- Email: [email protected]
Purification and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326
FEMS Microbiology Letters 167 (1998) 63^68
Puri¢cation and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326 Ruslina Kadri b
1;a
, Deirdre Devine b , William Ashraf a; *
a Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK Division of Oral Biology, Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK
Received 18 June 1998; revised 7 August 1998; accepted 7 August 1998
Abstract This study examined heat shock proteins (hsps) of the periodontal pathogen Prevotella intermedia and the closely related species, Prevotella nigrescens and Prevotella corporis. After heat shock at 45³C for 5 min, cell-free extracts were analysed by SDS-PAGE and Western blotting with polyclonal antibodies against Escherichia coli hsps. P. intermedia, P. nigrescens and P. corporis all expressed a DnaK homologue. The P. nigrescens DnaK was of a similar molecular mass to E. coli DnaK (70 kDa), whilst those of P. intermedia and P. corporis were approximately 69 kDa. DnaJ homologues were expressed in each species ; however, no homologue of GrpE was detected. P. intermedia DnaK was purified to homogeneity by ion-exchange and affinitychromatography, and was shown to restore activity of denatured luciferase. This molecular chaperone activity was enhanced by E. coli DnaJ and GrpE which are components of the Hsp70 molecular chaperone machine. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Prevotella ; Molecular chaperone ; DnaK; Periodontal pathogen; Heat shock protein
1. Introduction All organisms respond in a highly conserved manner to unfavourable conditions such as heat shock and stress (for example exposure to ethanol, heavy metals, amino acid analogues and viral infection), by the vigorous and transient acceleration in the rate of expression of a small number of speci¢c genes. The * Corresponding author. Tel.: +44 (1274) 383589; Fax: +44 (1274) 309742; E-mail: [email protected] 1
Present address: Department of Biomedical Sciences, University Putra Malaysia, 43400, UPM Serdang, Selangor Darul Ehsan, Malaysia.
products of these genes are commonly known as stress or heat shock proteins (hsps). Some of these proteins are also synthesised constitutively underlining their importance in normal cellular metabolism. The expression of hsps is also up-regulated in cells of the host and pathogen during infection. For general reviews see [1,2]. Stress responses have been extensively studied in Escherichia coli; for example, two of the main hsps, DnaK (hsp70) and DnaJ (hsp40), are also molecular chaperones and together with GrpE (hsp20) form the so called `Hsp70 chaperone machine' [3]. This machine mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes.
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 7 4 - 7
FEMSLE 8379 25-9-98
64
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Knowledge of such functions in obligately anaerobic bacteria is signi¢cantly more limited. The few studies which have examined responses of anaerobic bacteria have indicated fundamental di¡erences between these organisms and E. coli [4^6] and no heat shock response was observed at all in Treponema pallidum [7]. Recently, inter-species di¡erences were indicated in expression of hsps in a group of periodontal anaerobic bacteria using DNA probes and anti-hsp antibodies [9,10]. The periodontal anaerobe Prevotella intermedia has increasingly been implicated in the aetiology of periodontal disease [11^13]. The closely related Prevotella nigrescens is phenotypically indistinguishable by conventional tests but is more prevalent in health [14]. The reasons for the di¡erences between strains of these closely related species in respect to pathogenicity and isolation sites are unknown. Certainly, physiological stresses occur with the onset of periodontal disease: temperature and pH increase, gingival cerevicular £uid £ow increases and phagocytic cells provide oxidative stresses [15^17]. Responses to these stresses may be important in the survival of P. intermedia during the transition from health to disease and may contribute to its relative pathogenicity. We examined the heat shock response of strains of P. intermedia and P. nigrescens with respect to production of the DnaK chaperone. Therefore, the aim of this study was to examine the heat shock response in these closely related anaerobes and then to purify the DnaK homologue from P. intermedia which is an important periodontal pathogen. This work represents an important starting point in our e¡orts to explore the role of bacterial hsps and their relationship to dental health and disease at a molecular level.
2. Methods 2.1. Organisms and growth conditions Prevotella intermedia OMZ 326 and Prevotella nigrescens OMZ 327 were kindly provided by R. Gmuër (Department of Oral Microbiology and General Immunology, Dental Institute, University of Zurich, Switzerland), and were originally isolated from subgingival plaque. They were identi¢ed as P. intermedia
or P. nigrescens by enzyme electrophoresis, reactions with monoclonal antibodies and ribotyping [18,19]. Prevotella corporis (a closely related but phenotypically distinguishable species which is rarely isolated from oral sites) ATCC 33547 was included for comparison. All isolates were stored in glycerol (30% v/v) at 380³C. They were maintained on Columbia blood agar base (Oxoid) containing 5% (v/v) horse blood, and broth culture was in modi¢ed FUM [20]. All incubations were in an Anaerobic Work Station (Don Whitley Scienti¢c, Shipley, West Yorkshire, UK) under an atmosphere of 10% H2 , 10% CO2 and 80% N2 and at a temperature of 37³C. For heat stress, bacteria were grown to mid-exponential phase of growth and 1-ml aliquots were placed for 5 min in a heating block, placed within the Anaerobic Work Station, set at 45³C. Aliquots were then placed on ice and proteins were extracted. 2.2. SDS-PAGE and Western blotting SDS-PAGE analysis was carried out using 12.5% (w/v) polyacrylamide gels which were then electroblotted onto nitrocellulose. Immunological detection of DnaK, DnaJ and GrpE homologues was achieved using our laboratory's polyclonal anti-DnaK, -DnaJ and -GrpE antibodies and a peroxidase conjugate anti-rabbit IgG system [21]. 2.3. Preparation of cell-free extracts Frozen bacterial pellets containing 10% (w/v) sucrose in 50 mM Tris-HCl (pH 8.0) were allowed to thaw on ice. After thawing, Bu¡er K (180 mM spermidine-HCl (pH 8.0), 50 mM dithiothreitol, 50 mM EDTA, 0.9 M ammonium sulfate) containing fresh lysozyme (2 mg ml31 ) was added (0.5 ml Bu¡er K (g pellets)31 ). The suspension was left on ice for 45 min, followed by incubation at 37³C for 4 min and then returned to ice for a further 10 min. Following sonication on ice at maximum power for 5 s (U5) with 20-s intervals the extract was centrifuged at 12 000Ug for 60 min at 0³C. 2.4. Puri¢cation of DnaK The puri¢cation of Prevotella intermedia DnaK was based on a method previously described [22].
FEMSLE 8379 25-9-98
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Fig. 1. Western blot analysis of SDS-PAGE of cell-free extracts of Prevotella species grown at 30 and 37³C using antibodies against puri¢ed E. coli DnaK. Lane 1: P. intermedia OMZ326 at 30³C; lane 2: P. intermedia at 45³C; lane 3: P. nigrescens at 30³C; lane 4: Prevotella nigrescens at 45³C; lane 5: P. corporis at 30³C; lane 6: P. corporis at 45³C; lane 7: E. coli at 37³C. 100 Wg protein in each track.
100 ml cell free extract (94 mg protein) was applied to a pre-packed DEAE-Sepharose ion-exchange column (Pharmacia; 16/10 HiLoad1 column) attached to a Pharmacia Gradifrac1 low pressure protein puri¢cation system. The column had been previously washed with Bu¡er B (20 mM Tris-acetate (pH 7.5), 20 mM NaCl, 0.1 mM EDTA, 15 mM L-mercaptoethanol and 10% (v/v) glycerol). Bound proteins were eluted o¡ the column with a 20^350-mM linear gradient of NaCl in Bu¡er B. Collected fractions were analysed by SDS-PAGE and peak fractions containing the majority of protein of approximately 70 kDa were pooled. After adjustment to 3 mM MgCl2 the pooled fractions were applied to 1 ml ATP-agarose (Sigma) a¤nity column which had been equilibrated with Bu¡er D (20 mM Tris-acetate (pH 7.5), 0.1 mM EDTA, 15 mM L-mercaptoethanol, 3 mM MgCl2 ). The column was then washed with Bu¡er D containing 0.5 M NaCl to remove non-speci¢cally bound proteins, followed by Bu¡er D containing 3 mM ATP. The ATP-eluted fractions were dialysed overnight against Bu¡er B. All steps were performed at 4³C.
65
Fig. 2. Western blot analysis of SDS-PAGE of cell-free extracts of Prevotella species grown at 30 and 37³C using antibodies against puri¢ed E. coli DnaJ. Lane 1: P. intermedia OMZ326 at 30³C; lane 2: Prevotella intermedia at 45³C; lane 3: P. nigrescens at 30³C; lane 4: Prevotella nigrescens at 45³C ; lane 5: P. corporis at 30³C ; lane 6: P. corporis at 45³C. 100 Wg protein in each track.
7.4; Sigma) was diluted to 10 WM in un-folding bu¡er (25 mM HEPES-KOH (pH 7.6), 50 mM KCl, 5 mM MgCl2 , 5 mM L-mercaptoethanol, 6 M guanidium-HCl) and incubated for 1 h at 25³C. Denatured luciferase was then diluted to a ¢nal concentration of 80 nM in re-folding bu¡er (25 mM HEPES-KOH (pH 7.6), 50 mM KCl, 5 mM MgCl2 , 5 mM dithiothreitol, 1 mM ATP) containing combinations of 460 nM DnaK, 200 nM GrpE and 160 nM DnaJ (see Fig. 4). Puri¢ed E. coli DnaK, DnaJ and GrpE were obtained from Stressgene (Victoria, B.C., Canada). Ten Wl of the reaction mixture were
2.5. Luciferase re-folding assays Chaperone directed protein re-folding assays were performed as previously described [23]. A stock solution of luciferase (64 WM in 1 M glycylglycine, pH
Fig. 3. SDS-PAGE showing the puri¢cation of Prevotella intermedia DnaK. Lane 1: Molecular weight markers ; lane 2: cellfree extract (Wg) ; lane 3: DEAE-Sepharose (Wg); lane 4: ATPagarose (Wg).
FEMSLE 8379 25-9-98
66
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Fig. 4. Luciferase protein re-folding assay showing the molecular chaperone properties of P. intermedia OMZ 326 DnaK ; 8, denatured luciferase with E. coli DnaK, DnaJ and GrpE ; F, denatured luciferase with E. coli DnaK ; R, denatured luciferase with P. intermedia DnaK and E. coli DnaJ and GrpE; U, denatured luciferase with P. intermedia DnaK; a, denatured luciferase.
withdrawn at regular intervals, diluted in 1 ml assay bu¡er (25 mM glycylglycine (Sigma), 15 mM MgSO4 , 5 mM ATP) and after the addition of 7.5 nM luciferin were placed in a Luminometer (LKBWallac 1250) for detection of bioluminescence. Luciferase activity was determined and plotted as a percentage of the maximum obtained with non-denatured enzyme. The results presented are the means of at least three independent experiments.
3. Results and discussion While DnaK homologues have been sought in periodontal organisms, the co-chaperones have not, and the function of DnaK in these organisms has not been investigated. SDS-PAGE and Western blotting using anti-E. coli DnaK polyclonal antibody demonstrated expression of DnaK homologues in all three species (Fig. 1). The molecular mass of the P. nigrescens homologue was similar to the E. coli homologue (70 kDa), whereas those of P. intermedia and P. corporis were approximately 69 kDa.
In a previous study DNA digests from strains of P. intermedia and P. corporis were shown to react with a dnaK/J gene probe [8], although the DNA fragments which reacted were of di¡erent sizes. Di¡erences in the genes, or their products, in these species were not examined, nor was a representative of P. nigrescens. Many characteristics of P. intermedia and P. nigrescens have been examined with the aim of understanding the di¡erences in their ecology, but consistent di¡erences between the two species are rare and the reasons for the greater association of P. intermedia with periodontal disease are not understood. SDS-PAGE and Western blotting using anti-E. coli DnaJ polyclonal antibody demonstrated expression in P. intermedia OMZ 326, P. nigrescens OMZ 327 and P. corporis ATCC 33547 of a homologue of E. coli DnaJ (Fig. 2), at both 37³C and 45³C, with a molecular mass of approximately 40 kDa. Conversely, GrpE homologues were not detected in any isolate (data not shown) using this method. To facilitate our understanding of whether or not the `heat' or `stress response' has implications in dis-
FEMSLE 8379 25-9-98
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
ease we puri¢ed and functionally characterised the DnaK homologue from P. intermedia OMZ 326. DnaK was puri¢ed to homogeneity and its molecular mass of 69 kDa was con¢rmed (Fig. 3). The ability of the puri¢ed DnaK homologue to function as a molecular chaperone was assessed using a luciferase re-folding assay. Maximum restoration of luciferase activity was obtained with a mixture of E. coli DnaK, DnaJ and GrpE proteins (approximately 50% of native luciferase activity). E. coli DnaK alone restored activity to approximately 35% in comparison with 25% for the P. intermedia DnaK homologue. The activity of P. intermedia DnaK was increased in the presence of E. coli DnaJ and GrpE (approximately 35% of native luciferase activity; Fig. 4). In summary, the closely related species P. intermedia, P. nigrescens and P. corporis all expressed heat shock proteins homologous to E. coli DnaK and DnaJ, but not GrpE. Di¡erences in the P. intermedia and P. nigrescens DnaK homologues were indicated by di¡ering molecular masses. The DnaK homologue from the periodontal pathogen P. intermedia functioned as a molecular chaperone in a manner analogous to E. coli DnaK. Molecular chaperone activity has not been demonstrated in these organisms before. Our results also indicate that the P. intermedia DnaK is functioning as part of a chaperone machine, as does E. coli DnaK. The role of DnaK in infection and immunity has yet to be fully de¢ned. However, the puri¢cation of P. intermedia DnaK to homogeneity and its characterisation will now aid future studies with respect to periodontal disease. These include the biochemical and physiological analysis of dnaK mutants, gene cloning and an investigation into the potential immunological cross reactivity of patient sera against DnaK.
Acknowledgments Many thanks to Salma Al-Herran and Madge Hollowood for the preparation of antibodies against DnaK and GrpE, respectively. A special thanks to Dr Steve Picksley for helping with the ¢gures. R.K. is grateful to UPM (Malaysia) for ¢nancial support during her studentship.
67
References [1] Morimoto, R., Tissieres, A., Georgopoulos, C. (Eds.) (1990) Stress Proteins in Biology and Medicine, 450 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [2] Morimoto, R., Tissieres, A., Georgopoulos, C. (Eds.) (1994) The Biology of Heat Shock Proteins and Molecular Chaperones, 610 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [3] Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92, 351^366. [4] Woods, D.R. and Jones, D.T. (1986) Physiological responses of Bacteroides and Clostridium strains to environmental stress factors. Adv. Microb. Physiol. 28, 1^64. [5] Narberhaus, F. and Bahl, H. (1992) Cloning, sequencing and molecular analysis of the groESL operon of Clostridium acetobutylicum. J. Bacteriol. 174, 3282^3289. [6] Naberhaus, F., Giebeler, K. and Bahl, H. (1992) Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J. Bacteriol. 174, 3290^3299. [7] Stamm, L.V., Gheraardini, F.C., Parrish, E.A. and Moomaw, C.Y. (1991) [8] Lu, B. and McBride, B.C. (1994) Stress response of Porphyromonas gingivalis. Oral Microbiol. Immunol. 9, 166^173. [9] Vayssier, C., Mayrand, D. and Grenier, D. (1994) Detection of stress proteins in Porphyromonas gingivalis and other oral bacteria by Western immunoblotting analysis. FEMS Microbiol. Lett. 121, 303^307. [10] Ando, T., Kato, T., Ishihara, K., Ogiuchi, H. and Okuda, K. (1995) Heat shock proteins in the human periodontal disease process. Microbiol. Immunol. 39, 321^327. [11] Moore, L.V.H., Moore, W.E.C., Cato, E.P., Smibert, R.M., Burmeister, J.A., Best, A.M. and Ranney, R.R. (1987) Bacteriology of human gingivitis. J. Dent. Res. 66, 989^995. [12] Dahleèn, G., Wikstroëm, M., Renvert, S., Gmuër, R. and Guggenheim, B. (1990) Biochemical and serological characterization of Bacteroides intermedius strains isolated from the deep periodontal pocket. J. Clin. Microbiol. 28, 2269^2274. [13] Matto, J., Saarela, M., von Troil-Linden, B., Kononen, E., Jousimies-Somer, H., Torkko, H., Alaluusua, S. and Asikainen, S. (1996) Distribution and genetic analysis of oral Prevotella intermedia and Prevotella nigrescens. Oral Microbiol. Immunol. 11, 96^102. [14] Gmuër, R. and Guggenheim, B. (1994) Interdental supragingival plaque ^ a natural habitat of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, and Prevotella nigrescens. J. Dent. Res. 73, 1421^1428. [15] Bickel, M. and Cimasoni, G. (1985) The pH of human crevicular £uid measured by a new microanalytical technique. J. Periodont. Res. 20, 35^40. [16] Kung, R.T.V., Ochs, B. and Goodson, J.M. (1990) Temperature as a periodontal diagnostic. J. Clin. Periodontol. 17, 557^563. [17] Fedi, P.F. and Killoy, W.J. (1992) Temperature di¡erences at periodontal sites in health and disease. J. Periodontol. 63, 24^ 27.
FEMSLE 8379 25-9-98
68
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
[18] Devine, D.A., Pearce, M.A., Gharbia, S.E., Shah, H.N., Dixon, R. and Gmuër, R. (1994) Species-speci¢city of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens. FEMS Microbiol. Lett. 120, 99^104. [19] Pearce, M.A., Dixon, R.A., Gharbia, S.E., Shah, H.N. and Devine, D.A. (1996) Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses. Oral Microbiol. Immunol. 11, 135^141. [20] Gmuër, R. and Guggenheim, B. (1983) Antigenic heterogeneity of Bacteroides intermedius as recognised by monoclonal antibodies. Infect. Immun. 42, 459^470.
[21] Harlow, E. and Lane, D. (1988) Antibodies : A Laboratory Manual, 610 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [22] Zylicz, M., Ang, D., Georgopoulos, C. (1987) The grpE protein of Escherichia coli: puri¢cation and properties. J. Biol. Chem. 262, 17437^17442. [23] Buchberger, A., Schroder, H., Buttner, M., Valencia, A. and Bukau, B. (1994) A conserved loop in ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Struct. Biol. 1, 95^101.
FEMSLE 8379 25-9-98
Puri¢cation and functional analysis of the DnaK homologue from Prevotella intermedia OMZ 326 Ruslina Kadri b
1;a
, Deirdre Devine b , William Ashraf a; *
a Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK Division of Oral Biology, Leeds Dental Institute, University of Leeds, Clarendon Way, Leeds LS2 9LU, UK
Received 18 June 1998; revised 7 August 1998; accepted 7 August 1998
Abstract This study examined heat shock proteins (hsps) of the periodontal pathogen Prevotella intermedia and the closely related species, Prevotella nigrescens and Prevotella corporis. After heat shock at 45³C for 5 min, cell-free extracts were analysed by SDS-PAGE and Western blotting with polyclonal antibodies against Escherichia coli hsps. P. intermedia, P. nigrescens and P. corporis all expressed a DnaK homologue. The P. nigrescens DnaK was of a similar molecular mass to E. coli DnaK (70 kDa), whilst those of P. intermedia and P. corporis were approximately 69 kDa. DnaJ homologues were expressed in each species ; however, no homologue of GrpE was detected. P. intermedia DnaK was purified to homogeneity by ion-exchange and affinitychromatography, and was shown to restore activity of denatured luciferase. This molecular chaperone activity was enhanced by E. coli DnaJ and GrpE which are components of the Hsp70 molecular chaperone machine. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Prevotella ; Molecular chaperone ; DnaK; Periodontal pathogen; Heat shock protein
1. Introduction All organisms respond in a highly conserved manner to unfavourable conditions such as heat shock and stress (for example exposure to ethanol, heavy metals, amino acid analogues and viral infection), by the vigorous and transient acceleration in the rate of expression of a small number of speci¢c genes. The * Corresponding author. Tel.: +44 (1274) 383589; Fax: +44 (1274) 309742; E-mail: [email protected] 1
Present address: Department of Biomedical Sciences, University Putra Malaysia, 43400, UPM Serdang, Selangor Darul Ehsan, Malaysia.
products of these genes are commonly known as stress or heat shock proteins (hsps). Some of these proteins are also synthesised constitutively underlining their importance in normal cellular metabolism. The expression of hsps is also up-regulated in cells of the host and pathogen during infection. For general reviews see [1,2]. Stress responses have been extensively studied in Escherichia coli; for example, two of the main hsps, DnaK (hsp70) and DnaJ (hsp40), are also molecular chaperones and together with GrpE (hsp20) form the so called `Hsp70 chaperone machine' [3]. This machine mediates in a variety of highly conserved cellular processes including protein-folding reactions and the assembly/disassembly of protein complexes.
0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 3 7 4 - 7
FEMSLE 8379 25-9-98
64
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Knowledge of such functions in obligately anaerobic bacteria is signi¢cantly more limited. The few studies which have examined responses of anaerobic bacteria have indicated fundamental di¡erences between these organisms and E. coli [4^6] and no heat shock response was observed at all in Treponema pallidum [7]. Recently, inter-species di¡erences were indicated in expression of hsps in a group of periodontal anaerobic bacteria using DNA probes and anti-hsp antibodies [9,10]. The periodontal anaerobe Prevotella intermedia has increasingly been implicated in the aetiology of periodontal disease [11^13]. The closely related Prevotella nigrescens is phenotypically indistinguishable by conventional tests but is more prevalent in health [14]. The reasons for the di¡erences between strains of these closely related species in respect to pathogenicity and isolation sites are unknown. Certainly, physiological stresses occur with the onset of periodontal disease: temperature and pH increase, gingival cerevicular £uid £ow increases and phagocytic cells provide oxidative stresses [15^17]. Responses to these stresses may be important in the survival of P. intermedia during the transition from health to disease and may contribute to its relative pathogenicity. We examined the heat shock response of strains of P. intermedia and P. nigrescens with respect to production of the DnaK chaperone. Therefore, the aim of this study was to examine the heat shock response in these closely related anaerobes and then to purify the DnaK homologue from P. intermedia which is an important periodontal pathogen. This work represents an important starting point in our e¡orts to explore the role of bacterial hsps and their relationship to dental health and disease at a molecular level.
2. Methods 2.1. Organisms and growth conditions Prevotella intermedia OMZ 326 and Prevotella nigrescens OMZ 327 were kindly provided by R. Gmuër (Department of Oral Microbiology and General Immunology, Dental Institute, University of Zurich, Switzerland), and were originally isolated from subgingival plaque. They were identi¢ed as P. intermedia
or P. nigrescens by enzyme electrophoresis, reactions with monoclonal antibodies and ribotyping [18,19]. Prevotella corporis (a closely related but phenotypically distinguishable species which is rarely isolated from oral sites) ATCC 33547 was included for comparison. All isolates were stored in glycerol (30% v/v) at 380³C. They were maintained on Columbia blood agar base (Oxoid) containing 5% (v/v) horse blood, and broth culture was in modi¢ed FUM [20]. All incubations were in an Anaerobic Work Station (Don Whitley Scienti¢c, Shipley, West Yorkshire, UK) under an atmosphere of 10% H2 , 10% CO2 and 80% N2 and at a temperature of 37³C. For heat stress, bacteria were grown to mid-exponential phase of growth and 1-ml aliquots were placed for 5 min in a heating block, placed within the Anaerobic Work Station, set at 45³C. Aliquots were then placed on ice and proteins were extracted. 2.2. SDS-PAGE and Western blotting SDS-PAGE analysis was carried out using 12.5% (w/v) polyacrylamide gels which were then electroblotted onto nitrocellulose. Immunological detection of DnaK, DnaJ and GrpE homologues was achieved using our laboratory's polyclonal anti-DnaK, -DnaJ and -GrpE antibodies and a peroxidase conjugate anti-rabbit IgG system [21]. 2.3. Preparation of cell-free extracts Frozen bacterial pellets containing 10% (w/v) sucrose in 50 mM Tris-HCl (pH 8.0) were allowed to thaw on ice. After thawing, Bu¡er K (180 mM spermidine-HCl (pH 8.0), 50 mM dithiothreitol, 50 mM EDTA, 0.9 M ammonium sulfate) containing fresh lysozyme (2 mg ml31 ) was added (0.5 ml Bu¡er K (g pellets)31 ). The suspension was left on ice for 45 min, followed by incubation at 37³C for 4 min and then returned to ice for a further 10 min. Following sonication on ice at maximum power for 5 s (U5) with 20-s intervals the extract was centrifuged at 12 000Ug for 60 min at 0³C. 2.4. Puri¢cation of DnaK The puri¢cation of Prevotella intermedia DnaK was based on a method previously described [22].
FEMSLE 8379 25-9-98
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Fig. 1. Western blot analysis of SDS-PAGE of cell-free extracts of Prevotella species grown at 30 and 37³C using antibodies against puri¢ed E. coli DnaK. Lane 1: P. intermedia OMZ326 at 30³C; lane 2: P. intermedia at 45³C; lane 3: P. nigrescens at 30³C; lane 4: Prevotella nigrescens at 45³C; lane 5: P. corporis at 30³C; lane 6: P. corporis at 45³C; lane 7: E. coli at 37³C. 100 Wg protein in each track.
100 ml cell free extract (94 mg protein) was applied to a pre-packed DEAE-Sepharose ion-exchange column (Pharmacia; 16/10 HiLoad1 column) attached to a Pharmacia Gradifrac1 low pressure protein puri¢cation system. The column had been previously washed with Bu¡er B (20 mM Tris-acetate (pH 7.5), 20 mM NaCl, 0.1 mM EDTA, 15 mM L-mercaptoethanol and 10% (v/v) glycerol). Bound proteins were eluted o¡ the column with a 20^350-mM linear gradient of NaCl in Bu¡er B. Collected fractions were analysed by SDS-PAGE and peak fractions containing the majority of protein of approximately 70 kDa were pooled. After adjustment to 3 mM MgCl2 the pooled fractions were applied to 1 ml ATP-agarose (Sigma) a¤nity column which had been equilibrated with Bu¡er D (20 mM Tris-acetate (pH 7.5), 0.1 mM EDTA, 15 mM L-mercaptoethanol, 3 mM MgCl2 ). The column was then washed with Bu¡er D containing 0.5 M NaCl to remove non-speci¢cally bound proteins, followed by Bu¡er D containing 3 mM ATP. The ATP-eluted fractions were dialysed overnight against Bu¡er B. All steps were performed at 4³C.
65
Fig. 2. Western blot analysis of SDS-PAGE of cell-free extracts of Prevotella species grown at 30 and 37³C using antibodies against puri¢ed E. coli DnaJ. Lane 1: P. intermedia OMZ326 at 30³C; lane 2: Prevotella intermedia at 45³C; lane 3: P. nigrescens at 30³C; lane 4: Prevotella nigrescens at 45³C ; lane 5: P. corporis at 30³C ; lane 6: P. corporis at 45³C. 100 Wg protein in each track.
7.4; Sigma) was diluted to 10 WM in un-folding bu¡er (25 mM HEPES-KOH (pH 7.6), 50 mM KCl, 5 mM MgCl2 , 5 mM L-mercaptoethanol, 6 M guanidium-HCl) and incubated for 1 h at 25³C. Denatured luciferase was then diluted to a ¢nal concentration of 80 nM in re-folding bu¡er (25 mM HEPES-KOH (pH 7.6), 50 mM KCl, 5 mM MgCl2 , 5 mM dithiothreitol, 1 mM ATP) containing combinations of 460 nM DnaK, 200 nM GrpE and 160 nM DnaJ (see Fig. 4). Puri¢ed E. coli DnaK, DnaJ and GrpE were obtained from Stressgene (Victoria, B.C., Canada). Ten Wl of the reaction mixture were
2.5. Luciferase re-folding assays Chaperone directed protein re-folding assays were performed as previously described [23]. A stock solution of luciferase (64 WM in 1 M glycylglycine, pH
Fig. 3. SDS-PAGE showing the puri¢cation of Prevotella intermedia DnaK. Lane 1: Molecular weight markers ; lane 2: cellfree extract (Wg) ; lane 3: DEAE-Sepharose (Wg); lane 4: ATPagarose (Wg).
FEMSLE 8379 25-9-98
66
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
Fig. 4. Luciferase protein re-folding assay showing the molecular chaperone properties of P. intermedia OMZ 326 DnaK ; 8, denatured luciferase with E. coli DnaK, DnaJ and GrpE ; F, denatured luciferase with E. coli DnaK ; R, denatured luciferase with P. intermedia DnaK and E. coli DnaJ and GrpE; U, denatured luciferase with P. intermedia DnaK; a, denatured luciferase.
withdrawn at regular intervals, diluted in 1 ml assay bu¡er (25 mM glycylglycine (Sigma), 15 mM MgSO4 , 5 mM ATP) and after the addition of 7.5 nM luciferin were placed in a Luminometer (LKBWallac 1250) for detection of bioluminescence. Luciferase activity was determined and plotted as a percentage of the maximum obtained with non-denatured enzyme. The results presented are the means of at least three independent experiments.
3. Results and discussion While DnaK homologues have been sought in periodontal organisms, the co-chaperones have not, and the function of DnaK in these organisms has not been investigated. SDS-PAGE and Western blotting using anti-E. coli DnaK polyclonal antibody demonstrated expression of DnaK homologues in all three species (Fig. 1). The molecular mass of the P. nigrescens homologue was similar to the E. coli homologue (70 kDa), whereas those of P. intermedia and P. corporis were approximately 69 kDa.
In a previous study DNA digests from strains of P. intermedia and P. corporis were shown to react with a dnaK/J gene probe [8], although the DNA fragments which reacted were of di¡erent sizes. Di¡erences in the genes, or their products, in these species were not examined, nor was a representative of P. nigrescens. Many characteristics of P. intermedia and P. nigrescens have been examined with the aim of understanding the di¡erences in their ecology, but consistent di¡erences between the two species are rare and the reasons for the greater association of P. intermedia with periodontal disease are not understood. SDS-PAGE and Western blotting using anti-E. coli DnaJ polyclonal antibody demonstrated expression in P. intermedia OMZ 326, P. nigrescens OMZ 327 and P. corporis ATCC 33547 of a homologue of E. coli DnaJ (Fig. 2), at both 37³C and 45³C, with a molecular mass of approximately 40 kDa. Conversely, GrpE homologues were not detected in any isolate (data not shown) using this method. To facilitate our understanding of whether or not the `heat' or `stress response' has implications in dis-
FEMSLE 8379 25-9-98
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
ease we puri¢ed and functionally characterised the DnaK homologue from P. intermedia OMZ 326. DnaK was puri¢ed to homogeneity and its molecular mass of 69 kDa was con¢rmed (Fig. 3). The ability of the puri¢ed DnaK homologue to function as a molecular chaperone was assessed using a luciferase re-folding assay. Maximum restoration of luciferase activity was obtained with a mixture of E. coli DnaK, DnaJ and GrpE proteins (approximately 50% of native luciferase activity). E. coli DnaK alone restored activity to approximately 35% in comparison with 25% for the P. intermedia DnaK homologue. The activity of P. intermedia DnaK was increased in the presence of E. coli DnaJ and GrpE (approximately 35% of native luciferase activity; Fig. 4). In summary, the closely related species P. intermedia, P. nigrescens and P. corporis all expressed heat shock proteins homologous to E. coli DnaK and DnaJ, but not GrpE. Di¡erences in the P. intermedia and P. nigrescens DnaK homologues were indicated by di¡ering molecular masses. The DnaK homologue from the periodontal pathogen P. intermedia functioned as a molecular chaperone in a manner analogous to E. coli DnaK. Molecular chaperone activity has not been demonstrated in these organisms before. Our results also indicate that the P. intermedia DnaK is functioning as part of a chaperone machine, as does E. coli DnaK. The role of DnaK in infection and immunity has yet to be fully de¢ned. However, the puri¢cation of P. intermedia DnaK to homogeneity and its characterisation will now aid future studies with respect to periodontal disease. These include the biochemical and physiological analysis of dnaK mutants, gene cloning and an investigation into the potential immunological cross reactivity of patient sera against DnaK.
Acknowledgments Many thanks to Salma Al-Herran and Madge Hollowood for the preparation of antibodies against DnaK and GrpE, respectively. A special thanks to Dr Steve Picksley for helping with the ¢gures. R.K. is grateful to UPM (Malaysia) for ¢nancial support during her studentship.
67
References [1] Morimoto, R., Tissieres, A., Georgopoulos, C. (Eds.) (1990) Stress Proteins in Biology and Medicine, 450 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [2] Morimoto, R., Tissieres, A., Georgopoulos, C. (Eds.) (1994) The Biology of Heat Shock Proteins and Molecular Chaperones, 610 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [3] Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92, 351^366. [4] Woods, D.R. and Jones, D.T. (1986) Physiological responses of Bacteroides and Clostridium strains to environmental stress factors. Adv. Microb. Physiol. 28, 1^64. [5] Narberhaus, F. and Bahl, H. (1992) Cloning, sequencing and molecular analysis of the groESL operon of Clostridium acetobutylicum. J. Bacteriol. 174, 3282^3289. [6] Naberhaus, F., Giebeler, K. and Bahl, H. (1992) Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene. J. Bacteriol. 174, 3290^3299. [7] Stamm, L.V., Gheraardini, F.C., Parrish, E.A. and Moomaw, C.Y. (1991) [8] Lu, B. and McBride, B.C. (1994) Stress response of Porphyromonas gingivalis. Oral Microbiol. Immunol. 9, 166^173. [9] Vayssier, C., Mayrand, D. and Grenier, D. (1994) Detection of stress proteins in Porphyromonas gingivalis and other oral bacteria by Western immunoblotting analysis. FEMS Microbiol. Lett. 121, 303^307. [10] Ando, T., Kato, T., Ishihara, K., Ogiuchi, H. and Okuda, K. (1995) Heat shock proteins in the human periodontal disease process. Microbiol. Immunol. 39, 321^327. [11] Moore, L.V.H., Moore, W.E.C., Cato, E.P., Smibert, R.M., Burmeister, J.A., Best, A.M. and Ranney, R.R. (1987) Bacteriology of human gingivitis. J. Dent. Res. 66, 989^995. [12] Dahleèn, G., Wikstroëm, M., Renvert, S., Gmuër, R. and Guggenheim, B. (1990) Biochemical and serological characterization of Bacteroides intermedius strains isolated from the deep periodontal pocket. J. Clin. Microbiol. 28, 2269^2274. [13] Matto, J., Saarela, M., von Troil-Linden, B., Kononen, E., Jousimies-Somer, H., Torkko, H., Alaluusua, S. and Asikainen, S. (1996) Distribution and genetic analysis of oral Prevotella intermedia and Prevotella nigrescens. Oral Microbiol. Immunol. 11, 96^102. [14] Gmuër, R. and Guggenheim, B. (1994) Interdental supragingival plaque ^ a natural habitat of Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, and Prevotella nigrescens. J. Dent. Res. 73, 1421^1428. [15] Bickel, M. and Cimasoni, G. (1985) The pH of human crevicular £uid measured by a new microanalytical technique. J. Periodont. Res. 20, 35^40. [16] Kung, R.T.V., Ochs, B. and Goodson, J.M. (1990) Temperature as a periodontal diagnostic. J. Clin. Periodontol. 17, 557^563. [17] Fedi, P.F. and Killoy, W.J. (1992) Temperature di¡erences at periodontal sites in health and disease. J. Periodontol. 63, 24^ 27.
FEMSLE 8379 25-9-98
68
R. Kadri et al. / FEMS Microbiology Letters 167 (1998) 63^68
[18] Devine, D.A., Pearce, M.A., Gharbia, S.E., Shah, H.N., Dixon, R. and Gmuër, R. (1994) Species-speci¢city of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens. FEMS Microbiol. Lett. 120, 99^104. [19] Pearce, M.A., Dixon, R.A., Gharbia, S.E., Shah, H.N. and Devine, D.A. (1996) Characterization of Prevotella intermedia and Prevotella nigrescens by enzyme production, restriction endonuclease and ribosomal RNA gene restriction analyses. Oral Microbiol. Immunol. 11, 135^141. [20] Gmuër, R. and Guggenheim, B. (1983) Antigenic heterogeneity of Bacteroides intermedius as recognised by monoclonal antibodies. Infect. Immun. 42, 459^470.
[21] Harlow, E. and Lane, D. (1988) Antibodies : A Laboratory Manual, 610 pp. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [22] Zylicz, M., Ang, D., Georgopoulos, C. (1987) The grpE protein of Escherichia coli: puri¢cation and properties. J. Biol. Chem. 262, 17437^17442. [23] Buchberger, A., Schroder, H., Buttner, M., Valencia, A. and Bukau, B. (1994) A conserved loop in ATPase domain of the DnaK chaperone is essential for stable binding of GrpE. Struct. Biol. 1, 95^101.
FEMSLE 8379 25-9-98