PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
 
Infect Immun. 1994 August; 62(8): 3424–3433.
PMCID: PMC302974

Cloning and sequence analysis of a chymotrypsinlike protease from Treponema denticola.

Abstract

A clone expressing a Treponema denticola chymotrypsinlike protease from recombinant plasmid pSA2 was identified in a genomic library of T. denticola ATCC 35405. Nucleotide sequencing of the insert identified an open reading frame, designated the prtB gene, which codes for the protease. Two potential inverted repeat sequences are present both upstream and downstream from the prtB gene. The prtB gene would code for a putative protein of 273 amino acids with a calculated molecular mass of 30.4 kDa and an estimated pI of 7.0. The G+C content of the gene is 40.3%. The results of maxicell analysis are consistent with the expression of a 30-kDa protease from the prtB gene. Preliminary characterization of the protease indicated that it was inhibited by the protease inhibitors phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and N-tosyl-L-phenylalanine chloromethyl ketone but not by N alpha-p-tosyl-L-lysine chloromethyl ketone. Purification of the protease was accomplished with the PinPoint protein purification system following construction of site-directed mutagenized plasmid pXa-3:2. The purified protease degraded human and bovine serum albumins as well as casein. Furthermore, hemolysis of sheep erythrocytes by the protease was observed. Northern (RNA) blot analysis of mRNA extracted from strain 35405 indicated a single 1.9-kb mRNA species containing the prtB transcript. In addition, the results of primer extension analysis indicated that transcription was initiated primarily at a T residue. However, no corresponding -10 and -35 sequences related to Escherichia coli promoter sequences were identified. The availability of the purified protein and its gene will aid in evaluating the potential role of the protease in the physiology and virulence of T. denticola since proteases may play a key role in oral treponemal pathogenicity.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.4M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Baehni PC, Song M, McCulloch CA, Ellen RP. Treponema denticola induces actin rearrangement and detachment of human gingival fibroblasts. Infect Immun. 1992 Aug;60(8):3360–3368. [PMC free article] [PubMed]
  • Boehringer H, Berthold PH, Taichman NS. Studies on the interaction of human neutrophils with plaque spirochetes. J Periodontal Res. 1986 May;21(3):195–209. [PubMed]
  • Boehringer H, Taichman NS, Shenker BJ. Suppression of fibroblast proliferation by oral spirochetes. Infect Immun. 1984 Jul;45(1):155–159. [PMC free article] [PubMed]
  • Cheng SL, Chan EC. The routine isolation, growth, and maintenance of the intermediate-size anaerobic oral spirochetes from periodontal pockets. J Periodontal Res. 1983 Jul;18(4):362–368. [PubMed]
  • Dzink JL, Socransky SS, Haffajee AD. The predominant cultivable microbiota of active and inactive lesions of destructive periodontal diseases. J Clin Periodontol. 1988 May;15(5):316–323. [PubMed]
  • Frank RM. Bacterial penetration in the apical pocket wall of advanced human periodontitis. J Periodontal Res. 1980 Nov;15(6):563–573. [PubMed]
  • Grenier D. Characteristics of hemolytic and hemagglutinating activities of Treponema denticola. Oral Microbiol Immunol. 1991 Aug;6(4):246–249. [PubMed]
  • Grenier D, Uitto VJ, McBride BC. Cellular location of a Treponema denticola chymotrypsinlike protease and importance of the protease in migration through the basement membrane. Infect Immun. 1990 Feb;58(2):347–351. [PMC free article] [PubMed]
  • Haapasalo M, Müller KH, Uitto VJ, Leung WK, McBride BC. Characterization, cloning, and binding properties of the major 53-kilodalton Treponema denticola surface antigen. Infect Immun. 1992 May;60(5):2058–2065. [PMC free article] [PubMed]
  • Koopman MB, de Leeuw OS, van der Zeijst BM, Kusters JG. Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein. Infect Immun. 1992 Jul;60(7):2920–2925. [PMC free article] [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • LISTGARTEN MA. ELECTRON MICROSCOPIC OBSERVATIONS ON THE BACTERIAL FLORA OF ACUTE NECROTIZING ULCERATIVE GINGIVITIS. J Periodontol. 1965 Jul-Aug;36:328–339. [PubMed]
  • Listgarten MA. Direct microscopy of periodontal pathogens. Oral Microbiol Immunol. 1986 Nov;1(1):31–38. [PubMed]
  • Listgarten MA, Helldén L. Relative distribution of bacteria at clinically healthy and periodontally diseased sites in humans. J Clin Periodontol. 1978 May;5(2):115–132. [PubMed]
  • Listgarten MA, Levin S. Positive correlation between the proportions of subgingival spirochetes and motile bacteria and susceptibility of human subjects to periodontal deterioration. J Clin Periodontol. 1981 Apr;8(2):122–138. [PubMed]
  • Loesche WJ. The role of spirochetes in periodontal disease. Adv Dent Res. 1988 Nov;2(2):275–283. [PubMed]
  • Loesche WJ, Giordano J, Hujoel PP. The utility of the BANA test for monitoring anaerobic infections due to spirochetes (Treponema denticola) in periodontal disease. J Dent Res. 1990 Oct;69(10):1696–1702. [PubMed]
  • Loesche WJ, Syed SA, Stoll J. Trypsin-like activity in subgingival plaque. A diagnostic marker for spirochetes and periodontal disease? J Periodontol. 1987 Apr;58(4):266–273. [PubMed]
  • MacDougall JH, Beighton D, Russell RR. Cloning and expression of protease genes from Treponema denticola in Escherichia coli. Oral Microbiol Immunol. 1991 Oct;6(5):270–274. [PubMed]
  • Mayrand D, Holt SC. Biology of asaccharolytic black-pigmented Bacteroides species. Microbiol Rev. 1988 Mar;52(1):134–152. [PMC free article] [PubMed]
  • Mikx FH, de Jong MH. Keratinolytic activity of cutaneous and oral bacteria. Infect Immun. 1987 Mar;55(3):621–625. [PMC free article] [PubMed]
  • Mikx FH, Jacobs F, Satumalay C. Cell-bound peptidase activities of Treponema denticola ATCC 33520 in continuous culture. J Gen Microbiol. 1992 Sep;138(9):1837–1842. [PubMed]
  • Mikx FH, Maltha JC, van Campen GJ. Spirochetes in early lesions of necrotizing ulcerative gingivitis experimentally induced in beagles. Oral Microbiol Immunol. 1990 Apr;5(2):86–89. [PubMed]
  • Mikx FH, Ngassapa DN, Reijntjens FM, Maltha JC. Effect of splint placement on black-pigmented Bacteroides and spirochetes in the dental plaque of beagle dogs. J Dent Res. 1984 Nov;63(11):1284–1288. [PubMed]
  • Miyamoto M, Noji S, Kokeguchi S, Kato K, Kurihara H, Murayama Y, Taniguchi S. Molecular cloning and sequence analysis of antigen gene tdpA of Treponema denticola. Infect Immun. 1991 Jun;59(6):1941–1947. [PMC free article] [PubMed]
  • Moore LV, Moore WE, Cato EP, Smibert RM, Burmeister JA, Best AM, Ranney RR. Bacteriology of human gingivitis. J Dent Res. 1987 May;66(5):989–995. [PubMed]
  • Moore WE, Holdeman LV, Cato EP, Smibert RM, Burmeister JA, Palcanis KG, Ranney RR. Comparative bacteriology of juvenile periodontitis. Infect Immun. 1985 May;48(2):507–519. [PMC free article] [PubMed]
  • Nitzan D, Sperry JF, Wilkins TD. Fibrinolytic activity of oral anaerobic bacteria. Arch Oral Biol. 1978;23(6):465–470. [PubMed]
  • Ohta K, Makinen KK, Loesche WJ. Purification and characterization of an enzyme produced by Treponema denticola capable of hydrolyzing synthetic trypsin substrates. Infect Immun. 1986 Jul;53(1):213–220. [PMC free article] [PubMed]
  • Olsen I. Attachment of Treponema denticola to cultured human epithelial cells. Scand J Dent Res. 1984 Feb;92(1):55–63. [PubMed]
  • Oosterwaal PJ, Matee MI, Mikx FH, van 't Hof MA, Renggli HH. The effect of subgingival debridement with hand and ultrasonic instruments on the subgingival microflora. J Clin Periodontol. 1987 Oct;14(9):528–533. [PubMed]
  • Pabo CO, Lewis M. The operator-binding domain of lambda repressor: structure and DNA recognition. Nature. 1982 Jul 29;298(5873):443–447. [PubMed]
  • Pabo CO, Sauer RT. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. [PubMed]
  • Park Y, McBride BC. Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product. FEMS Microbiol Lett. 1992 May 1;71(3):273–278. [PubMed]
  • Que XC, Kuramitsu HK. 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. Infect Immun. 1990 Dec;58(12):4099–4105. [PMC free article] [PubMed]
  • Reijntjens FM, Mikx FH, Wolters-Lutgerhorst JM, Maltha JC. Adherence of oral treponemes and their effect on morphological damage and detachment of epithelial cells in vitro. Infect Immun. 1986 Feb;51(2):642–647. [PMC free article] [PubMed]
  • Saglie R, Newman MG, Carranza FA, Jr, Pattison GL. Bacterial invasion of gingiva in advanced periodontitis in humans. J Periodontol. 1982 Apr;53(4):217–222. [PubMed]
  • Sancar A, Hack AM, Rupp WD. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. [PMC free article] [PubMed]
  • Sancar A, Rupert CS. Determination of plasmid molecular weights from ultraviolet sensitivities. Nature. 1978 Mar 30;272(5652):471–472. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PubMed]
  • Savitt ED, Socransky SS. Distribution of certain subgingival microbial species in selected periodontal conditions. J Periodontal Res. 1984 Mar;19(2):111–123. [PubMed]
  • Schmidt EF, Bretz WA, Hutchinson RA, Loesche WJ. Correlation of the hydrolysis of benzoyl-arginine naphthylamide (BANA) by plaque with clinical parameters and subgingival levels of spirochetes in periodontal patients. J Dent Res. 1988 Dec;67(12):1505–1509. [PubMed]
  • Slots J, Genco RJ. 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. [PubMed]
  • Socransky SS, Haffajee AD. Effect of therapy on periodontal infections. J Periodontol. 1993 Aug;64(8 Suppl):754–759. [PubMed]
  • Uitto VJ, Chan EC, Quee TC. Initial characterization of neutral proteinases from oral spirochetes. J Periodontal Res. 1986 Mar;21(2):95–100. [PubMed]
  • Uitto VJ, Grenier D, Chan EC, McBride BC. Isolation of a chymotrypsinlike enzyme from Treponema denticola. Infect Immun. 1988 Oct;56(10):2717–2722. [PMC free article] [PubMed]
  • Wallich R, Moter SE, Simon MM, Ebnet K, Heiberger A, Kramer MD. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect Immun. 1990 Jun;58(6):1711–1719. [PMC free article] [PubMed]
  • Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)