INFECTION AND IMMUNITY, Nov. 1997, p. 4888–4891 0019-9567/97/$04.0010 Copyright © 1997, American Society for Microbiology
Vol. 65, No. 11
Cloning, Expression, and Sequencing of a Protease Gene from Bacteroides forsythus ATCC 43037 in Escherichia coli T. SAITO,1,2* K. ISHIHARA,2 T. KATO,2
AND
K. OKUDA2
Research & Development Department, Oral Care Business Headquarters, Sunstar Inc., Takatsuki, Osaka 569-111 and Oral Health Science Center and Department of Microbiology, Tokyo Dental College, Mihama, Chiba 261,2 Japan Received 5 May 1997/Returned for modification 17 June 1997/Accepted 14 August 1997
We have isolated and characterized an N-benzoyl-Val-Gly-Arg-p-nitroanilide-specific protease gene, designated prtH, from Bacteroides forsythus ATCC 43037. Nucleotide sequencing of the DNA insert from the clone (hereafter referred to as clone FST) revealed that the protease activity corresponded to an open reading frame consisting of 1,272 bp coding for a 47.8-kDa protein. When plasmid pFST was used as a probe in Southern hybridization, Sau3AI-digested chromosomal DNA of B. forsythus ATCC 43037 as well as the chromosomal DNAs of the isolated strains Ta4, TR5, and YG2 showed 0.6- and 0.8-kb hybridizing bands. The cell-free extracts of clone FST showed hemolytic activity on human blood cells. The hydrolytic activity of cell extracts of the pFST clone was inhibited by p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride, leupeptin, N-ethylmaleimide, iodoacetic acid, iodoaceteamide, and EDTA. ria-Bertani agar plates containing 1% skim milk (Difco Laboratories, Detroit, Mich.) and 60 mg of ampicillin per ml at 37°C overnight and then incubated anaerobically at 37°C overnight. Positive clones were detected as clear areas around colonies. Screening 2,000 recombinant clones yielded a single colony capable of hydrolyzing milk proteins. A clear zone appeared after 1 day of anaerobic incubation at 37°C following initial aerobic incubation at 37°C for 1 day. The positive clone was designated FST. To confirm the requirement for reducing conditions for activity of the cloned protease, the transformant was aerobically incubated on Luria-Bertani agar plates containing 1% skim milk with 1 or 10 mM dithiothreitol (DTT) and also without DTT. After aerobic incubation for 2 days, clear zones appeared and the diameter of the cleared zones increased in a dose-dependent manner (Fig. 1). These results suggested that the protease activity was enhanced under reducing conditions. Southern hybridization was performed by standard procedures (18, 20). Chromosomal DNA from B. forsythus ATCC 43037 and clinical strains Ta4, TR5, and YG2, completely digested with Sau3AI, all possessed 0.6- and 0.8-kb bands (data not shown). From the resulting DNA sequence, plasmid pFST was found to contain a 2,233-bp insert and 10 Sau3AI sites. The sizes of these bands agree with sequence data. Other smaller DNA fragments may not be detected on 1% electrophoresis gels. No hybridized bands were detected with a chromosomal DNA sample of a Porphyromonas gingivalis FDC381 strain. DNA was sequenced with double-stranded plasmid DNA as a template by the dideoxynucleotide chain termination method (19) with a dye primer sequencing kit (Applied Biosystems, Foster City, Calif.) or a dye terminator sequencing kit (Applied Biosystems) together with a synthetic oligonucleotide primer. Template DNA was prepared with the Wizard miniprep system (Promega, Madison, Wis.). The sequence was determined with an Applied Biosystems model 373A automated DNA sequencer. Both DNA strands were sequenced, with all sequences containing overlaps of the adjacent sequence. Nucleotide sequences were assembled and analyzed with the DNASIS software package (Hitachi, Tokyo, Japan). The sequence data indicated the presence of an open reading frame (ORF) on the insert contained in the plasmid (Fig. 2). The
A number of species of bacteria have been isolated from the human oral cavity, and several of these species have been implicated in periodontal diseases (13, 14). Some of these periodontopathic microorganisms have been shown to produce multiple proteolytic enzymes such as potential virulence factors, which are thought to participate in the pathogenesis of the disease via several mechanisms (8, 9, 22, 23). Bacteroides forsythus is an anaerobic, gram-negative, and fusiform organism (21) which is associated with advanced periodontitis as well as recurrent periodontitis (3, 11). The organism produces a trypsin-like protease (21) and a sialidase (6) that may be of importance in the pathogenesis of periodontal disease. Recently, a trypsin-like protease of B. forsythus was characterized, and the enzyme may be involved in the degradation of small peptides (5). However, currently available information regarding the virulence factors of this microorganism is limited. In this study, recombinant DNA technology was used to further characterize a protease produced by B. forsythus. The bacterial strains used in this study were B. forsythus ATCC 43037 from our culture collection and clinical isolates of B. forsythus Ta4, TR5, and YG2, which were kindly provided by I. Ishikawa, School of Dentistry, Tokyo Medical and Dental University. For isolation of genomic DNA, inocula from Trypticase soy agar plates were grown in Trypticase soy broth containing 5 mg of hemin per ml, 0.5 mg of menadione per ml, and 0.001% N-acetylmuramic acid (Sigma Chemical Co., St. Louis, Mo.). Escherichia coli HB101 was used in cloning and expression experiments. Extraction of chromosomal DNA from B. forsythus was performed as previously described (7). Chromosomal DNA from B. forsythus was partially digested with Sau3AI to yield fragments of 2 to 10 kb, the fragments were ligated with the plasmid vector pBluescript SK II1, and ligated plasmids were transformed into E. coli HB101. Standard procedures for recombinant DNA manipulation were used as previously described (18). Transformants were incubated aerobically on Lu* Corresponding author. Mailing address: Research & Development Department, Oral Care Business Headquarters, Sunstar Inc., 5-30-1, Kamihamuro, Takatsuki, Osaka 569-11, Japan. Phone: 81-726-94-7773. Fax: 81-726-95-0766. E-mail:
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FIG. 1. Effect of DTT on hydrolytic activity of clone FST on skim milk agar plates. Control lacks DTT.
ORF codes for a putative 41-kDa protein and was designated prtH (protease with hemolytic activity) gene. The prtH gene, which contains 1,272 bp, codes for a putative protease of 423 amino acids with a calculated molecular mass of 47,843 Da and an estimated pI of 9.69. The G1C content of the gene was 43.7% and closely resembled that estimated for chromosomal DNA from B. forsythus strains (45 to 47%) (21). The product of the prtH gene was designated the prtH protease. A ShineDalgarno sequence was observed upstream from the ATG
FIG. 2. DNA sequencing and deduced amino acid sequence of the prtH gene. A potential Shine-Dalgarno sequence is underlined.
codon at bases 177 to 182, and potential promoter elements homologous to the 210 and 235 consensus sequence could be identified at bases 83 to 111. The composition and spacing of the putative promoter and ribosome binding site are in very close agreement with published consensus sequences of E. coli (12, 17). However, determination of the transcriptional start site is necessary to accurately identify the promoter region for this gene. Two fragments were subcloned into E. coli HB101 with pBluescript and analyzed for the presence of protease activity. The plasmid pFST was digested with EcoO109 (which eliminated a 1.1-kb fragment) or ClaI (which eliminated a 1.0-kb fragment). These digested pFST fragments were self-ligated and remained upstream of both restriction enzyme sites. Selfligated pFST digested with EcoO109 or ClaI, resulting in the loss of the 39 ends of the ORF, failed to retain protease activity (data not shown). A comparison of the amino acid sequence encoded by the prtH gene with those of other cysteine or serine proteases in the DDBJ database with the FASTA and BLAST programs revealed no similarity with any protease domain or active site. Various protease substrates were used to test the extent of proteolytic activity by clone FST. Proteolytic activity was determined by mixing 50 ml of a cell-free extract of the clone FST (15 mg of protein) with 50 ml of chromogenic substrate (2 mM in dimethyl sulfoxide) and 150 ml of 50 mM Tris-HCl buffer (pH 8.0). The assay mixtures were then incubated at 37°C for 1 h. After the enzyme reaction was terminated, the release of p-nitroanilide was determined by measuring its A405. E. coli HB101 harboring pBluescript SK II1 was used as a control. The optimal pH of the cloned protease was found to be approximately 7.0 to 8.0. The extract specifically hydrolyzed N-benzoyl-Val-Gly-Arg-p-nitroanilide but not N-a-benzoylDL-Arg-p-nitroanilide (BAPNA), the chymotrypsin substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (SAAPNA), or Nbenzoyl-Pro-Phe-Arg-p-nitroanilide. As described previously (8), the effects of various protease inhibitors were examined with N-benzoyl-Val-Gly-Arg-p-nitroanilide as the substrate. The activity was drastically affected by the cysteine protease inhibitors p-toluenesulfonyl-L-lysine chloromethyl ketone hydrochloride (TLCK), leupeptin, N-ethylmaleimide, iodoacetic acid, and iodoaceteamide (Table 1). The activity was also reduced by EDTA and ZnCl2. However, serine protease inhibitors, including diisopropylfluorophosphate (DFP) and phenylmethylsulfonyl fluoride, had no significant effects. The activity was enhanced by CaCl2 and DTT. The ability to hydrolyze natural substrates was evaluated as described previously (8). Human plasma fibronectin, human immunoglobulin G, bovine serum albumin (all obtained from Sigma), and skim milk were used as substrates. Reaction mixtures were subjected to so-
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TABLE 1. Effects of chemical agents of enzyme activity of the cell extracts of clone FST Chemical agent
None Phenylmethylsulfonyl fluoride DFP Benzamidine Soybean trypsin inhibitor Iodoacetic acid Iodoacetamide N-ethylmaleimide TLCK Leupeptin DTT EDTA CaCl2 MgCl2 ZnCl2
Concn (mM)
Relative activitya (% 6 SD)
5 5 5 10b 5 5 5 5 5 5 5 5 5 5
100 88.6 6 13.8 100.3 6 4.8 27.3 6 12.1 104.1 6 3.5 3.4 6 2.0 2.2 6 1.7 2.0 6 1.3 3.3 6 0.4 1.2 6 0.3 105.8 6 12.9 1.1 6 0.2 108.3 6 4.9 94.3 6 3.2 10.2 6 0.8
a E. coli HB101 harboring pBluescript SK II1 used as a control had a relative activity of 0.8% 6 0.2%. b The concentration of soybean trypsin inhibitor is expressed in micrograms per milliliter.
dium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis. After electrophoresis, protein bands were stained with Coomassie brilliant blue. No proteolytic activity against native proteins (except skim milk) could be demonstrated in the clone extract. Because some clinical strains of B. forsythus show hemolytic activity on blood agar, we conducted the following examinations with clone FST to evaluate the hemolytic activity of the prtH protease. Erythrocyte lysis was determined as described by Kimizuka et al. (10). Determination of the level of hemolytic activity was carried out by SDS-PAGE analysis with a horse blood agar plate detection system. Duplication of the cell-free extract of clone FST were done by SDS-PAGE. One of the duplicate lanes was excised and renatured by two washes with water for 5 min and then two washes with 3 mM sodium citrate buffer (pH 6.8) containing 0.9% NaCl and 0.1% Triton X-100 for 5 min. Subsequently, the gel was washed five times (5 min each time) with 3 mM sodium citrate buffer (pH 6.8) containing 0.9% NaCl. The renatured gel was placed on a 2-mm-thick agarose gel containing 10% horse blood and incubated at 37°C overnight. Another lane was stained with Coomassie brilliant blue and used as a reference for localizing the functional protein. E. coli HB101 harboring pBluescript SK II1 was used as a control. Cell extracts of clone FST demonstrated hemolytic activities of 10.8 hemolytic units/mg on horse blood and 14.2 hemolytic units/mg on human blood, but no hemolytic activity was detected in the control sample. Overlaying an SDS-PAGE gel with a 10% horse blood agar gel resulted in a hemolytic band (Fig. 3). Hemolysis was clearly observed approximately at 41 kDa but not in any other area in the test or in control lanes. This molecular mass is smaller than that of the prtH protease (47,843 Da), suggesting that the protease may migrate anomalously in SDS-PAGE gels or that it processed in E. coli. The only characterized protease in B. forsythus is a trypsinlike protease (5). The proteolytic activity of clone FST was inhibited by cysteine protease inhibitor, while the trypsin-like proteases are inhibited by serine protease inhibitors, such as DFP. The prtH gene codes for a protease with a calculated molecular mass of 47,843 Da. Previously, Grenier (5) demonstrated that incubation of a cell envelope extract of B. forsythus
FIG. 3. The localization of hemolytic activity on horse blood agar plate following SDS-PAGE with cell envelope extracts. Lane 1, clone FST; lane 2, E. coli HB101 harboring pBluescript SK II1.
in the presence of 3H-labeled DFP formed two bands at 70 and 81 kDa. These results indicated that the prtH protease in the present study differs from trypsin-like protease. We revealed that the protease expressed in clone FST is an N-benzoyl-ValGly-Arg-p-nitroanilide-specific cysteine protease. B. forsythus as well as other periodontopathic microorganisms are present in increased numbers in active sites of subgingival plaque from subjects exhibiting actively destructive periodontal disease (3). Proteases from these periodontopathic microorganisms play a role in periodontitis (8, 9, 15, 22). The protease in our study may participate in the peptidolytic processing of human bioactive peptides. In addition, we found it to possess hemolytic activity. Hemolysins have been proven to be an important virulence factor of many pathogenic microorganisms (1). Substances in erythrocytes, such as the Fe ion, are known to be required by many periodontopathic bacteria (2, 4, 16, 24). It is suggested that the protease could promote the release of hemoglobin, which would increase the hemin concentration in periodontal sites. This protease was detected in the membranous fraction of B. forsythus (data not shown), indicating that it can react with erythrocytes and may be involved in the acquisition of iron from these cells. Although the role of this hemolytic protease needs to be investigated further, these results suggest that it can enhance the growth of the microorganism and other periodontopathic bacteria by supplying erythrocyte-derived substances. We are currently constructing a mutant of this protease in our laboratory to determine the importance of prtH in vivo. Nucleotide sequence accession number. The nucleotide sequence of prtH has been assigned DDBJ accession no. AB001892. We are grateful to I. Ishikawa for providing us with B. forsythus strains. This work was supported in part by Oral Health Science Center grants 961A01, 961A02, and 961C01.
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