- Email: [email protected]
Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product
FEMS Microbiolog$' Loners 9211992) 273-27S eJ lq02 Federation of European Microbiological Societic.,, 1~378-1097/t)2/$115.{)1) Published by Elsevier
273
FEMSLE 04861
Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product Yoonsuk Park a and Barry C. McBride '''" "lh,paronenr Of Microbioh)gy. and ~"D('Inmment t!f Ond Bitdo~g/. "Fht' Unit('r.~Hy ,)t"Britixh Cohmd)ia. Vtmcouw'r. Briti.~h ('olmnhia, (',mtuht Received 15 J:mmID' ttJ92 Revision received and accepted 15 Fch,'uaD' 1'1~2
Key words: Porphyromonas gingivalis; Protease; Cloning
I. SUMMAR'Y
A clone expressing a Porphyromonas gingiralis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between Bam Hi and Hindlll sites. When a 3.2-kb Hindlll fragment of oYS307 was used as a probe in Southern hybridization, Hindlll-digcsted chromosomal DNA of P. gingicalis W83, as well as those of W50 and WI2, showed a single 3.2-kb hybridizing band, whilc that of P. gingivalis 33277 showed a 5.l)-kb band, Colonies of E. coil containing pYS307 showed pronounced protcolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coil containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-depcn-
Correspondence tu: B.C. McBride, Faculty of Science. 6271.) University Boulevard, Vancouver, B.C'.. Canada V~T |Z4.
dent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coil JM83 containing pYS307 was prepared for electrophoresis in the absence of B" mercaptoethanol. When samples were solubilized in the presence of /J-mercaptoethanol prior to clcctrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The ac[ivity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCIv
2, INTRODUCTION A number of microbiological studies have provided strong evidence linking Porphyromonas (Bacteroides) gingivalLs to adult periodontitis [ i 4]. This black-pigmented Gram-negative anaerobe produces a number of potential virulence factors including cell associated proteases, collagenase and adhesins [5,6]. Several proteins, including collagen [7,8], immunoglobulins [~)]. iron-binding proteins [10], plasma proteins [ll] and complement factors
274 [12,13], are hydrolysed by P. gingif'alis. Various reports have suggested that proteases of P. gingirafts can inactivate host defense mechanisms by degrading host defense proteins. The proteases may a l ~ degrade matrix and intercellular proteins, directly as well as indirectly by activating host hydrolytic enzymes such as collagenases [14]. Proteases, as well as peptidases, have been isolated and characterized from P. gingicalis culture supernatants, outer membranes, cell extracts and extracellular vesicles [15-22]. In recent years, cloning of P. gingicalis protease genes has been attempted by several research groups [23,24]. In this paper, we report the cloning and characterization of a P. gingit'alis W83 protease gene and its product.
3. MATERIALS AND METHODS
3.1. Bacterial strahls and cultication P. gingicalis W83 and other black-pigmented Porphyromonas and Bacteroides (BPB) strains were grown in Bacto brain-heart infusion broth (Difco Laboratories, Detroit, MI) supplemented with haemin (5/xg/ml) and vitamin K (0.5/xg/ml) at 370C in an anaerobic chamber (Coy Manufacturing, Ann Arbor, MI) containing 5% CO 2, 10% H a and 85% N 2. Escherichia coli JM83 was grown in L-broth. 3.2. Construction of genomic library and screening of clones Rapid plasmid DNA preparations were made by an alkaline lysis method described previously [25]. Large-scale DNA purifications were performed by the method described by Sambmok et al. [26] for plasmids or by Silhavy et al. [27] for chromosomal DNA. All recombinant DNA procedures wt:re carried out as described by Sambrook et al. [26]. Purified chroLnosomal DNA from P. gingicalis W83 was partiali3, digested with endonuclease Sau3Al, and fractionated on a 10-40% (w/v) sucrose density gradient to yield size-selected DNA fragments from 2 to 10 kilobase pairs (kb). Size-selected fragments were ligated with dephosph~rylated pTZ18R [28] ~inearized with BamHi
using T4 DNA ligase. The ligation mixture was used to transform E. coli JM83 made competent by CaCi2 treatment. A representative P. gingit'alis genomic library was obtained by selecting for ampicillin-resistant white E. coil colonies from LB agar plates conte.ining 5-bromo-4-chloro-3-indolyl g-o-galactopyranoside (X-gaD. Proteasepositive clones were detected in LB agar plates supplemented with !% Carnation powdered skim milk. Transformants were transferred onto LB agar plates containing skim milk and incubated overnight at 37°C. After exposure to chloroform vapor for 1 h, the plates were incubated at 37°C and then examined for areas of clearing around the colonies.
3.3. Electrophoresis and BSA substrate zymography Sodium dodecyl sulfate (SDS)-I0% polyacrylamide gel electrophoresis (PAGE) was done by the Laemmli method [29], using a Mini PROTEAN il cell (BioRad Laboratories, Richmond, CA). E. coli cell lysates were obtained by solubilization in 4% SDS buffer (with or without 10% /~-mercaptoethano'.) at either 37°C (30 rain) or 100°C (5 min). To detect protcolytic activity, SDS-PAGE gels were modified by the addition of bovine serum albumin (BSA) conjugated to arcrylamide in a method described previously [30]. 3.4. bnmunoblot analysis Western blotting was done by the method of Renarr and Sandoval [31]. Immunoreactive proteins were detected by incubation of the filter with rabbit antiserum raised against P. gingit'alis W50 whole cells, followed by goat anti-rabbit lgG (H + L) alkaline phosphatase conjugate (humanadsorbed) (BRL, Gaithersburg, MD). 3.5. Southern hybridization A non-radioactive nucleic acid detection system (BluGENE TM,BRL, Gaithersburg, MD) was used to detect chromosomal DNA fragments homologous to the cloned DNA fragment. To prepare the probe DNA, the recombinant plasmid, pYS307, was digested with Hindlll, and a 3.2-kb fragment containing the cloned protease gene was extracted from an agarose gel after elec-
275
trophoresis by the methods described by Silhavy et al. [27]. The DNA was labelled with biotin-7dATP as described by the supplier, and chromosomal DNA samples were prepared by digestion with Hindlll. Hybridization and detection were performed by the standard methods described by Sambrook et al. [26] and by the supplier, respect ively.
4. RESULTS AND DISCUSSION
4. !. Cloning of a P. gingit'alis protease gene Screening 21100 recombinant clones yielded a single colony capable of hydrolysing milk proteins. The zone of clearing appeared only after 2 weeks of incubation at 37°C, Plasmid isolated from this clone was transformed in E. colt JM83. It was observed that colonies which grew deep within the agar developed zones of protein hydrolysis, while those inoculated only on the agar surface did not. This suggests that the protease was only active under the reducing conditions that would exist deep in the agar. To confirm the requirement of the reducing conditions for activity of the cloned protease, the transformants were incubated in an oxygen-free environment after treatment with chloroform vapor. Under these conditions, clear zones appeared around all the colonies after 3 days. These results suggest that the cloned protease is only active under reducing conditions. Arnott et aL [23] also reported that protease-positive clones were isolated only when the screening procedure was done under anaerobic conditions. 4.2 Subclon#ag from pYS307 The recombinant plasmid, named pYS307, contains a 5.2-kb DNA insert. Retransformation of pYS307 into E. colt JM83 confirmed that the proteolytie enzyme is expressed by the genc encoded in the plasmid. A restriction endonuclease map of pYS307 is shown in Fig. 1. Based on this restriction map, subclones were constructed. The plasmid vector pTZI9R [281 was used to construct pYS307-21.- ) and pYS307-3( + ). This vector differs from pTZI8R only in the orientation of the multiple cloning site. As shown in Fig. 1,
,,L
i
I
L
t
IDI~..,m.~_, !
,,I
Fig. I. The restriction-enzyme maps of oYS31|7 and its subclones. ( + ) and ( - ) indicate the right and opl~)Sile orienlalions, rcsl~eclivel.v, ~'ith resider to the lac promoter. T h e broad line indicates lh¢ vector piasmid, p T Z I g R . Abbreviations are as follows: Ac. A t c | : Bin. B a m i l l : E¢, EcoRI; ttc, tlmcll: Hd. t l m d l l l : Kp. Kidtl; Ps. P~,tl: So, Sacl: Sp. Sphl.
the cloned gene was localized to a 2.4-kb DNA fragment between BantHl and Hindlli sites (Fig. 1: pYS307-2(+ ) and -2(-)). The orientation of the 2.4-kb fragment did not influence expression of the protease suggesting that the DNA fragment contains a P. gingicalis promoter which is recognized by E. colt transeriptton and translation systems.
4.3. Southern hybri¢h'zation The origin of the cloned DNA fragment was confirmed by Southern hybridization using a 3.2kb HindIll fragment as a probe (Figs. 1 and 2). Chromosomal DNA isolated from P. gingiralis W83, W50, and Wl2 digested with Hindlll all possessed a single 3.2-kb fragment which hybridized with the probe. In the case of P. gingicalls 33277, the probe hybridized with a 5-kb fragment. No hybridizing bands were detected with chromosomal DNA samples of P. (Bacteroides) asaccharolyticus, Bacteroides corporis, B. demicola, B. intermedius, B. lecii, B. ioescheii and B. melan#zogenicus strains. This suggests that the Hindlll fragment containing the cloned protease gene may be species-specific, at least under high stringency washing conditions. 4.4. Characterization of the cloned protease The cloned gene product was identified by SDS-PAGE and BSA substrate zymography. Protein profiles of celt lysatcs in Coomassie blue-
27¢~
I
2
3
4
5
6
7
8
9
10
11
12
kb 23.7
-
9.50 6.664.26
-
2.251.96 -
~:
. . .
. ;
-
Fig. 2. Southern hybridization. A 3.2-kb HindllI fragment of pYS307 (Fig. I) was used :is a prohe. All of the chromosom~J DNA samples were digested with HindllI. lame t, pYS3tl7 digested with llindlll: hmcs 2-12 art: chromosomal DNAs of BPB strains digested with Hindltt. lane 2, P. gingirafis W83; lane 3. P. gingicalis 33277: lane 4. P. gingicalis WSIk lane 5. P. gb~girali., WI2: hme O, P. (Bacteroides) a.~archarolyticus 252fi0: lane 7. B. corporis 33547, lane 8, B. denticota 33185: hme 9, B. intennedius 15032" lane ll). B. lecii 122090: lane il, B. Ioescheii 15930: lane 12. B. mehminogenicus 25845.
I
2
3
4
224 109 72
46
29 ki~ Fig, 3. Detection of proteolytic activity in a BSA*conjugaled acrylamide gel. Lanes 1 and 3, E. colt JM83 (pTZ|SR); lanes 2 and 4, E. colt JM83 (pYS307), S:tmples of whole cell lysutes were solubilized at 37°C flrr 311 rain in buffer without ~8mercaploethanol prior to electrophoresis.
stained gels did not reveal significant differences between E. colt JM83 (pTZ18R) and the recombinant clone. The expression of the cloned protease with a molecular mass of approximately 80 kDa can be detected by BSA substrate zymography with samples of the clone prepared in the absence of /3-mercaptoethanol (Fig. 3). When samples were prepared in the presence of/~-mercaptoethanok no proteolytic activity could be detected. In lanes containing cell lysates of E. colt JM83 (pTZ18R), several host proteins appear as Coomassie bluestained bands (Fig. 3, lane 1 and 3), these bands disappear in lanes containing cell lysates of the clone (Fig. 3, lane 2 and 4). This suggests that the cloned protease can degrade E. colt proteins during solubilization. The subclones which show protease activity on skim-milk plates also show a proteolytic band in BSA substrate zymography similar to that shown in the original clone. The proteolytic band could be detected only when a reducing agent, cysteine (50 mM), was added to
277
fractions of P. gb~gicalis 33277 using BSA substratc zymography. Among the eight different bands, one of the protease bands (P4) showed a molecular mass of 80 kDa on BSA substrate zymography. This protease was also inhibited by TLCK, iodoacetic acid and heavy metal ions. but not b'y TPCK and EDTA. This suggests that the cloned protease and P4 have different characteristics.
the assay system following electrophoresis. This result suggests that the cloned protease is thioldependent, and confirms the results observed in the colony screening assay.
4.5. Effects of protease h+hibitors The effects of several protease inhibitors was determined by performing BSA substrate zymography in the presence of inhibitors. The cloned protease was inhibited by N-a-p-tosyi-t.-lysine chloromethyl ketone (TLCK, 4 mM), N-tosyl-t.phenylalanine chloromethyl ketone (TPCK, 4 mM), phenylmethylsulfonyl fluoride (PMSF, 4 raM), iodoacetic acid (8 mM), EDTA (40 mM) and ZnCI., (10 raM), but not by CaCI, (10 mM) or MgCi., (10 raM). In the study of Grenier et ai. [30], a total of eight distinct bands of protcolytic activity could be detected with different cellular
1
2
3
4.6. Western immtmoblott#lg The identity of the cloned P. gingiralis protein was determined by immunoblotting with the antiserum raised to P. g#lgil'alis W50 whole cells (Fig. 4). A single reactive band was detected v, hen cell lysates of the clone containing pYS307 were prepared for elcctrophoresis in the absence of /3-mercaptoethanol (Fig. 4, lane 5). The posi-
4
$
6
214
,91
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Fig. 4, Western immunoblot of the cloned protcase. Lane I. whole cellsof E. c+di JM83 (pTZISR) solubilizedat Itl~)~C:lanes 2 and 6, same as Lane I but solubilized al 37°C" lane 3, whole cells of E. coil JM83 (pYS307) st)lubilized at I(XFC; lanes 4 and 5, same as lane 3 but solubilized at 37°C. T h e samples of lanes 1-4 were solubilized in butler containing 10ek /~-mercaptoethanol. T h e samples of lanes 5 and 6 were solubilized in buffer without B-mercaptoethanol. Filters were reacted wilh antiserum raised against P. gingit'alis W50 whole cells. Secondary, anlibody was goat anti-rabbit lgG (H + L) alkaline phosphatase conjugate (human-adsorbed) (Gibco BRL, Gaithersburg. MD),
278
tion of the antibody staining hand was identical to the protcase activity sccn in the zymogram (Fig. 3, lane 2 and 4L A different result was observed when the sample was prepared in the presence of ~8-mercaptoethanol prior to dectrophorcsis. In this case, there was a major antibody reactive peptid¢ of 50 kDa and a minor peptide of 38 kDa (Fig. 4, lane 3 and 4). The minor band was not always detected. These resuits are noteworthy for comparison with the results of Smalley and Birss [t9]. They examined extracellular and vesicle-associated trypsin-like enzyme-containing fractions (VSF) of P. gingivails strains W50 and W50/BEI by gel-filtration chromatography, immunoblotting and zymography. Antiserum to the soluble trypsin-like enzyme of strain W50 reacted with a 50-kDa peptide of each subfraction but not with any peptide of molecular mass higher than 50 kDa, although gelatin substrate zymography following non-reducing SDS-PAGE revealed a major 80 kDa protease. It appears that in the presence of reducing agents there is auto-degradation of the proteasc. If this is the case, the site of attack is very specific as there is a very sharp band at 50 kDa.
ACKNOWLEDGEMENTS
This work was supported by the Medical Research Council.
References [1] Slots, L (19821 In: Host-Parasite Interactions in Periodontal Disease (Genco, R.J. and Mergenhagen, S.E.. Eds.). pp. 27-45. American Society for Microbiology, Washington. D.C. [2] Tanner, A.C.R., Huller, C.. Bratthall, G.T., Visconti. R.A. and Socransky, S.S. 119701 I. Clin, Periodontol. 6, 278-3117. [3] While, D. and Mayrand, D. (1981)J, Periodontal Res. 16, 259-265, [4] Zambon, J.J., Reynolds. H.S. and Slots. J. (19BI) Infect. Immun. 32, 198-203. [5l Mayrand. D. and Holt, S,C, (1988) Mierobiol, Rev. 52, 134-152.
[6] Van Winkclhoff.
A.J.. van Stecnhergen, T.J.M. and
Graaff. J.D. (1088)J. Clin. Periodontol. t5, 145-155. [7] Smalh:y. J.W., Birss. A.J. and Shuttleworth. C,A. 119881 Arch, Oral Biol. 33, 323-329. [8] Sundqvist. G.. Carlsson, J. and H~instri;,m, L. (19871 J. Periodonl. Res. 22, 31H)-31h% [9] Grenier, D.. Mayrand, D. and McBride. B.C. 119891 Oral MicmbioL Immunol. 4. 12-18. [111] Carlsson, J., Iliifling, J.F. and Sundqvisl, G,K. (19841 J. Med. Microbiol. I~. 39-46, [11] N ilsson, T,, (_arlsson. J. and Sundqvist. G. (1985) Infect. lmmun, 51k 467-471. [12] Schenkein, H.A. (19881J. Pcriodont. Res. 23, 187 192. [13] Schcnkein, H.A. (1991) Oral Microbiol. lmmunol. 6, 216- 221l. [14] Uitlo. V.-J., Larjava, tl., tteino. J. and Sorsa. T. (19891 Infect. Immun. 57, 2|3-218. [15] Abiko. Y.. tlayakawa. M., Mural. S. and Takiguchi. II. (1995) J. Dent. Res. 64. 106-11 I. [[6] Fujimura, S. and Nakamura, T. (19891 Oral Micmhiol. ImmunoL 4, 227-229. [17] Grcnier, D. and McBride, B.C. (|989) Infect. Immun, 57, 3265-3269. [18] Olsuka. M., Endo, J., Ilinode, D,, Nagata, A.. Maehara, R., Salo, M. and Nakamura, R. (1987) ,I. Periodont. Rcs. 22, 491-498. [19] Smallcy, J,W, and Birss. A.J. (19911 Oral Microbiol, Immunol. 6, 21)2-208. [20] Sorsa, T., Uitto, V.-J., Suomalaine. K., Turlo, tL and Lindy. S. 119871 J. Periodont. Res. 22, 375-381I. [21] Suido, H., Nciders, M.E., Barus, P,K., Nakamura, M., Mashimo, P.A. and Genco, R.J. ( 19871 J. Perit~dont. Rcs. 22. 4i2-418, [22] Tsutsui, H., Kinouchi, T.. W=.kano, Y. and Ohnishi. Y. (19871 Infect. Immun. 55, 420-427. [23] Arm)n, M.A., Rigg. G., Shah, it., Willi:lms, D., Wallace, A. and Roberts, I.S. (19901 Arch. Oral Bk)l. 35, 97S-99S. [24] Takahashi, N,, Kato, T. and Kuramitsu, i[.K. (1¢;~11 FEMS Microbiol, Left. 94, 135-t38. [25] Rodrlgucz. R.L. and Tail, R.C. ( 19831 Recombinant DNA Techniques. An Intnaluction. Addimn-Wesley Publishing Company, Reading, MA. [26] Sambrtmk, J., Fritsch, E.F. and Maniatis, T. (1989I Molecular Ckming: a Laboratory Manual (2nd edn). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [27] Silhavy, T,J,, Berman, M.J. and Enquist, L.W. (1984) Experiments with gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [28] Mead, D.A.. Szczesna-Skorupa. E. and Kcmper, B, ( 1986} Prof. Eng. I, 67-74. [29] Laemmli, U.K. (197()) Nature (London) 277, 680-685. [30] Grenier, D., Chao, G. and McBride, B.C. {19891 Infect, lmmun. 57, 95-99. [31] Renart, J. and S:mdoval, I.V, 119841 Methods Enz~ymol. 1(}4, 455-46a.
273
FEMSLE 04861
Cloning of a Porphyromonas (Bacteroides) gingivalis protease gene and characterization of its product Yoonsuk Park a and Barry C. McBride '''" "lh,paronenr Of Microbioh)gy. and ~"D('Inmment t!f Ond Bitdo~g/. "Fht' Unit('r.~Hy ,)t"Britixh Cohmd)ia. Vtmcouw'r. Briti.~h ('olmnhia, (',mtuht Received 15 J:mmID' ttJ92 Revision received and accepted 15 Fch,'uaD' 1'1~2
Key words: Porphyromonas gingivalis; Protease; Cloning
I. SUMMAR'Y
A clone expressing a Porphyromonas gingiralis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between Bam Hi and Hindlll sites. When a 3.2-kb Hindlll fragment of oYS307 was used as a probe in Southern hybridization, Hindlll-digcsted chromosomal DNA of P. gingicalis W83, as well as those of W50 and WI2, showed a single 3.2-kb hybridizing band, whilc that of P. gingivalis 33277 showed a 5.l)-kb band, Colonies of E. coil containing pYS307 showed pronounced protcolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coil containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-depcn-
Correspondence tu: B.C. McBride, Faculty of Science. 6271.) University Boulevard, Vancouver, B.C'.. Canada V~T |Z4.
dent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coil JM83 containing pYS307 was prepared for electrophoresis in the absence of B" mercaptoethanol. When samples were solubilized in the presence of /J-mercaptoethanol prior to clcctrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The ac[ivity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCIv
2, INTRODUCTION A number of microbiological studies have provided strong evidence linking Porphyromonas (Bacteroides) gingivalLs to adult periodontitis [ i 4]. This black-pigmented Gram-negative anaerobe produces a number of potential virulence factors including cell associated proteases, collagenase and adhesins [5,6]. Several proteins, including collagen [7,8], immunoglobulins [~)]. iron-binding proteins [10], plasma proteins [ll] and complement factors
274 [12,13], are hydrolysed by P. gingif'alis. Various reports have suggested that proteases of P. gingirafts can inactivate host defense mechanisms by degrading host defense proteins. The proteases may a l ~ degrade matrix and intercellular proteins, directly as well as indirectly by activating host hydrolytic enzymes such as collagenases [14]. Proteases, as well as peptidases, have been isolated and characterized from P. gingicalis culture supernatants, outer membranes, cell extracts and extracellular vesicles [15-22]. In recent years, cloning of P. gingicalis protease genes has been attempted by several research groups [23,24]. In this paper, we report the cloning and characterization of a P. gingit'alis W83 protease gene and its product.
3. MATERIALS AND METHODS
3.1. Bacterial strahls and cultication P. gingicalis W83 and other black-pigmented Porphyromonas and Bacteroides (BPB) strains were grown in Bacto brain-heart infusion broth (Difco Laboratories, Detroit, MI) supplemented with haemin (5/xg/ml) and vitamin K (0.5/xg/ml) at 370C in an anaerobic chamber (Coy Manufacturing, Ann Arbor, MI) containing 5% CO 2, 10% H a and 85% N 2. Escherichia coli JM83 was grown in L-broth. 3.2. Construction of genomic library and screening of clones Rapid plasmid DNA preparations were made by an alkaline lysis method described previously [25]. Large-scale DNA purifications were performed by the method described by Sambmok et al. [26] for plasmids or by Silhavy et al. [27] for chromosomal DNA. All recombinant DNA procedures wt:re carried out as described by Sambrook et al. [26]. Purified chroLnosomal DNA from P. gingicalis W83 was partiali3, digested with endonuclease Sau3Al, and fractionated on a 10-40% (w/v) sucrose density gradient to yield size-selected DNA fragments from 2 to 10 kilobase pairs (kb). Size-selected fragments were ligated with dephosph~rylated pTZ18R [28] ~inearized with BamHi
using T4 DNA ligase. The ligation mixture was used to transform E. coli JM83 made competent by CaCi2 treatment. A representative P. gingit'alis genomic library was obtained by selecting for ampicillin-resistant white E. coil colonies from LB agar plates conte.ining 5-bromo-4-chloro-3-indolyl g-o-galactopyranoside (X-gaD. Proteasepositive clones were detected in LB agar plates supplemented with !% Carnation powdered skim milk. Transformants were transferred onto LB agar plates containing skim milk and incubated overnight at 37°C. After exposure to chloroform vapor for 1 h, the plates were incubated at 37°C and then examined for areas of clearing around the colonies.
3.3. Electrophoresis and BSA substrate zymography Sodium dodecyl sulfate (SDS)-I0% polyacrylamide gel electrophoresis (PAGE) was done by the Laemmli method [29], using a Mini PROTEAN il cell (BioRad Laboratories, Richmond, CA). E. coli cell lysates were obtained by solubilization in 4% SDS buffer (with or without 10% /~-mercaptoethano'.) at either 37°C (30 rain) or 100°C (5 min). To detect protcolytic activity, SDS-PAGE gels were modified by the addition of bovine serum albumin (BSA) conjugated to arcrylamide in a method described previously [30]. 3.4. bnmunoblot analysis Western blotting was done by the method of Renarr and Sandoval [31]. Immunoreactive proteins were detected by incubation of the filter with rabbit antiserum raised against P. gingit'alis W50 whole cells, followed by goat anti-rabbit lgG (H + L) alkaline phosphatase conjugate (humanadsorbed) (BRL, Gaithersburg, MD). 3.5. Southern hybridization A non-radioactive nucleic acid detection system (BluGENE TM,BRL, Gaithersburg, MD) was used to detect chromosomal DNA fragments homologous to the cloned DNA fragment. To prepare the probe DNA, the recombinant plasmid, pYS307, was digested with Hindlll, and a 3.2-kb fragment containing the cloned protease gene was extracted from an agarose gel after elec-
275
trophoresis by the methods described by Silhavy et al. [27]. The DNA was labelled with biotin-7dATP as described by the supplier, and chromosomal DNA samples were prepared by digestion with Hindlll. Hybridization and detection were performed by the standard methods described by Sambrook et al. [26] and by the supplier, respect ively.
4. RESULTS AND DISCUSSION
4. !. Cloning of a P. gingit'alis protease gene Screening 21100 recombinant clones yielded a single colony capable of hydrolysing milk proteins. The zone of clearing appeared only after 2 weeks of incubation at 37°C, Plasmid isolated from this clone was transformed in E. colt JM83. It was observed that colonies which grew deep within the agar developed zones of protein hydrolysis, while those inoculated only on the agar surface did not. This suggests that the protease was only active under the reducing conditions that would exist deep in the agar. To confirm the requirement of the reducing conditions for activity of the cloned protease, the transformants were incubated in an oxygen-free environment after treatment with chloroform vapor. Under these conditions, clear zones appeared around all the colonies after 3 days. These results suggest that the cloned protease is only active under reducing conditions. Arnott et aL [23] also reported that protease-positive clones were isolated only when the screening procedure was done under anaerobic conditions. 4.2 Subclon#ag from pYS307 The recombinant plasmid, named pYS307, contains a 5.2-kb DNA insert. Retransformation of pYS307 into E. colt JM83 confirmed that the proteolytie enzyme is expressed by the genc encoded in the plasmid. A restriction endonuclease map of pYS307 is shown in Fig. 1. Based on this restriction map, subclones were constructed. The plasmid vector pTZI9R [281 was used to construct pYS307-21.- ) and pYS307-3( + ). This vector differs from pTZI8R only in the orientation of the multiple cloning site. As shown in Fig. 1,
,,L
i
I
L
t
IDI~..,m.~_, !
,,I
Fig. I. The restriction-enzyme maps of oYS31|7 and its subclones. ( + ) and ( - ) indicate the right and opl~)Sile orienlalions, rcsl~eclivel.v, ~'ith resider to the lac promoter. T h e broad line indicates lh¢ vector piasmid, p T Z I g R . Abbreviations are as follows: Ac. A t c | : Bin. B a m i l l : E¢, EcoRI; ttc, tlmcll: Hd. t l m d l l l : Kp. Kidtl; Ps. P~,tl: So, Sacl: Sp. Sphl.
the cloned gene was localized to a 2.4-kb DNA fragment between BantHl and Hindlli sites (Fig. 1: pYS307-2(+ ) and -2(-)). The orientation of the 2.4-kb fragment did not influence expression of the protease suggesting that the DNA fragment contains a P. gingicalis promoter which is recognized by E. colt transeriptton and translation systems.
4.3. Southern hybri¢h'zation The origin of the cloned DNA fragment was confirmed by Southern hybridization using a 3.2kb HindIll fragment as a probe (Figs. 1 and 2). Chromosomal DNA isolated from P. gingiralis W83, W50, and Wl2 digested with Hindlll all possessed a single 3.2-kb fragment which hybridized with the probe. In the case of P. gingicalls 33277, the probe hybridized with a 5-kb fragment. No hybridizing bands were detected with chromosomal DNA samples of P. (Bacteroides) asaccharolyticus, Bacteroides corporis, B. demicola, B. intermedius, B. lecii, B. ioescheii and B. melan#zogenicus strains. This suggests that the Hindlll fragment containing the cloned protease gene may be species-specific, at least under high stringency washing conditions. 4.4. Characterization of the cloned protease The cloned gene product was identified by SDS-PAGE and BSA substrate zymography. Protein profiles of celt lysatcs in Coomassie blue-
27¢~
I
2
3
4
5
6
7
8
9
10
11
12
kb 23.7
-
9.50 6.664.26
-
2.251.96 -
~:
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. ;
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Fig. 2. Southern hybridization. A 3.2-kb HindllI fragment of pYS307 (Fig. I) was used :is a prohe. All of the chromosom~J DNA samples were digested with HindllI. lame t, pYS3tl7 digested with llindlll: hmcs 2-12 art: chromosomal DNAs of BPB strains digested with Hindltt. lane 2, P. gingirafis W83; lane 3. P. gingicalis 33277: lane 4. P. gingicalis WSIk lane 5. P. gb~girali., WI2: hme O, P. (Bacteroides) a.~archarolyticus 252fi0: lane 7. B. corporis 33547, lane 8, B. denticota 33185: hme 9, B. intennedius 15032" lane ll). B. lecii 122090: lane il, B. Ioescheii 15930: lane 12. B. mehminogenicus 25845.
I
2
3
4
224 109 72
46
29 ki~ Fig, 3. Detection of proteolytic activity in a BSA*conjugaled acrylamide gel. Lanes 1 and 3, E. colt JM83 (pTZ|SR); lanes 2 and 4, E. colt JM83 (pYS307), S:tmples of whole cell lysutes were solubilized at 37°C flrr 311 rain in buffer without ~8mercaploethanol prior to electrophoresis.
stained gels did not reveal significant differences between E. colt JM83 (pTZ18R) and the recombinant clone. The expression of the cloned protease with a molecular mass of approximately 80 kDa can be detected by BSA substrate zymography with samples of the clone prepared in the absence of /3-mercaptoethanol (Fig. 3). When samples were prepared in the presence of/~-mercaptoethanok no proteolytic activity could be detected. In lanes containing cell lysates of E. colt JM83 (pTZ18R), several host proteins appear as Coomassie bluestained bands (Fig. 3, lane 1 and 3), these bands disappear in lanes containing cell lysates of the clone (Fig. 3, lane 2 and 4). This suggests that the cloned protease can degrade E. colt proteins during solubilization. The subclones which show protease activity on skim-milk plates also show a proteolytic band in BSA substrate zymography similar to that shown in the original clone. The proteolytic band could be detected only when a reducing agent, cysteine (50 mM), was added to
277
fractions of P. gb~gicalis 33277 using BSA substratc zymography. Among the eight different bands, one of the protease bands (P4) showed a molecular mass of 80 kDa on BSA substrate zymography. This protease was also inhibited by TLCK, iodoacetic acid and heavy metal ions. but not b'y TPCK and EDTA. This suggests that the cloned protease and P4 have different characteristics.
the assay system following electrophoresis. This result suggests that the cloned protease is thioldependent, and confirms the results observed in the colony screening assay.
4.5. Effects of protease h+hibitors The effects of several protease inhibitors was determined by performing BSA substrate zymography in the presence of inhibitors. The cloned protease was inhibited by N-a-p-tosyi-t.-lysine chloromethyl ketone (TLCK, 4 mM), N-tosyl-t.phenylalanine chloromethyl ketone (TPCK, 4 mM), phenylmethylsulfonyl fluoride (PMSF, 4 raM), iodoacetic acid (8 mM), EDTA (40 mM) and ZnCI., (10 raM), but not by CaCI, (10 mM) or MgCi., (10 raM). In the study of Grenier et ai. [30], a total of eight distinct bands of protcolytic activity could be detected with different cellular
1
2
3
4.6. Western immtmoblott#lg The identity of the cloned P. gingiralis protein was determined by immunoblotting with the antiserum raised to P. g#lgil'alis W50 whole cells (Fig. 4). A single reactive band was detected v, hen cell lysates of the clone containing pYS307 were prepared for elcctrophoresis in the absence of /3-mercaptoethanol (Fig. 4, lane 5). The posi-
4
$
6
214
,91
ki]
Fig. 4, Western immunoblot of the cloned protcase. Lane I. whole cellsof E. c+di JM83 (pTZISR) solubilizedat Itl~)~C:lanes 2 and 6, same as Lane I but solubilized al 37°C" lane 3, whole cells of E. coil JM83 (pYS307) st)lubilized at I(XFC; lanes 4 and 5, same as lane 3 but solubilized at 37°C. T h e samples of lanes 1-4 were solubilized in butler containing 10ek /~-mercaptoethanol. T h e samples of lanes 5 and 6 were solubilized in buffer without B-mercaptoethanol. Filters were reacted wilh antiserum raised against P. gingit'alis W50 whole cells. Secondary, anlibody was goat anti-rabbit lgG (H + L) alkaline phosphatase conjugate (human-adsorbed) (Gibco BRL, Gaithersburg. MD),
278
tion of the antibody staining hand was identical to the protcase activity sccn in the zymogram (Fig. 3, lane 2 and 4L A different result was observed when the sample was prepared in the presence of ~8-mercaptoethanol prior to dectrophorcsis. In this case, there was a major antibody reactive peptid¢ of 50 kDa and a minor peptide of 38 kDa (Fig. 4, lane 3 and 4). The minor band was not always detected. These resuits are noteworthy for comparison with the results of Smalley and Birss [t9]. They examined extracellular and vesicle-associated trypsin-like enzyme-containing fractions (VSF) of P. gingivails strains W50 and W50/BEI by gel-filtration chromatography, immunoblotting and zymography. Antiserum to the soluble trypsin-like enzyme of strain W50 reacted with a 50-kDa peptide of each subfraction but not with any peptide of molecular mass higher than 50 kDa, although gelatin substrate zymography following non-reducing SDS-PAGE revealed a major 80 kDa protease. It appears that in the presence of reducing agents there is auto-degradation of the proteasc. If this is the case, the site of attack is very specific as there is a very sharp band at 50 kDa.
ACKNOWLEDGEMENTS
This work was supported by the Medical Research Council.
References [1] Slots, L (19821 In: Host-Parasite Interactions in Periodontal Disease (Genco, R.J. and Mergenhagen, S.E.. Eds.). pp. 27-45. American Society for Microbiology, Washington. D.C. [2] Tanner, A.C.R., Huller, C.. Bratthall, G.T., Visconti. R.A. and Socransky, S.S. 119701 I. Clin, Periodontol. 6, 278-3117. [3] While, D. and Mayrand, D. (1981)J, Periodontal Res. 16, 259-265, [4] Zambon, J.J., Reynolds. H.S. and Slots. J. (19BI) Infect. Immun. 32, 198-203. [5l Mayrand. D. and Holt, S,C, (1988) Mierobiol, Rev. 52, 134-152.
[6] Van Winkclhoff.
A.J.. van Stecnhergen, T.J.M. and
Graaff. J.D. (1088)J. Clin. Periodontol. t5, 145-155. [7] Smalh:y. J.W., Birss. A.J. and Shuttleworth. C,A. 119881 Arch, Oral Biol. 33, 323-329. [8] Sundqvist. G.. Carlsson, J. and H~instri;,m, L. (19871 J. Periodonl. Res. 22, 31H)-31h% [9] Grenier, D.. Mayrand, D. and McBride. B.C. 119891 Oral MicmbioL Immunol. 4. 12-18. [111] Carlsson, J., Iliifling, J.F. and Sundqvisl, G,K. (19841 J. Med. Microbiol. I~. 39-46, [11] N ilsson, T,, (_arlsson. J. and Sundqvist. G. (1985) Infect. lmmun, 51k 467-471. [12] Schenkein, H.A. (19881J. Pcriodont. Res. 23, 187 192. [13] Schcnkein, H.A. (1991) Oral Microbiol. lmmunol. 6, 216- 221l. [14] Uitlo. V.-J., Larjava, tl., tteino. J. and Sorsa. T. (19891 Infect. Immun. 57, 2|3-218. [15] Abiko. Y.. tlayakawa. M., Mural. S. and Takiguchi. II. (1995) J. Dent. Res. 64. 106-11 I. [[6] Fujimura, S. and Nakamura, T. (19891 Oral Micmhiol. ImmunoL 4, 227-229. [17] Grcnier, D. and McBride, B.C. (|989) Infect. Immun, 57, 3265-3269. [18] Olsuka. M., Endo, J., Ilinode, D,, Nagata, A.. Maehara, R., Salo, M. and Nakamura, R. (1987) ,I. Periodont. Rcs. 22, 491-498. [19] Smallcy, J,W, and Birss. A.J. (19911 Oral Microbiol, Immunol. 6, 21)2-208. [20] Sorsa, T., Uitto, V.-J., Suomalaine. K., Turlo, tL and Lindy. S. 119871 J. Periodont. Res. 22, 375-381I. [21] Suido, H., Nciders, M.E., Barus, P,K., Nakamura, M., Mashimo, P.A. and Genco, R.J. ( 19871 J. Perit~dont. Rcs. 22. 4i2-418, [22] Tsutsui, H., Kinouchi, T.. W=.kano, Y. and Ohnishi. Y. (19871 Infect. Immun. 55, 420-427. [23] Arm)n, M.A., Rigg. G., Shah, it., Willi:lms, D., Wallace, A. and Roberts, I.S. (19901 Arch. Oral Bk)l. 35, 97S-99S. [24] Takahashi, N,, Kato, T. and Kuramitsu, i[.K. (1¢;~11 FEMS Microbiol, Left. 94, 135-t38. [25] Rodrlgucz. R.L. and Tail, R.C. ( 19831 Recombinant DNA Techniques. An Intnaluction. Addimn-Wesley Publishing Company, Reading, MA. [26] Sambrtmk, J., Fritsch, E.F. and Maniatis, T. (1989I Molecular Ckming: a Laboratory Manual (2nd edn). Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [27] Silhavy, T,J,, Berman, M.J. and Enquist, L.W. (1984) Experiments with gene fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [28] Mead, D.A.. Szczesna-Skorupa. E. and Kcmper, B, ( 1986} Prof. Eng. I, 67-74. [29] Laemmli, U.K. (197()) Nature (London) 277, 680-685. [30] Grenier, D., Chao, G. and McBride, B.C. {19891 Infect, lmmun. 57, 95-99. [31] Renart, J. and S:mdoval, I.V, 119841 Methods Enz~ymol. 1(}4, 455-46a.