with regard to exopeptidase specificity and sensitivity to p roteinase inhibitors. Lys-amidolytic activity, but not Arg-amidolytic activ ity, was detected. PrtP was activated by cysteine and, to a lesser extent, dithiothreitol, and it was stimulated by glycine containing compounds. It a lso was inhibited by N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) an d, to a lesser extent, H-D-Tyr-L-Pro-L-arginyl chloromethyl ketone (YPRCK) and was relatively insensitive to EDTA and leupeptin. Neither B. fragilis A TCC 25285(pFD340-prtP) cells nor the CHAPS extract effected hemagglutinatio n of sheep red blood cells or collagen cleavage, but the cells did cleave g elatin. Furthermore, P. gingivalis W12, ATCC 33277, KDP110, and HG66 with k nockout mutations in prtP were constructed by allelic replacement. Unlike t he parent strains, the mutant strains produced beige colonies on plates con taining sheep blood. These strains also were affected in their ability to e ffect hemagglutination, cleave collagen, and cleave a Lys-specific peptide substrate. This report presents the results of the first characterization o f the PrtP proteinase clearly in the absence of any influence by other P. g ingivalis proteins and describes the properties of P. gingivalis cells defe ctive in the production of PrtP.