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. 1990 Dec;58(12):4099–4105. doi: 10.1128/iai.58.12.4099-4105.1990

Isolation and characterization of the Treponema denticola prtA gene coding for chymotrypsinlike protease activity and detection of a closely linked gene encoding PZ-PLGPA-hydrolyzing activity.

X C Que 1, H K Kuramitsu 1
PMCID: PMC313782  PMID: 2174832

Abstract

The chymotrypsinlike protease gene (prtA) from Treponema denticola ATCC 35405 was isolated from a lambda gt11 clone bank as one of several clones expressing protease activity. The DNA from one positive clone capable of hydrolyzing type IV collagen was subcloned into plasmid vector pUC119 for further analysis. Deletion analysis of subclone pXQ27.2 revealed the approximate location of the prtA gene on the DNA insert. DEAE-Sephadex chromatography of crude cell extracts of the subclone revealed two distinct T. denticola enzymes, one hydrolyzing SAAPNA (succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide [chymotrypsin substrate]) and the other hydrolyzing PZ-PLGPA (phenylazobenzyl-oxycarbonyl-L-leucylglycyl-L-prolyl-D -arginine [collagenase substrate]). Each activity was purified to near homogeneity and exhibited by sodium dodecyl sulfate-polyacrylamide gel electrophoresis estimated molecular sizes of 67 and 36 kDa, respectively. Western blot (immunoblot) analysis demonstrated that only the 67-kDa SAAPNA-hydrolyzing enzyme reacted with antibody against the T. denticola chymotrypsinlike protease. The purified SAAPNA-hydrolyzing enzyme degraded type IV collagen, laminin, and fibronectin, but not type I collagen. These results indicate that the prtA gene coding for the chymotrypsinlike protease from T. denticola has been isolated. Another distinct gene encoding an enzyme hydrolyzing PZ-PLGPA appears to be adjacent to the prtA gene.

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Selected References

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  1. Allenspach-Petrzilka G. E., Guggenheim B. Bacterial invasion of the periodontium; an important factor in the pathogenesis of periodontitis? J Clin Periodontol. 1983 Nov;10(6):609–617. doi: 10.1111/j.1600-051x.1983.tb01299.x. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Chavira R., Jr, Burnett T. J., Hageman J. H. Assaying proteinases with azocoll. Anal Biochem. 1984 Feb;136(2):446–450. doi: 10.1016/0003-2697(84)90242-2. [DOI] [PubMed] [Google Scholar]
  4. Cheng S. L., Siboo R., Quee T. C., Johnson J. L., Mayberry W. R., Chan E. C. Comparative study of six random oral spirochete isolates. Serological heterogeneity of Treponema denticola. J Periodontal Res. 1985 Nov;20(6):602–612. doi: 10.1111/j.1600-0765.1985.tb00844.x. [DOI] [PubMed] [Google Scholar]
  5. Doi E., Shibata D., Matoba T. Modified colorimetric ninhydrin methods for peptidase assay. Anal Biochem. 1981 Nov 15;118(1):173–184. doi: 10.1016/0003-2697(81)90175-5. [DOI] [PubMed] [Google Scholar]
  6. Grenier D., Chao G., McBride B. C. Characterization of sodium dodecyl sulfate-stable Bacteroides gingivalis proteases by polyacrylamide gel electrophoresis. Infect Immun. 1989 Jan;57(1):95–99. doi: 10.1128/iai.57.1.95-99.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jacobs W. R., Barrett J. F., Clark-Curtiss J. E., Curtiss R., 3rd In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries. Infect Immun. 1986 Apr;52(1):101–109. doi: 10.1128/iai.52.1.101-109.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kaminishi H., Hagihara Y., Hayashi S., Cho T. Isolation and characteristics of collagenolytic enzyme produced by Candida albicans. Infect Immun. 1986 Aug;53(2):312–316. doi: 10.1128/iai.53.2.312-316.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Mayrand D., Grenier D. Detection of collagenase activity in oral bacteria. Can J Microbiol. 1985 Feb;31(2):134–138. doi: 10.1139/m85-026. [DOI] [PubMed] [Google Scholar]
  11. Mäkinen K. K., Syed S. A., Loesche W. J., Mäkinen P. L. Proteolytic profile of Treponema vincentii ATCC 35580 with special reference to collagenolytic and arginine aminopeptidase activity. Oral Microbiol Immunol. 1988 Sep;3(3):121–128. doi: 10.1111/j.1399-302x.1988.tb00096.x. [DOI] [PubMed] [Google Scholar]
  12. Nethery A., Lyons J. G., O'Grady R. L. A spectrophotometric collagenase assay. Anal Biochem. 1986 Dec;159(2):390–395. doi: 10.1016/0003-2697(86)90358-1. [DOI] [PubMed] [Google Scholar]
  13. Ohta K., Makinen K. K., Loesche W. J. Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect Immun. 1986 Jul;53(1):213–220. doi: 10.1128/iai.53.1.213-220.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Reynolds E. C., Riley P. F. Protein dissimilation by human salivary-sediment bacteria. J Dent Res. 1989 Feb;68(2):124–129. doi: 10.1177/00220345890680020501. [DOI] [PubMed] [Google Scholar]
  15. Simonson L. G., Goodman C. H., Bial J. J., Morton H. E. Quantitative relationship of Treponema denticola to severity of periodontal disease. Infect Immun. 1988 Apr;56(4):726–728. doi: 10.1128/iai.56.4.726-728.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Slots J., Genco R. J. Black-pigmented Bacteroides species, Capnocytophaga species, and Actinobacillus actinomycetemcomitans in human periodontal disease: virulence factors in colonization, survival, and tissue destruction. J Dent Res. 1984 Mar;63(3):412–421. doi: 10.1177/00220345840630031101. [DOI] [PubMed] [Google Scholar]
  17. Uitto V. J., Grenier D., Chan E. C., McBride B. C. Isolation of a chymotrypsinlike enzyme from Treponema denticola. Infect Immun. 1988 Oct;56(10):2717–2722. doi: 10.1128/iai.56.10.2717-2722.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Uitto V. J., Haapasalo M., Laakso T., Salo T. Degradation of basement membrane collagen by proteases from some anaerobic oral micro-organisms. Oral Microbiol Immunol. 1988 Sep;3(3):97–102. doi: 10.1111/j.1399-302x.1988.tb00092.x. [DOI] [PubMed] [Google Scholar]
  19. WUENSCH E., HEIDRICH H. G. ZUR QUANTITATIVEN BESTIMMUNG DER KOLLAGENASE. Hoppe Seylers Z Physiol Chem. 1963;333:149–151. doi: 10.1515/bchm2.1963.333.1.149. [DOI] [PubMed] [Google Scholar]
  20. Whaley D. N., Dowell V. R., Jr, Wanderlinder L. M., Lombard G. L. Gelatin agar medium for detecting gelatinase production by anaerobic bacteria. J Clin Microbiol. 1982 Aug;16(2):224–229. doi: 10.1128/jcm.16.2.224-229.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]

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