Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies

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1 ELSEVIER FEMS Microbiology Letters 145 (1996) MICROBIOLOGY LETTERS Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies Yukihide Tomita aab, Xia Zhu a, Kuniyasu Ochiai a, Yasuji Namiki b, Tamami Okada b, Takuji Ikemi b, Kazuo Fukushima al* a Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo. Chiba 271, Japan b Department of Operative Dentistry, Nihon University School of Dentistry at Matsudo, Maisudo, Chiba 271, Japan Received 5 June 1996; revised 8 October 1996; accepted 15 October 1996 Abstract We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyltransferases (I, SI and S) of Streptococcus mutuns which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans. The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-si and glucosyltransferase-i enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates. Keywords: Streptococcus mutans; Monoclonal antibody; Immune-dot assay 1. Introduction Among the seven species comprising the mutans streptococci, S. mutans is implicated in the etiology of human dental decay [I]. This organism produces three extracellular glucosyltransferases (EC S) that synthesize water-insoluble and -soluble glucans from dietary sucrose [2,3]. The ability of this bacterium to firmly adhere to tooth surfaces through de novo water-insoluble glucan synthesis by the three glucosyltransferases is considered to be one of the * Corresponding author. Tel. : +8 1 (473) ; Fax: +81 (473) ; kfuku@mascat,nihon-u.ac.jp most important pathogenic factors leading to cariogenie plaque formation and subsequent dental caries [l]. The gtfb, gtfc and gtfd genes encoding glucosyltransferase-i, -SI and -S enzymes, respectively. have been isolated from S. mutans [2,4,5], and the mechanisms of cariogenic plaque and dental caries formation by S. mutans are being clarified by molecular genetic approaches [6-a]. Epidemiological and microbiological studies have revealed different cariogenie potentials and genetic diversity among fresh S. mutans isolates 19,101. However, there is little information about the glucosyltransferase production by S. mutans, because there are no useful tools available for determining the individual amounts present in /96/$12.00 Copyright Federation of European Microbiological Societies. Published by Elsevier Science B.V. PIZSO (96)

2 428 Y. Tomita e! ul. IFEMS MtcrobiologJ Lettrrs 145 (1996) st 2nd 3rd P32 ia 1 I P72 I P4 N ' ' Fig 1. Extraction of the three glucosyltransferases from a S. rrwans PS14 culture using 8 M urea. The ethanol precipitate from a 5 ml G-THB culture of PS14 cells was extracted three times with 250 ul UP buffer and analyzed by the cross-dot assay as described in Section 2.4. cells. Polyclonal antibodies cannot distinguish between the glucosyltransferase-i and -SI enzymes, which have extensive amino acid homology [II]. We established murine hybridomas producing a monoclonal antibody (mab) monospecific for each of the three glucosyltransferases, and confirmed their usefulness through Western blotting of glucosyltransferase samples from S. mutans isolates Here we describe a new immunochemical technique (cross-dot system) using monospecific mabs, which can semi-quantify the relative amount of each glucosyltransferase. We used it to compare the levels of the three glucosyltransferases expressed by the reference strain PS14 and six clinical isolates of S. mutans possessing different in vitro artificial plaque forming abilities. 2. Materials and methods 2.1. Bacteria, media and culture conditions S. mutans strain PS14 (serotype c) was mainly used A B C P32 P72 P ' Fig. 2. Cross-dot determination of the three giucosyltransferases produced by S. mutans PS14 grown in THB (A), G-THB (B), BHI (C), TSY (D) and M4 (E). The ethanol precipitate from each 5 ml of PS14 culture with the five media was extracted twice with 250 pl UP buffer and the pooled extract was analyzed by the cross-dot assay as described in Section 2.4.

3 Y. Tomita et al. IFEMS Microbiology Letters 145 (1996) #l #2 P32 P72 P #@I #5 P32#!m m mllat P72 1 P4 me # s, Fig. 3. Cross-dot determination of the three glucosyltransferases produced by six clinical isolates (l-6) of S. mutans. The ethanol precipitate from each 5 ml culture of the six isolates grown for 18 h in G-THB medium was extracted twice with 250 ~1 UP buffer and the pooled extract was analyzed by the cross-dot assay as described in Section 2.4. throughout this study. Six S. mutans isolates were routinely isolated from the saliva of six individuals, identified, and used in the enzyme expression study. Cultured bacteria were inoculated into 5 ml of Todd- Hewitt broth (THB, BBL), THB supplemented with 1% glucose (G-THB), Brain Heart Infusion broth (BHI, Difco), Trypticase Soy broth containing yeast extract (TSY), or M4 semi-synthetic medium (M4) [12], then cultured anaerobically at 37 C for 18 h in candle jars Preparation of glucosyltransferase extracts In order to recover the total glucosyltransferases (extracellular and cell-associated forms), chilled ethanol (5 ml) was added to each 5 ml culture at the post-stationary phase and left for 30 min. The resultant precipitate was collected by centrifugation at Xg for 5 min, suspended in 250 l.tl of 8 M urea in 10 mm potassium phosphate buffer, ph 7.2 (UP buffer), then incubated at 25 C for 1 h under vigorous stirring with a Twin Mixer TM-282 (Iuchi, Osaka). After centrifugation ( Xg, 5 min), the precipitate was again extracted with the UP buffer as described above. The supernatants were pooled and used in the cross-dot assay as the sample containing total glucosyltransferases. In some experiments, the urea extraction was repeated three times and each supernatant was separately assayed Preparation of primary antibodies Culture supernatants of the hybridomas producing mabs P4, P32 and P72 were prepared as described [3] and used as primary antibodies. The specificity and potency of these mabs were confirmed by an enzyme-linked immunosorbent assay (ELISA) as described [3], using crude glucosyltransferase-i from S. milleri transformant KSB8, crude glucosyltransferase-si from S. milleri transfonnant KSC43, and pure glucosyltransferase-s from S. mutans PS Cross-dot assay The assay was performed essentially as described by Altic et al. [13], using a cross-blot/cross-dot apparatus (Boite-a-Blot ; Sebia, France). Briefly, a Clear Blot Membrane-P (Atto, Tokyo) was rinsed

4 430 Y. Tomilu ( I ul. I FEMS Microhiolo~)~ Letters 145 (1996) OOr and incubated for 15 min in 20 ml of the blocking buffer. After blocking, the membrane was incubated for 2 h in 20 ml of a solution of horseradish peroxidase conjugated goat anti-mouse immunoglobulin (Amersham, 1:2000 dilution in 0.1% bovine serum albumin-tbs), washed three times in 20 ml of TBS, followed by incubation in the substrate solution containing 0.05% 4-chloro-1-naphthol. The results were visualized using a Densitograph AE MLR (Atto, Tokyo) hi vitro artiji ciu1 plaque formation PSI4 #I #2 #3 #4 #5 #6 Fig. 4. Artificial plaque formation by PSI4 stram and six clinical isolates (l-6) of S. mutans. The cariogenic plaqueforming ability of PSI4 cells and the six fresh isolates was compared by evaluating firmly adherent artificial plaque formation in S-THB as described in Section 2.5. The data are the means I? S.D. of triplicate determinations. *Significant difference relative to PSI4 at PC (by Student s r-test). with methanol, washed with distilled water and assembled into the cross-dot apparatus using a grid D. After the apparatus was filled with UP buffer and individual lanes were aspirated, each lane was filled with 50 ~1 of the enzyme preparations diluted from 2 - to 2 j-fold (some to 212-fold) with UP buffer. The membrane was then incubated at 4 C overnight. Following three washes with 200 mm NaCl in 50 mm Tris-HCl buffer, ph 7.4 (TBS), the membrane was removed from the apparatus, incubated for 5 min in 20 ml of blocking buffer (10% skimmed milk in TBS), and re-assembled into the apparatus using grid A (in which the configuration of the wells is perpendicular to that of grid D). The apparatus was then filled with blocking buffer. Individual lanes were aspirated and each was refilled with a dilution of hybridoma culture supernatant containing mabs P4 (1:50), P32 (1: 10) and P72 (1:20) diluted to 500 ~1 in the blocking buffer, followed by incubation for 1 h at room temperature. The membrane was washed three times with TBS, removed from the apparatus, Cultured bacteria were inoculated into 2 ml THB containing 5% sucrose (S-THB) in disposable culture tubes (12 x 75 mm, Iwaki Glass, Chiba), and cultured anaerobically at 37 C for 18 h at a 30 angle. The tubes with artificial plaque were vortex-mixed for 10 s at maximal speed on a Touch Mixer MT- 51 (Yamato, Tokyo), decanted and washed with 2 ml of phosphate buffered saline (PBS). The decanted and wash solutions were pooled as the non- and loose-adhered cell fraction. The cells remaining on the tube surface were suspended by sonication in 4 ml of PBS and are referred to as the adherent fraction. The turbidity at 550 nm of both fractions was determined immediately after sonication (50 W, 10 s). The percentage of firmly adhered cells per total cells (! adherence) was calculated and used as an indication of the in vitro artificial plaque forming ability of S, mutans isolates. 3. Results and discussion Using the cross-dot assay with three mabs, the experimental conditions preparing total glucosyltransferases were examined. The solubilization of cell-associated glucosyltransferases was performed primarily by the 8 M urea extraction method described by Hamada et al. [14]. The ethanol precipitate from a G-THB overnight culture of PS14 cells was extracted repeatedly with UP buffer, and each extract was analyzed with the cross-dot assay. As shown in Fig. 1, large portions of the three glucosyltransferases were solubilized by the first extraction, and the remaining portions were recovered by the secondary extraction. This result suggests that the

5 Y. Tomita et al. IFEMS Microbiology Letters 145 (1996) complete extraction of glucosyltransferases can be achieved by repeating the treatment in which an ethanol precipitate from a 5 ml culture is suspended in 250 ul of UP buffer and incubated at 25 C for 1 h under vigorous stirring. The total glucosyltransferases produced by PS 14 cells grown for 18 h in five different media were extracted with UP buffer, and analyzed with the cross-dot assay. Fig. 2A-E shows the results of a 2-fold dilution series of the five samples prepared from the THB, G-THB, BHI, TSY and M4 cultures that were dotted and immunostained. The mab P32 (anti-si) reacted up to 23-, 24-, 2 -, 23- and 27-fold dilutions, mab P72 (anti-i) up to 2 -, 22-, 2l-, 22- and 27-fold dilutions, and mab P4 (anti-s) up to 22-, 23-, 25-, 23- and 27-fold dilutions. The results showed that the amounts of the three glucosyltransferases produced by PS14 cells differed with the culture medium. In particular, the differences between the four complex media and the M4 semi-synthetic medium were remarkable. The data indicated that the levels of three glucosyltransferases produced in the M4 medium were 8-60-fold higher than those produced in the complex media. Motoda [15] reported that the extracellular water-insoluble glucan synthesizing activity of S. sobrinus strain B13N is over 5-fold higher in M4 than in THB, TSY or FMC medium. Therefore, it seems of interest to identify the nutritional factor(s) in the M4 medium that stimulates the production of extracellular glucosyltransferases from mutans streptococci. We examined the glucosyltransferase production and in vitro artificial plaque forming ability of six fresh S. mutans isolates by means of the cross-dot assay and by estimating firmly adherent artificial plaque formation. Figs. 3 and 4 show the results of the glucosyltransferase production and artificial plaque formation by the clinical isolates grown in G-THB and S-THB, respectively. Although the profiles of glucosyltransferase production and firmly adherent plaque formation greatly differed among the isolates, they were classified approximately into three groups. Isolates 1 and 6, which produced relatively high levels of the three glucosyltransferases, showed the maximal adherence, whereas isolates 2, 4 and 5, which produced relatively high levels of glucosyltransferase-si and gluco- syltransferase-s but relatively low levels of glucosyltransferase-i, showed moderate adherence, in agreement with reference strain PS14. In contrast, isolate 3, which produced a relatively high level of glucosyltransferase-s but little glucosyltransferase-i and no glucosyltransferase-si, did not form firmly adherent plaque on the glass surface. Similar glucosyltransferase profiles were also obtained when these isolates were cultured in other complex media (data not shown). These findings are comparable to those of an in vitro cariogenicity test with S. milleri transformants indicating that the transformant expressing gtfc (encoding glucosyltransferase-si) can firmly colonize glass surfaces, whereas the transformants expressing gtfb (encoding glucosyltransferase-i) or gtfd (encoding glucosyltransferase-s) cannot, and that the co-cultures of the transformants expressing gtfc and gtfb result in the maximal sucrose-dependent colonization [7,16]. These results and those of the present study suggest that the glucosyltransferase-si enzyme plays the most important role in the process of in vitro artificial plaque formation and possibly in cariogenicity, and that the glucosyltransferase-i enzyme also functions as an important accelerating factor in that process. Thus, the cariogenie potential of S. mutans producing high levels of both glucosyltransferase-si and glucosyltransferase-i enzymes is most likely higher than that of S. mutans producing low levels of those enzymes. In conclusion, the cross-dot assay system employed in this study should be a useful method of semi-quantifying the individual levels of the three glucosyltransferases expressed by S. mutans, and of estimating the cariogenie potential of S. mutans isolates. Currently, in vitro and in vivo studies to clarify the detailed relationship between glucosyltransferase producibility and the exact cariogenicity are in progress in our laboratory using this assay system with many S. mutans isolates. Acknowledgments This study was supported in part by research grants from Nihon University, the Hamaguchi Foundation, and the Ministry of Education, Science and Culture of Japan.

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