Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities

Aduse-Opoku, Joseph; Davies, Nyama N.; Gallagher, Alex; Hashim, Ahmed; Evans, Helen E. A.; Rangarajan, Minnie; Slaney, J. M.; Curtis, Michael A.

doi:10.1099/00221287-146-8-1933

Cited by 62 publications

(64 citation statements)

References 37 publications

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“…It was found that leupeptin inhibited the cleavage of all substrates, except for BikKam14, the only substrate in which no arginine residue is present (Table 4). This finding is in agreement with findings by Aduse-Opoku and coworkers, who observed inhibition of Arg-gingipain activity in the presence of leupeptin, whereas no effect on Lys-gingipain activity was observed (1). A literature search revealed that the amidolytic activity of both Argand Lys-gingipain on L-BApNA is stimulated by the addition of glycyl-glycine (5, 7).…”

Section: Figsupporting

confidence: 82%

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Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

et al. 2012

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Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain D-amino acids, led to the discovery of five P. gingivalisspecific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and L-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10 5 CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of L-amino acids and Bz-L-Arg-NHPhNO 2 showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single D-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of D-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

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Section: Figsupporting

confidence: 82%

“…1). In addition to the absence of Arg-gingipain in ⌬Rgp, it is known that ⌬Rgp strains secrete Lys-gingipain less efficiently (1,41). This was confirmed by the decreased proteolytic activity of ⌬Rgp culture supernatant on the BikKam14 substrate (Fig.…”

Section: Figmentioning

confidence: 54%

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

et al. 2012

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“…or 1i10"" c.f.u. of P. gingivalis W50, the isogenic luxS mutant or the kgp mutant K1A (Aduse-Opoku et al, 2000). K1A is known to be attenuated in this animal model (M. A. Curtis, unpublished data).…”

Section: N a Burgess And Othersmentioning

confidence: 99%

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence

et al. 2002

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Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either Nacylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5α. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the midexponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5α transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45 % less Rgp and 30 % less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.

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“…53978) and the mutant W50 strain K1A (kgp : :erm), lacking the Lys-Xaaspecific proteinase Kgp [19], were obtained from the culture collection of the School of Dental Science, The University of Melbourne. Strains W50, K1A and W50AB (see below) were grown anaerobically and maintained ( 10 passages) on horse blood agar (HBA ; Oxoid Blood Agar Base 2, Oxoid, Basingstoke, U.K.) supplemented with 10 % (v\v) defibrinated horse blood, with weekly subculturing.…”

Section: Bacterial Strains and Batch Culture Growth Conditionsmentioning

confidence: 99%

“…The residue preceding the N-terminus of most of the domains is arginine, suggesting the involvement of the arginine-specific proteinases, RgpA and RgpB, in N-terminal processing. To investigate the involvement of these enzymes in N-and Cterminal processing, OMs were prepared from mutant strains lacking either a functional Kgp (K1A) [19] or both RgpA and RgpB in a double mutant (W50AB) that was constructed by insertional inactivation for this study. The correct insertion of the antibiotic-resistance cassettes into the rgpA and rgpB genes in W50AB was confirmed by Southern blot analysis (results not shown).…”

Section: Proteolytic Processing Of Rgpa Kgp and Hagamentioning

confidence: 99%

Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50

Veith

Talbo

Slakeski

et al. 2002

Biochemical Journal

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Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16–57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13–42kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.

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Generation of Lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities

Cited by 62 publications

References 37 publications

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

Highly Specific Protease-Based Approach for Detection of Porphyromonas gingivalis in Diagnosis of Periodontitis

LuxS-dependent quorum sensing in Porphyromonas gingivalis modulates protease and haemagglutinin activities but is not essential for virulence

Major outer membrane proteins and proteolytic processing of RgpA and Kgp of Porphyromonas gingivalis W50

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