Spirochetes were isolated from the lesions of periodontitis patients, identified them as Treponema denticola and their properties were investigated. These strains required serum for growth and demonstrated low sugar fermentation activity. Indole, hydrogen sulfide, acetic acid, propionic acid and butyric acid were detected in the metabolites of all the isolates. Proteases, esterase and neuraminidase were also produced by the isolates, of which, proteolytic enzymes appeared to contain at least three different kinds. These proteases were possible to separate from each other by column chromatography

FUJIMURA SETSUO

HOSHINO TERUMUNE

NAKAMURA TAKESHI

application/pdfSpirochetes were isolated from the lesions of periodontitis patients, identified them as Treponema denticola and their properties were investigated. These strains required serum for growth and demonstrated low sugar fermentation activity. Indole, hydrogen sulfide, acetic acid, propionic acid and butyric acid were detected in the metabolites of all the isolates. Proteases, esterase and neuraminidase were also produced by the isolates, of which, proteolytic enzymes appeared to contain at least three different kinds. These proteases were possible to separate from each other by column chromatography.journal articl

HOSHINO, TERUMUNE

FUJIMURA, SETSUO

NAKAMURA, TAKESHI

Matsumoto Dental University Repository

(Original) Matsumoto Shigaku 22 : 8 A-12, 1996key words : T. denticola - isolation - properties - proteasesIsolation and Biological Properties of Oral Spirochete, Treponema denticolaSpirocTERUMUNE HOSHINO SETSUO FUJIMURA and TAKESHI NAKAMURA     DePatrment of Oral Microbiology, ?lfatsumoto Dental                    (Chief : Prof T. Nakamura)NA AMURA CollegeSummary    Spirochetes were isolated from the lesions of periodontitis patients, identified them asTroponema denticola and their properties were investigated. These strains required serumfor growth and demonstrated low sugar fermentation activity. Indole, hydrogen sulfide,acetic acid, propionic acid and butyric acid were detected in the metabolites of all theisolates. Proteases, esterase and neuraminidase were also produced by the isolates, ofwhich, proteolytic enzymes appeared to contain at least three different kinds. Theseproteases were possible to separate from each other by column chromatography.                                     Introduction   Even though the spirochetes are the minor group of the normal subgingival microbial florai"'3),their numbers in the lesions of adult periodontitis and acute necrotizing ulcerative gingivitisincrease drastically, the proportion of the spirochetes accounts for 30 to 50% of all the microscopi-cally countable microbial population3'`}. Troponema denticola, a medium-sized spirochete is regarded                                                          ,as a potent periodontopathogen as well as Gram negative rods5'6). The recent progress of thecultivation methods facilitated in vitro growth of the oral spirochetes and resulted in increasingaccumulation of the taxonomic studies7) and the reports on the pathogenic factors includingprotease8-'i3). The aim of the present investigation was the isolation and the biological characteriza-tion of the spirohetes from the adult periodontitis patients.                                Materials and Methods   The samples taken using paper points from the lesions of 8 patients with adult periodontitis (36to 45 years of age) were suspended in TYGS brothii} and aliquots of the suspension were introducedonto the sterile filter membranes (cellulose nitrate Type TM-4, pore size O.2 ptm, Toyo Roshi Co.,Tokyo) placed on the TYGS agar plates. The plates were incubated at 370C in an anaerobic glovebox (Coy Laboratory Products, Ann Arbor, USA) filled with an mixture of gases (N2-H2uC02 ; 85 :10 : 5) for 5 days. Eight spirochetes strains isolated by this procedure were cloned from the coloniesformed beneath the membranes by the ordinary methods.   Morphological studies of the isolates were performed light and electron microscopicallyi`}. The(accepted for pulication on February 6, 1996)                                   M:Jztsta'\ 22(l) 1996 9biological properties of the isolated 8 spirochetes strains were determined by the methods ofHoldemani5) using the basal PY medium supplemented with 10% rabbit serum, O.5% TPP solutionand VFA solution.    Identification of the metabolic products was carried out by gas chromatography (Shimadzu Co.Ltd., Kyoto) according to Fukumoto et al.'). Phosphatases, esterases, lipases, arylamidases, trypsin,chymotrypsin, galactosidases, glucuronidase, glucosidases, glucosaminidase, mannosidase andfucosidase were detected using API ZYM system (API System S. A., Motalieu Vercieu, France).Neuraminidase activity was estimated by the methods of Potieri6) using a fluorescence substrate,2'-(4-methylumbelliferyl)-a-D-N-acetylneuramic acidi7) (MUA, Sigma Chem. Co. St. Louis, USA).Trypsin and chymotrypsin activity in the culture supernatant and cell extracts of an isolate strain(TN-2) were also measured using benzoyl-arginyl-P-nitroanilidei8) (BAPNA, Sigma) and succinyl-alanyl-alanyl-prolyl-phenylalnyl-P-nitroanilider9) (SAAPNA, Sigma), respectively. Collagenaseactivity was examined using 4-phenylazobenzyloxy-carbonyl-prolyl-leucyl-glycyl-prolyl-arginine(Pz-PLGPA, Sigma).                                 Results and Discussion    Eight isolated strains formed rough and opaque colonies on TYGS agar plates. The mor-phological and biochemical characteristics are summarized in Table1. All the strains exhibitedblunt end and spiral structure with O.20 to O.25 "m in width (Fig. 1). All the strains required serumfor the growth, however, volatile fatty acids were not essential. Acid production from 8 carbo-hydrates was not observed. Hydrolysis of esculin and gelatin was positive, indicating the spirocheteselaborate esteolytic and proteolytic enzymes. Indole and hydrogen sulfide production were positive.All the strains yielded acetic acid, propionic acid and butyric acid. Lactic acid and succinic acidwere detected from various strains. Production of neuraminidase, trypsin-like protease,chymotrypsin-like protease and collagenase in the spent medium and cell sonicate by one of theisolates (strain TN-2) is compared in Table 2. Neuraminidase was found in the culture supernatant,however, proteolytic enzymes located only in the cell extracts. All the strains produced acidphosphatase, alkaline phosphatase, C-8 esterase, leucine arylamidase, phosphoamidase and trypsin-like enzyme. C-4 esterase, varine arylamidase, cystine arylamidase, crgalactosidase, 6-galactosidase, P-glucosidase, chymotrypsin-like enzyme and neuraminidase were produced by themany strains. C-14 lipase, B-glucuronidase, N-acetyl-fi-glucosaminidase, ev-mannosidase and ev-fucosidase were negative in all the strains.    The morphological and biochemical properties of the isolated spirochetes from the periodontitispatients resembled the observations by Fukumoto et al.'}. These isolates were identified asTrePonema denticola. Proteolytic activities were not found in the supernatant fluid of the liquidculture obtained by centrifugation at 10,OOO g for 20 min, they were detected in the cell sonicate.Therefore, proteases Iocate in the cytoplasmic fraction or in the cell surface. In the syntheticprotease substrates, BAPNA was most actively hydrolyzed and then SAAPNA and Pz-PLGPAfollowed. MUA (neuraminidase substrate) was degraded by both culture supernatant and cellsonicate, but the former fraction contained about 14-fold total activity of the Iatter fraction. Thesefindings suggest that synthesized neuraminidase is actively secreted extracellularly.    The sample of cell sonicate, which had been dialyzed against 50 mM Tris-HCI buffer (pH 7.2)was applied to a column of Q-Sepharose equilibrated with the same buffer and eluted with a lineargradient concentration of NaCl. As illustrated in Fig. 2, collagenase and chymotrypsin-like enzyme10 Hoshino, et al.: Isolation and Biological Properties of OTable 1. Properties of the isolates of oral spirohetesral Spirochete, T7'ePonemadenticoga"iidth of cel](#ni)Cell endsRequirement for growth serum volatile fatty acidsFermentation  arabinose fructose glucose glycogen rhamnose ribose starch xy]oseEsculin hydrolysisIndole produtionGelatin liquefactionH2S productionEndproducts acetate proplonate butyrate lactate succmateO.15-O.25  blunt++++++++++Fig.1 : Electron micrographs of T. denticola TN-2  a [ transmission electron micrograph  b : scanning electron micrographTable 2. Enzyme activities of TrePonema denticola TN-2Enzynie Culture supernatant(1950 ml)Ceil sonicate from10.4 g cell(165 ml)Neuraminidase(MUA)Trypsin-like(BAPNA)Chymotrypsin-like(SAAPNA)Collagenase(Pz-PLGPA)151(#M/ml/min)N. D.N. D.N, D,I26("Mlml/min)O.69(ZÅrs ODIrn1/min)1.78(t(X ODIml!min)1 , 71(A ODIml/min)N, D.; not detected.                                                seluted in a single peak but trypsin-like enzyme appeared in a minor and a major peak. Collagenase,trypsin-Iike enzyme and chyomotrypsin-like enzyme eluted at NaCl concentrations of 150 to 200mM, 220 to 250 mM and 300 to 400 mM, respectively. These three enzymes are being purified usingQ-Sepharose chromatography as first step of procedure.  O.6LI'S O.4=oesz  O,2o o co N(O.8O.4ozalsits:Eii'F, 22(1) 1996o 20Eg. 2:1.0:  rt  gg,., g  g.  •s-  .-.o  40 60 8o 100   Fraction numberChromatography of proteases of TrePonemadenticola TN-2 on Q-Sepharose columnO.6z   T   'E   'xo•4 o-sE   g   g-   •go.2 ljo'   s   ooO.31  ?o.2 Lr  g  g   i   e   aO.1 S   -o  oo11                                     Acknowledgment    We thank Mr. S. Akahane for his technical assistance of electronmicroscopic work.                                        References1 ) Amitage, G. C., Dickson, W. R, Jenderseck, R. S., Levine, S. M. and Chambers, D. W. (1982) Relationship   between the percentage of subgingival spirochetes and the severity of periodontal disease. J. Per-   iodontol. 53 : 550-556.2) Listgarten, M.A. and Hellden, L. (1978) Relative distribution of bacteria at clinically healthy and   periodontally diseased sites in humans. J. CIin. Periodontol. 5 : 115-132.3) Loesche, W.J., Syed, A. S,, Schmidt, E. and Morrison, E. C. (1985) Bacterial profiles of subgingival   plaques in periodontitis. J. Periodontol. 56 : 447-450.4 ) Loesche, W. J., Syed, A. S., Laughon, B. E. and Stoll, J. (1982) The bacteriology of acute necrotizing   gingivitis. J. Periodontol. 53 : 223-230.5 ) Moore, W. E. C., Holdeman, L. V,, Cato, E, P., Smibert, R. M., Burmeister, J. A., Palcanis, K, G. and   Ranney, R. R. (1985) Comparative bacteriology of juvenile periodontitis. Infect. Immun. 48 : 507-519.6) Simonson, L.G., Goodmann, C.H., Bial, J.J. and Morton, H.E. (1988) Quantitative relationship of    TrePonema denticola to severity of periodontal disease. Infect. Immun. 56 : 726-728.7 ) Fukumoto, Y., Okuda, K., Kato, K., Ohta, T. and Takazoe, I. (1987) Taxonomic study of spirochetes   isolated from human periodontal lesions. Oral Microbiol. Immunol. 2: 82-87.8) Makinen, K. K., Makinen, P. L. and Syed, S.A. (1992) Purification and substrate specificity of an   endopeptidase from the human oral spirochete TrePonema denticola ATCC 35405, active on furacryloyl-   Leu-GIy-Pro-Ala and bradykinin, J. Biol. Chem. 267 : 14285-14293.9) Shibata, Y., Fujimura, S. and Nakamura, T. (1992) Purification and characterizatiori of an enzyme   produced by TrePonema denticola hydrolyzing synthetic collagenase substrates. Matsumoto Shigaku 8 :   138-143.10) Makinen, P. L., Makinen, K.K. and Syed, S.A. (1994) An endo-active proline-specific oligopeptidase   from TrePonema denticola ATCC 35405 : evidence of hydrolysis of human bioactive peptides. Infect.   Immun. 62 : 4938-4947.11) Ohta, K., Makinen, K, K. and Loesche, W. J. (1986) Purification and characterization of an enzyme   produced by TrePonema denticola capable of hydrolyzing synthetic trypsin substrates. Infect. 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Immun. 56 : 2717-2722.?J'st ] mtwx tO m .x-S Treponema denticola (7)"ss t Je cotstz:M3Lts'# ecEnyuaX, eststthikii, pitJSi• rt (tl}ZsttsJiÅqI• mtemeut) ntyK.{lktsrefi,$..ZOpa..ss lk O JJCL tf p Ala S Sr5iÅr'meL, )Z2L5eut Treponema denticola E ffESlh.tc.5År nt st o ee fi tr= etwt R ft ,L(Zi pt E L, fik zKIlt im oD 5År ne tsE eXIrk hÅr -) fe . -t y F" - 7v, calt JdÅq$, ff me, 7" pt` 1- vme, asme fi X' -tr- v-Åq(ofi ntstottrsgtzhts 6stM8 in.k. 7" p f7 - ti, =- j= 9 - le", = a - 7E = ,St" - Rt" cDgli1 tits ge. bt) tl) 2z, 70 p tr 7- -lt' eC -) LN -( e#1i7J ts ts Åq i5 3 ptffE g in. 6 e 2 bS fi•hts -. te. I 2zti)o 7" p 77- -lt" e: -r 1- 7.I32igei7 p -gr F `)t" iii 7 i - '(i`k21.;e 2L5Årme }Is 2L6 t 2 i5 \IJ HA Lt( .

Isolation and Biological Properties of Oral Spirochete, Treponema denticola

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口腔スピロヘータTreponema denticolaの分離とその生物的性状

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