Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis

Otsuka, Makoto; Hinode, Daisuke; Nagata, Atsushi; Maehara, Reiko; Sato, Mitsunobu; Nakamura, Ryô

doi:10.1111/j.1600-0765.1987.tb02060.x

Cited by 64 publications

(32 citation statements)

References 19 publications

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“…Several proteinases with specificities similar to Arg-gingipain have been purified from various P. gingivalis strains (20,28,30,(47)(48)(49)(50)(51)(52). In this study, we found that all of the enzymatic activity for arginine-specific cysteine proteinase in P. gingivalis ATCC33277 might be derived from the rgpA and rgpB, as suggested by the total loss of the activity in the rgpA rgpB mutant.…”

Section: Discussionmentioning

confidence: 63%

Construction and Characterization of Arginine-specific Cysteine Proteinase (Arg-gingipain)-deficient Mutants of Porphyromonas gingivalis

Nakayama

Kadowaki

Okamoto

et al. 1995

Journal of Biological Chemistry

229

247

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Arginine-specific cysteine proteinase (Arg-gingipain; formerly, argingipain) is one of the major extracellular proteinases produced by the oral anaerobic bacterium Porphyromonas gingivalis. To determine whether Arggingipain is important for periodontopathogenicity of the organism, Arg-gingipain-deficient mutants were constructed via gene disruption by use of suicide plasmid systems. First, Southern hybridization analyses suggested that two separate Arg-gingipain-encoding genes designated rgpA and rgpB existed on 12.5-and 7.8-kilobase pair HindIII chromosomal fragments of P. gingivalis ATCC33277, respectively. rgpA and rgpB single mutants were constructed by mobilization of a suicide plasmid. Then, an rgpA rgpB double mutant was isolated by electroporation with a second suicide plasmid. No proteolytic activity for Arg-gingipain was observed in either the cell extract or the culture supernatant of the rgpA rgpB mutant. The chemiluminescence response of polymorphonuclear leukocytes, which is closely related to their bactericidal function, was not inhibited by the culture supernatant of the rgpA rgpB mutant, while the wild type parent showed a significant inhibition of the response. The result suggests that Arggingipain is responsible for disruption of the function of polymorphonuclear leukocytes. In addition, the rgpA rgpB double mutations caused a marked decrease in the hemagglutination of P. gingivalis, indicating that a major part of the hemagglutinin activity of the organism is associated with the two genes. These findings demonstrate that Arg-gingipain makes a significant contribution to the virulence of P. gingivalis.

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Section: Discussionmentioning

confidence: 63%

Construction and Characterization of Arginine-specific Cysteine Proteinase (Arg-gingipain)-deficient Mutants of Porphyromonas gingivalis

Nakayama

Kadowaki

Okamoto

et al. 1995

Journal of Biological Chemistry

229

247

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“…Another consequence of the presence of B. gingivalis in saliva is the possible degradation of protective salivary proteins. Recently, it has been reported that B. gingivalis produces a protease which is capable of degradation and inactivation of salivary lysozyme (5). Therefore, the presence of B. gingivalis in the saliva may weaken the oral antibacterial properties.…”

Section: Discussionmentioning

confidence: 99%

Intra‐oral distribution of black‐pigmented Bacteroides species in periodontitis patients

Winkelhoff

Velden

Clement

et al. 1988

Oral Microbiology and Immunology

113

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In this study the occurrence of black‐pigmented Bacteroides on oral mucous membranes and in saliva was investigated. For this purpose untreated adult periodontitis patients were selected on the basis of clinical parameters. Bacteroides gingivalis was isolated from the subgingival area of all 8 patients. Other sites from which B. gingivalis was recovered were the tonsillar area, the gingiva and the tongue. Six of the 8 patients had B. gingivalis in saliva in numbers ranging from 6 × 106 to 20 × 106/ml. Bacteroides intermedius was also widespread through the oral cavity, including the saliva. The results show that these black‐pigmented Bacteroides species do occur outside the periodontal pocket and that the intra‐oral spread of these bacteria can take place via saliva.

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“…Three peaks of activity against the two substrates and reactivity to specific antibodies were found. The Rgpspecific peak fractions were pooled, concentrated, and applied to an arginineSepharose column according to the protocol described by Otsuka et al (30), with modified elution volumes of 75 ml. The fractions were pooled and used for further analysis.…”

Section: Methodsmentioning

confidence: 99%

Gingipain RgpB Is Excreted as a Proenzyme in the vimA -Defective Mutant Porphyromonas gingivalis FLL92

et al. 2003

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We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp-and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturationrenaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobic bacterium, is an important etiological agent of chronic adult periodontitis (reviewed in references 23, 25, and 43) and is associated with other systemic diseases, including atherosclerosis (reviewed in reference 11). While several virulence factors have been implicated in the pathogenicity of P. gingivalis, the high-level proteolytic abilities of this organism have been the focus of much attention and appear to play an important role in its virulence. The major proteases, called gingipains, are both extracellular and cell associated. They consist of arginine-specific protease (Arg-gingipain [Rgp]) and lysine-specific protease (Lys-gingipain [Kgp]) (35). The Rgp is encoded by two genes, rgpA and rgpB, while Kgp is encoded by a single gene, kgp (28,33). In addition to being essential for growth, proteases have other functions. Several reports (reviewed in reference 22) have documented the multiple effects of proteases, including degradation of complement and immunoglobulin, inactivation of cytokines and their receptors, aggregation of platelets, attenuation of neutrophil antibacterial activities, and increases in vascular permeability and blood clotting prevention. Furthermore, these gingipains, which have been suggested...

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Isolation and characterization of protease from culture supernatant of Bacteroides gingivalis

Cited by 64 publications

References 19 publications

Construction and Characterization of Arginine-specific Cysteine Proteinase (Arg-gingipain)-deficient Mutants of Porphyromonas gingivalis

Construction and Characterization of Arginine-specific Cysteine Proteinase (Arg-gingipain)-deficient Mutants of Porphyromonas gingivalis

Intra‐oral distribution of black‐pigmented Bacteroides species in periodontitis patients

Gingipain RgpB Is Excreted as a Proenzyme in the vimA -Defective Mutant Porphyromonas gingivalis FLL92

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