Oral Mici-obiol Imtmmol 1992: 7: 349-356

Expression of Porphyromonas gingivalis proteolytic activity in Eschericiiia coli

T. E. Madden, T. M. Thompson, V. L. Clark Department of Dental Research, University of Rochester, New York, USA

Madden TE, Tlwtnp.son TM, Clark VL. Expression of Porphyromonas gingivalis proteolytie activity in Fscherichia coli. Oral Mierobiol Inimutiol 1992: 7: 349-356. © 1992 Munksgaard Porphyrotnonas gingivalis (formerly Baeteroides gingivalis) degrades numerous protein substrates including collagen, fibrinogen, fibronectin, gelatin, casein, immunoglobulins and complement components. In order to clone one or more of these protease genes, a genomic library was constructed with Sau3AI restriction fragments of chromosomal DNA from P gingivalis ATCC 33277 ligated into the temperature-regulated vector pCQV2, and expressed in Eschericiiia coli DH5o(mcr. The electro-transformants (3 x 10") were screened for general protease activity on Luria broth agar containing ampicillin (50 mg/1) and sodium caseinate (2%). One casein-hydrolyzing clone was detected and subcultured, and the activity of the cell extracts was characterized. We were able to show that the protease-positive clone, (pTEMI), had broad substrate specificity. Colorimetric assays indicated the hydrolysis of azocoU, azocasein, collagen, elastin-congo red and artificial substrates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to confirm that collagen, casein, fibrinogen and fibronectin were degraded by the clone. ••

Since Porphyromonas gingivalis is cultured at elevated levels from advanced lesions of periodontitis, it is a suspected pathogen (11, 39). This conclusion is supported by the observation that significant alveolar bone loss has been demonstrated in nonhuman primates subsequent to the implantation of P gingivalis in the periodontal microflora (23). A gram-negative, asaccharolytic anaerobe, P gingivalis depends upon the amino acids from protein degradation for its nutritional source of carbon and energy. Its proteolytic nature is thought to be a major virulence determinant since it degrades connective tissue and immune components (40). P gingivalis hydrolyzes collagen, gelatin, keratin, fibronectin, fibrinogen, IgA, IgG, plasma proteinase inhibitors, fibrinolysin, haptoglobin, hemopexin, transferrin (28), casein, albumin and artificial peptides (14, 48), as well as the complement components C3, C4 and factor B (38). Of particular importance as a suspected periodontal pathogen, P. gitigivctlis extracts show greater collagenase activity than those of other oral bacteria (36). The number of P. gitigivalis proteases

remains unknown despite partial purification of serine, thiol, trypsin-like and mctallo-enzymcs. In addition, the cellular location of these enzymes remains unclear because activity has been detected in the membrane, cytoplasm and supernatant cell fractions as well as from membrane vesicles (18, 41, 43-45, 47). Taking a genetic approach to isolating and characterizing the proteases of P gingivalis presented several potential difficulties. There is a paucity of published reports of genetic manipulations in this organism and only a few genes have been cloned to date (1, 2, 6, 9, 10, 30, 34, 46). Because the RNA polymerase of P. gingivalis appears to be significantly different from that of Escherichia eoli, it is not clear that P gitigivalis genes will be expressed from their own promoters when cloned in E. coli (24). Since expression of a foreign protease gene in E. eoli could be a lethal event, it is also necessary to control expression of cloned P. gitigivalis genes in E. coli by using an expression vector system. We report here our efforts to clone P. gingivalis protease genes under the control of the lambda promoter, in

Key words: protease: Porphyromonas vatis\ cloning; protease

gingi-

Virginia L. Clark. Associate Professor of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Medical Box 672, 601 Elmwood Avenue, Rochester, NY 14642, USA Accepted for publication May 22, 1992

the presence of a thermo-labile lambda repressor to regulate expression from the promoter. Although evidence is presented that one or more P. gingivalis protease genes was cloned, expression of the protease(s) under non-permissive conditions occurred, resulting in instability of the cloned DNA. Despite these problems with stability, the substrate specificity of the cloned proteolytic activity was determined and indicated that cither we cloned a single protease with broad specificity, or more likely, several protease genes. Material and methods Bacterial strains and growth conditions

P. gingivalis ATCC 33277 was grown on pre-reduced paromomycin-vancomycin laked blood agar (Carr Scarborough Microbiologicals: Decatur, GA), Schaedler agar (Oxoid, Hampshire, UK) plates supplemented (per liter) with 5 mg of hemin, 1 mg of vitamin K and 15 mg of nonfat dry milk, and in peptone yeast extract broth (PY) supplemented (per liter) with 5.0 mg of hemin, LO mg of vitamin K, 0.42 g of NaHCO3 and LO g of soluble starch.

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Cultures were incubated at 37°C in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI) under an atmosphere of 5% CO,, 10% H2, and 85% Nj, E. coli strains DH5amcr [F'tncrA mcrB mrr q)80dlacZAMl5 A(laeZYAargF)U169 endAl recAl li.sdR (fv.m^) supE44 thi-1 X-gyrA96 relAI] and HBlOl [.•iupE44 hsdS20 (r^.m^) recA13 ara-14 proA2 lacYl galK2 rpsL20 xyl-5 mtl-1] were grown aerobically at 37°C in Luria broth (LB) with shaking (250 rpm), and on Luria agar plates. Purity of the cultures was assessed by Gram stain and phase-contrast microscopy. When screening the E. coli transformants for protease activity, selective plates were supplemented (per liter) with 50 mg ampicillin and 20 g sodium caseinate.

a fusion protein from the ATG codon or as a non-fusion protein if its own ribosome binding site and ATG start codon are present and functional. The vector is derived from pBR322 and confers ampicillin resistance (35), Constructing the genomic library

P. gingivalis chromosomal DNA was partially digested with the restriction enzyme Sau3Al (Bethesda Research Laboratories), The fragments were separated on a 1% agarose gel and stained with ethidium bromide (0,5 fj.g/m\). The area of the gel containing 1.5 to 5 kilobase fragments was excised and the DNA fragments were recovered using the Elutrap electro-separation system (Schleicher & Schuell; Keene, NH), The pCQV2 vector was digested with BamHl (Bethesda Research LaboraIsolation of P. gingivalis chromosomal DNA tories), and the overhanging ends were P. gingivalis was grown in supplemented treated with calf intestinal phosphatase PY broth (22) to an ODjgo of 0,6, and (Promega, Madison, Wl) to remove centrifuged at 8000 x g for 25 min. The phosphate groups from the 5' ends, cell pellets were washed with 2 mol/1 thereby inhibiting re-ligation of the vecNaCl and resuspended in 42 mmol/1 tor, T4 DNA Hgase (Bethesda Research tris-HCl (pH 8,0), Cells were lysed by a Laboratories) was used (according to combination of incubation with 40 U/ the manufacturer's instructions) to fi\ lysozyme (Sigma Chemical Co,, St, ligate the DNA fragments into pCQV2, Louis, MO) and 2 freeze-thaw cycles These constructions were electroporatin a dry-ice acetone bath and a SVC ed into E. eoli DH5amcr at a field water bath. Eight phenol extractions strength of 12,5 kV/cm (Bio-Rad Labwere followed by 2 extractions in oratories Gene-Pulser, Richmond, CA), chloroform: isoamyl alcohol (24:1), RNAse A (1 fig/m\) (Sigma Chemical Co,) was added, and the mixture was Screening the library incubated at 37°C for 20 min, Protein- Transformants were grown at 32°C on ase K (30 U/ml) (Bethesda Research LB plates containing ampicillin (50 mg/ Laboratories, Gaithersburg, MD) was 1) and sodium caseinate (20 g/1). Coloadded, and incubation continued over- nies were replica-plated using 82 mm night at 4"C, Phenol extraction was fol- diameter nitrocellulose cicles (Schleilowed by ethanol precipitation and re- cher & Schuell), To initiate protein exsuspension of the DNA pellet in TE pression from the cloned DNA, one set buffer (10 mmol/1 Tris-HCl, 1 mmol/1 of plates was shifted to 42°C, while coloEDTA, pH 7,4). nies on the master plates remained repressed at 32°C, After 24 to 48 h, the induced cells were lysed by exposure to Cloning vector chloroform vapor for 5 min and incuThe expression vector, pCQV2 (35), was bated at 37°C for several hours. Proused to construct the genomic library. tease-positive colonies were surrounded This multiple copy vector contains the by clear areas in the agar, which persiststrong PR promoter from bacteriophage ed after inverting plates over HCl vapor. -i, an ATG translational start site im- This last step insured that clear zones mediately upstream of the BamHl clon- were caused by true proteolysis and not ing site, and the gene for the cl tempera- by differential casein dissolution from ture-sensitive A-phage repressor. This pH changes in the agar, repressor prohibits transcription from the PR promoter at 32"C, At 42°C, transcription occurs from PR, through the Re-transtormation with pTEiMI ATG codon, A gerie inserted into the Plasmid preparations of positive clones BamHl site will either be translated as (pTEM 1) were obtained using the large-

scale CsCl/ethidium bromide gradient technique and the miniprep boiling method (5), In order to confirm that the chimeric plasmid carried the protease phenotype, pTEMl was introduced into E. coli HBlOl via CaCl, transformation (5), and again into DH5amcr using electroporation (5), and the transformants were screened on LB/Amp/casein plates, as previously described. Preparation of cell extracts

E. coli DH5amcr(pCQV2), DH5amcr(pTEMl), HB101(pCQV2) and HBlOl(pTEMl) were grown in LB broth with ampicillin (50 mg/1) at 32°C with shaking for 24 h. In order to induce expression of the cloned protease gene, the temperature of the cultures was then shifted to 42°C for 0, 2, 4 or 16 h (overnight). The cultures were centrifuged for 15 min at 2700 x^ and the supernatants collected. The cell pellets were resuspended in 50 mmol/1 (Tris-HCl (pH 7,4), and sonicated on ice 3 times for 20 s at 40 V (Vibra-cell, Sonics and Materials, Danbury, CT; model ASI 3 mm diameter probe). The whole-cell lysates were collected, and cellular debris was removed by centrifugation at 3000 x^ for 5 min. The cytoplasmic and membrane cell fractions were separated by centrifugation at 30,000 x^ for 30 min. The membrane preparations were resuspended in 50 mmol/1 Tris-HCl (pH 7,4), All extracts were kept on ice and then stored at -70°C, The protein concentrations of the extracts were determined by protein assay (Pierce BCA, Rockford, IL), using bovine serum albumin (Sigma Chemical Co.) as the standard. Protein substrate assays

The following colorimetric assays were chosen for preliminary analysis of protease activity of the gene product. Cell extracts of DH5amcr(pCQV2) and HB101(pCQV2) were used as negative controls, Azocasein. 2,5% sulfanilamide-azocasein (Sigma Chemical Co,) in 0,5% Na^COj (pH 7,0) was incubated with cell extracts at 37'C for 18 h, and an equal volume of cold 10%i trichloroacetic acid was then added. Following centrifugation to remove precipitated peptides, the OD44,, of the supernatants was determined spectrophotometrically. An increase in OD demonstrated increased azocasein hydrolysis.

Cloning of P. gingivalis protease

351

Artificial substrates. Five artificial pep- determined using the azocasein assay. tides were solubilized according to the The assay was run at pH values ranging manufacturer's (Sigma Chemical Co,) from 5,5 to 8,6, by addition of buffer recommendations. Fifty mg/ml Nabcn- containing appropriate volumes of 0,2 zoyl-DL-arg-p-nitroanilide (Bz-arg- mol/1 Tris-maleate and 0,2 mol/1 NaOH pNA or BAPNA) in dimethyl sulfoxide, (15), Experimental salt concentrations 50 mg/ml N-succinyl-ala-ala-val-p-ni- ranged from 0,0 to 1,0 mol/1 NaCl and troanilidc (Suc-ala-ala-val-pNA) in 0,1 KCl, mol/1 NaOH, 20 mg/ml N-benzoyl-prophe-arg-p-nitroanilide (Bz-pro-phe-argResults Elastiti-congo red. Twenty mg of elastin- pNA) in methanol, 50 mg/ml N-p-tosylgly-pro-lys-p-nitroanilide acetate (TosylP. gitigivalis colonies grown on Schaedcongo red (Sigma Chemical Co,) in 50 gly-pro-lys-pNA) in methanol and 10 ler plates containing casein produced mmol/1 HEPES buffer (pH 7,0) was inmg/ml D-val-leu-lys-p-nitroanilide (Dsignificant clear zones in the agar. Neicubated with cell extracts at 37°C in a roller drum for 3 h, Following centri- val-leu-lys-pNA) in H2O were used as ther E. co//controls, DH5amcr(pCQV2) fugation, the OD495 of the supernatants substrates for the cell extracts. The total nor HBI01(pCQV2) produced clear reaction volumes of 250 ft\ contained 25 zones when grown on LB/Amp/casein was determined and an increase in OD ft\ substrate, 20 ftg cell extract protein, plates. Of approximately 3x10'' demonstrated increased elastin hyand 0,1 mol/1 Tris HCl buffer (pH 8,0), DH5amcr transformants, one colony drolysis. Trypsin (20 ftg) (Sigma Chemical Co,) was associated with a zone of clearance was used as the positive control, and no in the casein agar. Change in agar pH enzyme was added to the set of negative from colony growth (resulting in casein Collagena.se as.wy (49). Rat type I col- controls. Duplicate samples were incu- dissolution) was ruled out as the cause lagen (Chemicon; El Segundo, CA) was bated at 37°C for 18 h and absorbances of clear zones by inverting the plates solubilized in 5 mmol/1 acetic acid, di- were read at 405 nm (43), over HCl vapor for 5 min. Precipitated luted with PBS (7 mmol/1 acetic acid, Following subtraction of the OD of by this method, the casein became more 7 mmol/1 K2HPO4, 0,17 mol/1 NaCl, the negative control from all samples, opaque and clear zones around positive 0,02% sodium azide (NaN,)), pH 7,5, colonies remained unchanged. One hundred ft\ of a solution containing calculation of specific activities was as follows: 200 ng of of collagen was added to each well of a flat-bottom microtiter plate that was then left undisturbed at 4°C OD (pTEM 1) - OD (pCQV2) for a minimum of 7 days, Nonadhesive Specific activity of clone cell extract = 0.02 mg protein collagen was discarded and the wells were rinsed, as they were between all OD trypsin (substrate) - OD trypsin (no substrate) steps, with 0.05'^ Tween 20 in 0,05 Specific activity of trypsin = 0.02 mg protein mol/1 Tris-HCl, 0,15 mol/1 NaCl and 5 mmol/1 CaCl, (pH 7,8), First collagenase buffer (0,05 mol/1 Tris-HCl, 0,15 Determination of substrate degradation by The plasmid DNA (pTEMl) recovmol/1 NaCl and 5 mmol/1 (CaCl,, pH pTEMI clone 7,4) and then cell extracts were added Cell extracts were incubated with the ered from the protease-positive clone to the wells. Type II collagenase from substrates casein, bovine fibronectin, was introduced into E. coli strain Clostridiutn histolyticittn (740 U/mg) and bovine fibrinogen (Sigma Chemical HBlOl and again into DH5o:mcr, Cell (Sigma Chemical Co,) was used as a Co.). Each 5Q-ft\ reaction mixture con- extracts from cultures of the proteasepositive control. After incubation for taining 0,5 ftg cell extract protein, 2.5 ftg positive clones were prepared and used 7,5 h at room temperature (RT), rabbit substrate in 35 mmol/1 Tris-HCl buffer in several protease assays. The values anti-type 1 collagen antibody (Chem- (pH 7.2) was incubated at 37 C for 20 presented in Table 1 represent optical icon), diluted 1 : 1000 in PBS-Tween 20, h. Rat type 1 collagen (Chemicon) was densities (OD) following incubation of 1% BSA, was added for 45 min at RT, solubilized in 5 mmol/1 acetic acid, 0,1% 4 different substrates with the cell exPolyclonal anti-rabbit IgG peroxidase sodium azide (NaN,), The collagen re- tracts. Since the collagenase plate assay conjugate (Sigma Chemical Co,) diluted action mixtures, containing 5,0 fig col- measured disappearance of native col1 : 1000 in PBS-Tween 20, 1%. BSA was lagen in 50 mmol/1 Tris, 150 mmol/1 lagen from the bottom of the well, the added to the wells for 45 min at RT, NaCi, 5 nimol/1 CaCK (pH 7,5), were reciprocal of the OD was calculated. followed by the addition of 8 mmol/1 incubated at 37"C with 68 fig total This was done so that the results were o-phenylene-diamine: 0,3% H2O2 (9:1) supernatant protein for 3 h. The reac- easily compared with the results of the for 45 min. The reaction was stopped tion mixtures were analyzed by sodium azocasein, azocoll and elastin-congo red with 2 mol/1 H2SO4, and the OD4,2 was dodecyl sulfate-polyacrylamide gel elec- assays for which OD was positively correlated with substrate hydrolysis. determined spectrophotometrically. trophoresis (SDS-PAGE) (25), Whole-cell lysates of DH5amcrHence, this assay measured the disap(pTEM 1) significantly degraded elastinpearance of native collagen from the congo red, azocoll and azocasein, bottom of the wells. Since greater col- Determination of optimal reaction Supernatants of DH5amcr(pTEMl) lagenase activity yielded decreased OD, conditions for the pTEM1 enzyme the reciprocal values were calculated, The effect of pH and salt concentration clearly degraded azocasein, azocoll and collagen. Cell extracts from some culfor ease of interpretation. on activity of the cloned protease was

Azocotl. Ten mg of azocoll (Sigma Chemical Co.), in 50 mmol/1 Tris-HCl and 0,1 mmol/1 EDTA (pH 8,0) was incubated with cell extracts at 37 C with shaking for 3 h. After centrifugation, the OD5go of the supernatants was determined and an increase in OD demonstrated increased azocoll hydrolysis.

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Table I. Colorimetric substrate assays Without induction*

With induction al 42"Ct

Substrate

Cell extract

Elastin-congo red

DH5(pCQV2)tSN§ DH5(pCQV2)WCE§§ DH5(pCQV2)Mem^ DH5(pCQV2)Cyt#

0.00 0.37 0.06 0.06

0.01 0.74 0.01 0.00

DH5(pTEMl)tSN DH5 (pTEMl)WCL DH5(pTEMl)Mem DH5(pTEMl)Cyt

0.06** 0.65 0.10 0.04

0,04 1,50 0.33 0.03

DH5(pCQV2)SN DH5(pCQV2)WCL DH5(pCQV2)Mem DH5(pCQV2)Cyt

0,07 1.21 0.23 0.11

0.01 0.62 0.18 0.22

DH5(pTEMl)SN DH5(pTEMl)WCE DH5(pTEMl)Mem DH5(pTEMl)Cyt

0.80 1.82 0.20 0.47

1.18 1.27 0.73 0.27

DH5(pCQV2)SN DH5(pCQV2)WCE DH5(pCQV2)Mem DH5(pCQV2)Cyt

0.10 0.16 0.00 0.47

0.30 0,12 0.22 0.26

DH5(pTEMl)SN DH5(pTEMl)WCL DH5(pTEMl)Mem DH5(pTEMl)Cyt

1.57 0.58 0.27 0.49

2.38 1.64 0.25 0.89

DH5(pCQV2)SN DH5(pCQV2)WCE DH5(pCQV2)Mem DH5(pCQV2)Cyt

4.03 4.12 3.85 4.15

3.80 4.20 3.60 4.02

DH5(pTEMl)SN DH5(pTEMl)WCE DH5(pTEMl)Mem DH5(pTEMl)Cyl

5.42tt 4.49 3.80 4.28

6.17 4.34 3.40 3.95

Azocoll

Azocasein

Type I collagentt

Degradation of rat type 1 collagen was confirmed by SDS-PAGE. Supernatants of both E. eoli clones DH5amcr(pTEMl) and HBIOI(pTEMl) were collagenolytic. This was demonstrated by the significant diminution of the al, a2, and the /? collagen bands visualized on SDSPAGE (Fig. 1). No degradation was seen when collagen was incubated with supernatants of DH5amcr(pCQV2) or HBlOl(pCQV2), used as controls. In Fig. 2 fibronectin, undigested by

TYPE 1 COLLAGEN 1 2

3

4

ol

* Growth of cells at 32°C, the non-inducing temperature, f After growth at 32°C, the cultures were shifted to 42°C, derepressing the A repressor, inducing expression of the cloned gene. See Table 2 for induction times. J Cell extracts of DH5amcr(pCQV2) were used as negative controls and 3 different cultures orDH5amcr(pTEMl) were used as the experimental extracts. § Supernatant of DH5amcr(pCQV2) culture. §§ Whole-cell lysate. '^ Membrane preparations. jt Cytoplasmic extract. ** All DH5amcr(pTEMl) assays were performed in triplicate and the mean appears here, t t Coliagenase plate assay (see Material and methods). JJ values expressed as 1/OD; negative control (Tris or EB) 1/OD = 3.7-4.2. Positive control (clostridial collagenase) 1/OD = 6,0-9,6.

tures maintained at 32°C degraded the colorimetric substrates. The specific activities of the colorimetric assays are found in Table 2, When expression of the DH5amcr-(pTEMl) cloned protease was induced overnight by incubation at 42"C, the supernatant had a specific activity of 16.80 (z(OD/mg protein) against azocasein. This was compared with a specific activity of 1.20 by the DH5amcr(pCQV2) supernatant. Four-hour induction of DH5amcr(pTEMl) and 2-h induction of HBIOI(pTEM 1) resulted in specific activities of 13,83 and 13,25, respectively, of the whole cell lysates, Azocoll, which is azo dye covalently bound to cowhide or denatured collagen, was hydrolyzed by the

supernatant of DH5amcr(pTEMl). Minimal hydrolysis of elastin-congo red was detected in the supernatant of DH5amcr(pTEMl), Using the microtiter plate collagenase assay, DH5o(mcr(pTEMl) supernatant from cultures which had been induced at 42"C overnight appeared to degrade native rat type I collagen. The control supernatant of DH5amcr(pCQV2) had no activity in this assay. The specific activity of the clone extract was 1,51, compared with a specific activity of 11,1 produced by 1,0 fig (0,74 units) of type II clostridial collagenase. Thus, the crude supernatant extract of the clone was approximately 14'/o as active as the purified, commercially available enzyme.

Ftg. 1. Degradation of rat type 1 collagen by the cloned protease (lanes 2 and 4). Intact ft, al and a2 collagen subunits are evident in control lanes (1 and 3). Collagen (5 fig) was incubated with supernatants (68 fig total protein) from DH5amcr(pTEMl) and HBIOl(pTEMl) cultures and separated by SDS-PAGE (lanes 2 and 4, respectively). Equal amounts of collagen incubated with supernatants of DH5c(mcr(pCQV2) and HB101(pCQV2) cultures {lanes 1 and 3, respectively).

Cloning of P. gingivalis protease Table 2. Specific activities of the cloned and control cell extracts against colorimetric substrates Induction Specific Substrate Cell extract time* Activityt Azocasein

DH5(pCQV2):|:SN§ DH5(pCQV2)WCE§§ DH5(pTEMl)"SN DH5(pTEMl)WCL

DH5(pCQV2)SN DH5(pCQV2)WCL DH5(pTEMl)SN DH5(pTEMl)WCL DH5(pCQV2)SN DH5(pTEMI)SN

overnight overnight overnight overnight 2 hours 2 hours 2 hours 2 hours 4 hours 4 hours 4 hours 4 hours overnight overnight

13.25 0,27 5.85 4,48 13.83 0.003 0.790

DH5(pCQV2)SN DH5(pTEMl)SN

overnight overnight

0.003 0.078

HB101/r(pCQV2)SN HB101(pCQV2)WCL HBlOKpTEMDSN HBlOl (pTEMl)WCL

Azocoll Elastin-congo red

1.20 0.39 16.80 1.61 0.35 2.07

FIBRONECTIN 1 2

3

4

200t

'% £

1 lo07.

1.39

0.000 overnight DH5(pCQV2)SN 1.51 overnight DH5(pTEMI)SN Incubation at 42 C lo induce expression of the cloned protein, t Change in optical densuy/ mg prolein. % E. coii DH5amcr containing the plasmid pCQV2. § Supernatant. §§ Whole-cell lysate. ^ E. coii DH5amcr containing the cloned plasmid pTEMl. // E. coli HBlOl.

66-

45-

Type 1 collagen

DH5amcr(pCQV2), was degraded by DH5amcr(pTEMI) supernatant and HBlOl(pTEMl) whole-cell lysate. The discrete degradation products of fibronectin were approximately 175, 116, 85 and 30 kDa. DH5c(mcr(pTEMl) supernatant and whole-cell lysate hydrolyzed fibrinogen (Fig, 3), Similarly, HBlOl(pTEMl) whole-cell extract and supernatant hydrolyzed fibrinogen. Casein was significantly degraded by supernatant and whole-cell lysate of DH5o(mcr(pTEMl), and HBlOl(pTEMl) (Fig, 4), Cell extracts of the pTEMl clone hydrolyzed BAPNA, stic-ala-alaval-pNA, bz-pro-phe-arg-pNA, tosylgly-pro-lys-pNA, and D-val-leu-lyspNA (Table 3), The results of the pH optimum experiments suggested that the cloned protease was most active against azocasein between pH 6,8 and 8,2, The salt optimum was 0,1 to 0,2 M,

353

ii

m

Discussion

We achieved expression of P gingivalis ATCC 33277 protcolytic activity in 2 strains of E. coli. The results of the substrate profile and the pH optimum experiments suggest that the cloned enzymatic activity is a neutral or slightly alkaline cndopeptidase with broad substrate specificity. The colorimetric assays demonstrated that cell extracts of DH5amcr(pTEMl) and HBlOl(pTEMl) hydrolyzed azocoll, azocasein, elastin and 5 artificial peptides. SDS-PAGE was used to monitor degradation of type 1 collagen, casein, fibrinogen, and fibronectin. Since azocoll and azocasein were degraded by the cultures of the clone that were maintained at 32 C, it appeared that the gene(s) was not under the control of the cl temperature-sensitive / repressor in pCQV2. Fig. 2. Degradation of fibronectin by the Characteristics of this cloned pro- cloned protease (lanes 3 and 4). Bovine fib-

Table 3. Artificial substrate degradation HBIOUpTEMI)WCL DH5(pTEMl)SN Substrate 5.40 4.25t Bz-arg-pNA (BAPNA)* Suc-ala-ala-val-pNAJ 25.80 7.45 Bz-pro-phe-arg-pNA§ 3.15 6.25 ND~ 19.85 Tosyl-gly-pio-lys-pNAiJS ND D-val-leu-lys-pNA# 23.40 Na-benzoyl-DL-arg-p-nitroanilide (BAPNA). t Calculation of specific activities described in Material and methods. % N-succinyl-ala-ala-val-p-nitroanilide. § N-benzoyl-pro-phe-arg-pnitroanilidc. S N-p-losyl-gly-pro-lys-p-nitioanilide acetate. ^ Not determined. » D-val-leulys-p-nitroanilide.

ronectin (2.5 fig) was incubated with supernatant from DH5amcr(pTEMl) and wholecell lysate from HBlOl(pTEMl) cultures and separated by SDS-PAGE (lanes 3 and 4, respectively). Following incubation with supernatant from DH5amcr(pCQV2) cultures (lane 2), protein bands appeared identical to intact nbronectin (lane 1). Standard protein molecular weight markers indicated in kilodaltons.

354

Madden et al.

tease were similar to P. gingivalis proteases which have previotisly been characterized. Others have shown degradation of native and denatured collagen by this organism (16, 17, 29), Our recombinantly derived E. eoli (pTEMl) clone degraded azocoll, a commonly used denatured collagen

substrate. Proteolytic activity reported by Smalley et al, (42), Robertson et al, (36), Abiko et al, (3), Mayrand & Grenier (29) and Grenier & Mayrand (16) supports the existence of a P. gingivalis collagenase. In the present study, disappearance of the (i, al, and a2 bands following incubation of type I collagen with extracts of E. coli (pTEMl) indicates that the coUagen-

FIBRINOGEN 1 2 3 4 5 6 7 8 9 200-

I

CASEIN 1 2 3 4 5 6 7 8 9

200

Fig. 3. Degradation of fibrinogen by the cloned protease (lanes 4, 5, 8 and 9). Bovine fibrinogen (2.5 ftg) was incubated with supernatant and whole-cell lysate from DH5amcr(pTEMI) cultures (lanes 4 and 5, respectively) as well as supernatant and whole-cell lysate from HBlOl(pTEMl) cultures (lanes 8 and 9, respectively). Following incubation with DH5o(mcr(pCQV2) supernatant (lane 2) or whole-cell lysate (lane 3) protein bands appeared identical to intact fibrinogen (lane 1). Similarly, following incubation with HBI01(pCQV2) supernatant (lane 6) or whole-cell lysate (lane 7), protein bands appeared identical to intact fibrinogen. Standard protein molecular weight markers indicated in kilodaltons.

Fig. 4. Degradation of casein by the cloned protease (lanes 4, 5, 8 and 9). Bovine casein (2.5 fig) was incubated with supernatant and whole-cell lysate from DH5amcr(pTEM I) cultures (lanes 4 and 5, respectively) as well as supernatant and whole-cell lysate from HBlOl(pTEMl) cultures (lanes 8 and 9, respectively). Following incubation with DH5imcr(pCQV2) supernatant (lane 2), whole-cell lysate (lane 3), or with HBlOl(pCQV2) supernatant (lane 6) or whole-cell lysate (lane 7), protein bands appeared identical to intact casein alone (lane I). Standard protein molecular weight lnarkers indicated in kilodaltons.

asc may have been cloned. Visualizing multiple small bands in the pTEMl lanes of the SDS-PAGE suggests that the cloned product cleaved multiple sites within the collagen molecule. Similarly, Birkedal-Hansen et al, (7) demonstrated multiple collagen degradation products after incubation with detergent extracts of P. gitigivalis 381, Since our preparations also contained E. coli proteins, further comparisons of the size and number of degradation products cannot be made. Four strains of P. gitigivalis, including ATCC 33277, have been shown to degrade fibrinogen (20). Lantz et al. (26) demonstrated that two thiol-dependent, trypsin-like proteases of M^ 120,000 and 150,000 are found on the cell surface and are able to bind and cleave fibrinogen into 2 major fragments. In contrast, our cloned product completely degraded fibrinogen. Fibronectin, another serum protein, was degraded by cell extracts of E. coli (pTEMl) into products of approximately 175, 116, 85 and 30 kPa. A similar pattern of degradation was previously obtained when fibronectin was exhaustively digested by the trypsin-like enzyme and the membrane vesicles of P. gingivalis W50 (42). Trypsin-like proteases from P. gingivalis ATCC 33277 and 381 hydrolyze lysine- and arginine-containing substrates (12, 43). Our recombinantly derived proteolytic product demonstrated similar activity by degrading the artificial substrates tosyl-gly-prolys-pNA, BAPNA, bz-pro-phc-argpNA and D-val-leu-lys-pNA. The activity was optimal at a salt concentration (0.1-0.2 mol/1) similar to the enzyme prepared by Sorsa et al. (43). However, cleavage of suc-ala-ala-valpNA and eiastin-congo red (both substrates of elastase) by the clone were perplexing, since there is little evidence in the literature for the existence of a P. gitigivalis elastase (28). Casein, a substrate with diverse protein sequence, has been previously used to detect non-specific protease activity in Bacteroides tiielaninogenicus and P. gingivalis (13, 21, 33), In this study, significant degradation of casein by E. coli (pTEMl) was determined by SDSPAGE and azocasein assay. These data strengthen the conclusion that broad substrate specificity is a feature of our cloned gene product. P. gingivalis protease activity has been localized to every isolated cell fraction

B-,

Clonitig of P. gingivdVis protease

(8, 12, 17, 19, 20, 31). Incubation of DE00159, both from the National Instithis organism for greater than 4 days, tutes of Health, however, showed protease activity predominantly in the supernatant (32, 14). References Similarly, while azocasein degradation 1. Abiko Y. Hayakawa M, Aoki H. Kikuby the pTEMl clone was initially cellchi T, Shimatake H, Takiguchi H. Clonassociated, when induction at 42"C was ing of a Bacteroides gingivaiis outer extended overnight, the majority of the membrane protein gene in Fsdierielita coli. Arch Oral Biol 1990: 35: 689-695. activity was found in the supernatant. 2. Abiko Y, Hayakawa M, Aoki H, TakiIt is most likely in both cases that the guchi H. Gene cloning and expression of enzymes were released into the medium a Bacteroides gii!givaii.s-spedfic protein by cell lysis rather than via active export antigen in Escherichia coli. Adv Dent Res through the membrane. 1988:2: 310-314. Since we were unable to analyze the 3. Abiko Y, Hayakawa M, Murai S, Takiinsert DNA of pTEMl because of its guchi H, Glycylprolyl dipeptidylaminogenetic instability, we cannot definitely peptidase from Bacteroides gingivalis. J Dent Res 1985: 64: 106-111. conclude whether we cloned a single P. 4. Arnott MA, Rigg G, Shah H, Williams gingivalis protease gene, encoding a D, Wallace A, Roberts IS. Cloning and broad spectrum protease, or several expression of a ForfjliyronioiuLs {Bacterprotease genes. It is likely, however, that oides) gingivalis protease gene in Fscherseveral protease genes were cloned. ichia 'eoii. Arch Oral Biol 1990: 35: Takahashi et al. (46) have reported clon97S-99S. ing a P gitigivalis DNA fragment con5. Ausebel FM, Brent R, Kingston RE et taining two distinct protease genes. This al, ed. Current protocols in molecular indicates that at least 2 P. gingivalis probiology. Vol. 1. Boston: Greene Pubtease genes are genetically linked. Adlishers and Wiley-Interscience, 1987: K6.1-1.6.2and 1.8.1-1,8,8. ditionally, in our laboratory we have 6. Banas JA, Ferrelti JJ, Progulske-Eox A. found that purified P. gingivalis proIdentification and sequence analysis of a teases with activity against matrix promethylase gene in Porf)hyronwna.s gingiteins, such as type IV collagen, gelatin vaiis. Nucleic Acids Res 1991: 15: and fibronectin, each have a narrow 4189^192. substrate specificity with little activity 7. Birkedal-Han,sen H, Taylor RE, Zanibon against casein (J. D. Grubb & V L, JJ, Barwa PK.. Neiders ME. CharacterClark, in preparation). ization of collagenolytic acitivity from strains of Bacteroides gingtvali.s. J PerioProteases capable of degrading coldont Res 1988: 23: 258-264. lagen, fibrinogen, fibronectin and other 8. Carlsson JJ, Hofiing JF, Sunqvist GK. host proteins have a central role in the Degradation of albumin, haemopexin, bacterial infection and tissue destruchaptoglobin and transferrin, by blacktion of periodontai diseases. Since puripigmented Bacteroides species. J Med fication of these bacterial enzymes has Microbiol 1984: 18: 39-46. been difficult and incomplete, molecular 9. Choi J, Takahashi N, Kato T, Kuramitsu biology offers alternative methods for HK. Isolation, expression, and nucleotheir study (27), In this study we have tide sequence of the sod gene from Porcloned, expressed and characterized P. piivromonas gingivaiis. Infect Immun 1991: 59: 1564-1566. gitigivalis proteolytic activity with broad 10. Dickenson DP, Kubiniec MA, Yoshisubstrate specificity. However, it is clear mura F, Genco RJ. Molecular cloning that when high levels of this proteolytic and sequencing of the gene encoding the activity are expressed in E. coli, it is a fimbrial subunit protein of Bacteroide.t lethal event, resulting in instability of gingivalis. J Bacteriol 1988: 170: the clone. This suggests that shotgun 'i 658-1665. approaches, in which clones are iden11. Dzink JL, Socransky SS, Haffaje AD. tified based on enzymatic activity may The predominant cultivable microbiota not easily lead to isolation of stable P. of active and inactive destructive periogitigivalis clones, and other cloning dontai diseases. J Clin Periodontol 1988: 15: 316-323. strategies should be considered.

Acknowledgement

This investigation was supported by USPHS Research Grant 5R01 DEO8512 and TEM is supported by Dentist-Scientist Award 5K16

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