JOURNAL OF BACTERIOLOGY, Jan. 2005, p. 795–799 0021-9193/05/$08.00⫹0 doi:10.1128/JB.187.2.795–799.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 187, No. 2
Cloning, Purification, and Enzymatic Properties of Dipeptidyl Peptidase IV from the Swine Pathogen Streptococcus suis Marie-Claude Jobin,1 Gabriela Martinez,2 Julie Motard,1 Marcelo Gottschalk,2,3 and Daniel Grenier1,3* ´ cologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, Quebec City,1 and Groupe de Recherche en E Groupe de Recherche sur les Maladies Infectieuses du Porc, Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montre´al,2 and Canadian Research Network on Bacterial Pathogens of Swine, Natural Sciences and Engineering Research Council of Canada,3 Saint-Hyacinthe, Quebec, Canada Received 5 July 2004/Accepted 15 October 2004
In this study, the dipeptidyl peptidase IV (DPP IV) of the swine pathogen Streptococcus suis was cloned, overexpressed in Escherichia coli, and characterized. The coding region comprises 2,268 nucleotides containing an open reading frame that codes for a 755-amino-acid protein with a calculated molecular mass of 85 kDa. The amino acid sequence contained the sequence Gly-X-Ser-X-X-Gly, which is a consensus motif flanking the active-site serine shared by serine proteases. The recombinant DPP IV showed a high affinity for the synthetic peptide glycine–proline–p-nitroanilide and was strongly inhibited by Hg2ⴙ and diprotin A. S. suis S735 (European virulent reference strain) and 89–999 (North American virulent field strain) (19), both of which belong to serotype 2, were routinely grown in Todd-Hewitt broth (BBL Microbiology Systems, Cockeysville, Mass.) or on Todd-Hewitt agar at 37°C under aerobiosis. Escherichia coli TOP10, which was purchased from Invitrogen (Burlington, Ontario, Canada), was grown in Luria-Bertani medium (15) at 37°C under aerobiosis. When required, ampicillin (100 g/ml) was added to the culture medium. S. suis S735 DNA was isolated as described previously (22). Chromosomal DNA of 16 additional S. suis serotype 2 strains (31533, 94–623, 89–1591, 90–1330, 91–1804, 93-614-3, 94–3037, 95–8242, 89-3977b, 96– 52466, ITA228, T15, TD10, 4/3H1, 4/39H1, and 4/40H2), prepared in a previous study (4), were also used to screen for the presence of the S. suis S735 DPP IV gene. Genomic DNA was amplified by PCR using KlenTaq DNA polymerase (AB Peptides, St. Louis, Mo.) or Vent DNA polymerase (New England Biolabs, Mississauga, Ontario, Canada) for its high fidelity, following the manufacturer’s instructions. The primers N1 (5⬘ ATTTCACTCCTCAGTCAT 3⬘) and N2 (5⬘ GTTCACTCTA TCTCCAACCTTC 3⬘) were designed following the identification of a consensus sequence from several streptococci DPP IV genes and a BLAST against the S. suis P1/7 (serotype 2) genomic sequence produced by the Sanger Institute (www.sanger .ac.uk). They amplified a 2.6-kb fragment containing the DPP IV gene (Ss-xPDPP gene) from chromosomal DNA of S. suis which was directly used for DNA sequencing. Internal sequencing of the gene was performed using primers N3 (5⬘ CTATGGCAACCTTTTCAAC 3⬘) and N4 (5⬘ GGCAGTTG GTAGTTGTTC 3⬘). The above primers were purchased from Invitrogen. The cloned fragment presented a coding region of 2,268 nucleotides and had a G⫹C content of 49%, which was much higher than the usual proportion found in the S. suis genome (38 to 42%) (18). The sequence (approximately 3,000 nucleotides) before and after the gene has a G⫹C content of 36 and 37%, respectively, and a palindrome of 10 nucleotides was identified at ⫺316 from the start codon and at position 2,325
Streptococcus suis, an early colonizer of the upper respiratory tract, is a major swine pathogen worldwide (13). Thirtyfive serotypes (1 to 34 and 1/2) have been identified so far and serotype 2 is the one most commonly isolated from diseased pigs (8). Infections caused by this pathogen include meningitis, arthritis, pneumonia, and septicemia (9). S. suis is also an important zoonotic agent that can cause severe infections in workers in the pig industry (5, 24). The mechanisms by which S. suis invades and infects the host are still unclear even though numerous studies on potential virulence factors have been carried out over the past decade. Many virulence candidate determinants produced by S. suis have been identified (21). Among these virulence factors, the polysaccharide capsule, which provides protection against phagocytosis (3), appears critical for the pathogenicity of S. suis. Proteases are important virulence factors for a variety of microbial pathogens and may contribute to tissue degradation and perturbation of the host defense system (14, 23). Recently, our laboratory reported that S. suis produces four major proteases (arginine aminopeptidase, chymotrypsin-like, caseinase, and dipeptidyl peptidase IV [DPP IV]) (11). DPP IV (EC 3.4.14.5) is a highly specific protease that cleaves after the X-Pro and X-Ala residues at the N terminus of polypeptide chains. The DPP IV produced by eukaryotic cells, also called CD26, has been associated with several biological functions (16). Many bacterial species, including Lactobacillus helveticus (26), Streptococcus gordonii (7), Streptococcus thermophilus (25), Porphyromonas gingivalis (12), and Prevotella albensis (27) produce DPP IV. Interestingly, a P. gingivalis mutant deficient in DPP IV was found to be much less virulent than the parent strain in a mouse model, indicating that DPP IV may contribute to the pathogenicity of this microorganism (28). Since DPP IV may play a critical role in virulence, we cloned, purified, and characterized the DPP IV produced by S. suis. * Corresponding author. Mailing address: Groupe de Recherche en ´ cologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, E Quebec City, Quebec, Canada G1K 7P4. Phone: (418) 656-7341. Fax: (418) 656-2861. E-mail:
[email protected]. 795
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TABLE 1. Motif of the serine active site and nucleotide and amino acid sequence homologies of S. suis S735 DPP IV with other bacterial DPP IVs Bacterial species
S. suis S. pyogenes S. gordonii L. lactis subsp. lactis S. pneumoniae P. gingivalis S. thermophilus L. delbrueckii
Homologous gene
Ss-xPDPP pepXP Sg-xPDPP pepXP pepXP dpp pepX pepX
Homologous protein
Motif of the active-site serine (position)
% Nucleotide identity with Ss-xPDPP
% Amino acid identity with S. suis recombinant DPP IV
Accession no.
Dipeptidyl aminopeptidase IV Putative X-Pro dipeptidyl peptidase IV X-prolyl dipeptidyl peptidase X-prolyl dipeptidyl aminopeptidase X-Pro dipeptidyl peptidase Dipeptidyl aminopeptidase IV X-prolyl dipeptidyl aminopeptidase X-prolyl dipeptidyl aminopeptidase
G-I-S-Y-L-G (345–350) G-K-S-Y-L-G (347–352) G-I-S-Y-L-G (345–350) G-K-S-Y-L-G (346–351) G-L-S-Y-L-G (384–389) G-W-S-Y-G-G (591–596) G-L-S-Y-L-G (346–351) G-R-S-Y-L-G (355–360)
100 60 63 47 61 0 57 21
100 55 62 50 64 6 59 31
AY533504 AE006611 AY032733 AE006435 AE008455 AE017173 AY055853 AJ012302
(stop codon at position 2,268). This suggests that the SsxPDPP gene may have been acquired from another microorganism evolving in the same environment of S. suis and that the gene was likely inserted in the genome by transformation. A potential promoter sequence and a ribosome binding site were found upstream of the cloned gene based on the homology with published consensus sequences (6). The Ss-xPDPP (strain S735) sequence showed a high degree of homology (99 and 93% identity) with that of strain P1/7 (Sanger Institute) and 89/1591 (National Center for Biotechnology) for which the genomes were recently sequenced. A search in the GenBank database revealed that the derived amino acid and nucleotide sequences of Ss-xPDPP shared identity with the X-Pro dipeptidyl aminopeptidases of Streptococcus pneumoniae, S. thermophilus, Lactobacillus delbrueckii, Streptococcus pyogenes, Lactococcus lactis subsp. lactis, and S. gordonii but not with that of the gram-negative bacterium P. gingivalis (Table 1). This analysis was performed with GenBank sequences using the BLAST network service and ClustalW program (clustalw.genome.ad.jp). The deduced amino acid sequence from the nucleotide sequence revealed that it contained a complete open reading frame coding for a 755-aminoacid protein with a calculated molecular mass of 85 kDa. This correlates well with the molecular masses previously reported for other DPP IVs, including those from P. albensis (27), S. gordonii (7), Lactobacillus sakei (20), and S. thermophilus (25). Chromosomal DNA (30 g) from S. suis S735 (European origin) and 89-999 (North American origin) was digested with EcoRI and KpnI to investigate the copy number of the gene and possible differences related to strain origin. The digested DNA was separated on a 0.8% agarose gel and transferred to a nylon membrane as previously described (15). A DNA probe was produced using primers N5 (5⬘ CCAGCATCACCAAGT TAGA 3⬘) and N6 (5⬘ TGGAAGGCAGAGTAGGATTTG 3⬘) to amplify an internal segment (nucleotides 728 to 1,757) of the DPP IV gene. The PCR product (4 l) was labeled with digoxigenin using a digoxigenin DNA labeling and detection kit from Boehringer-Mannheim (Laval, Quebec, Canada), following the manufacturer’s instructions. The hybridization was performed at 68°C with the previously amplified probe (25 ng/ ml). As shown in Fig. 1, only one copy of the gene was present in the genome of both strains although the profiles were not the same. For this reason, the region was further characterized in different S. suis serotype 2 strains by amplification with internal and external primers for the Ss-xPDPP gene. All
strains (17 S. suis strains) except one (91-1804) presented the expected band when the internal primers N7 and N8 (sequences presented below) were used (data not shown). When primers (N1 and N2) external to the Ss-xPDPP gene were used to analyze the same isolates, the number of positive strains remained the same (data not shown). These observations suggest that the gene is highly conserved. The strain 91-1804, which was negative for the presence of the Ss-xPDPP gene, showed the capacity to hydrolyze the chromogenic substrate for DPP IV. Further studies may be required to determine the extent of the variability in the open reading frame of the strain 91-1804 and the other strains tested. The primers N7 (5⬘ CGCTTTAATCAATTTTCTTTCATA AAAAAAGAGAC 3⬘) and N8 (5⬘ TTTGGATTTTCATTGA
FIG. 1. Southern analysis of digested total DNA of S. suis S735 and 89–999. Lane 1, total DNA of S735 digested with EcoRI; lane 2, total DNA of S735 digested with KpnI; lane 3, total DNA of 89–999 digested with EcoRI; lane 4, total DNA of 89–999 digested with KpnI.
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FIG. 2. SDS-PAGE analysis of fractions obtained during the purification of the recombinant DPP IV. Proteins were stained with Coomassie blue. Lane 1, crude extract of transformed E. coli following induction with 0.2% arabinose; lane 2, membranes of transformed E. coli; lane 3, cytoplasm of transformed E. coli; lane 4, purified recombinant DPP IV.
GTATTAGTGCG 3⬘) were designed and used to produce a fragment containing the Ss-xPDPP gene without putative start and stop codons in concordance with the pBAD/thio-TOPO system (Invitrogen). This system produces a recombinant protein with six histidine residues at the C terminus and thioredoxin at the N terminus. A total of 90 E. coli clones were obtained and 10 were screened for the presence of the gene by PCR. All 10 clones were positive following screening; one was chosen for further analysis and a second was kept as backup. The chosen clone overexpressed the protease responsible for S. suis S735 DPP IV activity in the following induction with 0.2% arabinose. The cytosol extract of the E. coli TOP10 clone was prepared according to the manufacturer’s instructions. Glycerol (10%) was added to the cytoplasmic fraction, which was then loaded on a Ni2⫹-nitrilotriacetic acid affinity chromatography column. Fractions (1 ml) were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue staining and were tested for DPP IV activity using the chromogenic substrate glycine–proline–p-nitroanilide (pNa). The release of p-nitroaniline was quantified by measuring the absorbance at 415 nm (A415). One unit was defined as the amount of enzyme that generated 1 mol of p-nitroaniline per minute. Fractions with DPP IV activity were pooled and stored at ⫺20°C. Approximately 1 g of recombinant DPP IV was recovered from 1 mg of E. coli cells (wet weight). The SDS-PAGE analysis of the purified recombinant DPP IV indicated a molecular mass of approximately 100 kDa, which represents the total molecular mass taking into account the His tag and the thioredoxin attached to the protein (Fig. 2). Various synthetic peptides labeled with pNA (Table 2) were tested for their susceptibility to the recombinant DPP IV. The reaction mixtures contained 90 l of diluted (in phosphatebuffered saline [PBS]) enzyme preparation (324 U) and 10 l of the tested substrate (20 mM). Prior to adding the substrate, the enzyme was preincubated for 10 min at 37°C. The recombinant DPP IV had a high affinity for the synthetic substrates Gly-Pro-pNa and Ala-Pro-pNa. To a lesser extent, other dipep-
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tides, including Lys-Ala-pNa, Arg-Pro-pNa, Ala-Ala-pNa, and Asp-Pro-pNa, were also hydrolyzed by the protease. A similar observation was previously reported for other bacterial DPP IVs (2, 7, 17, 25). Graph Prism 4.0 software was used to study the enzyme kinetics. The Km and Vmax values of the Michaelis-Menten curves of the recombinant DPP IV preparation were determined using Gly-Pro-pNA, Ala-Pro-pNa, and Lys-Ala-pNa as substrates. The substrate showing the highest affinity for the recombinant DPP IV was Gly-Pro-pNa, with a Km of 0.26 mM and a Vmax of 7.8 mol mg⫺1 min⫺1. This value is comparable to those reported for P. albensis (27) and L. helveticus (26). The recombinant DPP IV also had significant affinity for Lys-AlapNa (Km of 0.87 mM) and Ala-Pro-pNa (Km of 12 mM) with Vmax values of 5.9 and 30 mol mg⫺1 min⫺1, respectively. By using the synthetic peptide Gly-Pro-pNa as substrate, several inhibitors and compounds were tested for their effect on the activity of the recombinant DPP IV (Table 3). The assay was done as described above. The recombinant S. suis DPP IV was strongly inhibited by Hg2⫹, diprotin A, and SDS. The sulfhydryl group reagent iodoacetamide caused some inhibition, suggesting the involvement of functional sulfhydryl group(s) at or near the active site of the enzyme. This was also observed for the S. thermophilus DPP IV (25). Two serine protease inhibitors (aminoethyl-benzene sulfonyl fluoride hydrochloride and 3,4-dichloroisocoumarin) that strongly inhibit other bacterial DPP IVs had little or no effect on the recombinant S. suis DPP IV. However, this does not imply that the S. suis DPP IV is a nonserine protease, especially since it contains the conserved Gly-X-Ser-X-X-Gly sequence, which is a consensus motif flanking the active-site serine shared by serine proteases, serine esterases, and other DPPs. As shown in Table 1, the recombinant S. suis DPP IV possessed the Gly-Lys-Ser-Tyr-Leu-Gly motif, which is the same found in S. pyogenes and L. lactis subsp. lactis. The motif was located at residue positions 345 to 350, as for S. gordonii.
TABLE 2. Hydrolysis of synthetic peptides by the recombinant S. suis DPP IV Synthetic peptide
Relative activity (%)
Gly-Pro-pNaa ...............................................................................100 ⫾ 1b Ala-Pro-pNaa ............................................................................... 80 ⫾ 2 Lys-Ala-pNaa ............................................................................... 51 ⫾ 1 Arg-Pro-pNaa ............................................................................... 42 ⫾ 0 Ala-Ala-pNaa ............................................................................... 36 ⫾ 0 Asp-Pro-pNaa .............................................................................. 11 ⫾ 1 Met-pNa ....................................................................................... 2 ⫾ 5 Ala-pNa ........................................................................................ 0 ⫾ 1 Ala-Ala-Pro-pNaa ........................................................................ 0 ⫾ 1 Leu-pNa........................................................................................ 0 ⫾ 1 Arg-pNa........................................................................................ 0 ⫾ 0 Gly-Arg-pNaa ............................................................................... 0 ⫾ 0 Pro-pNa ........................................................................................ 0 ⫾ 0 Succinyl-Gly-Pro-pNaa ................................................................ 0 ⫾ 0 Lys-pNa ........................................................................................ 0 ⫾ 1 Glu-pNa........................................................................................ 0 ⫾ 1 Tosyl-Gly-Pro-Lys-pNa ............................................................... 1 ⫾ 2 a These synthetic chromogenic peptides were obtained from Bachem. All others were from Sigma-Aldrich Canada. b The highest activity obtained with 324 U of enzyme was given a value of 100%. Means of three assays ⫾ standard deviations.
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TABLE 3. Effect of protease inhibitors on the activity of the recombinant S. suis DPP IV Inhibitorsa
Specificity
Concentration
% of residual activityb
Diprotin A o-Phenanthroline IAN 3,4-DIC AEBSF E-64 SDS Triton X-100 Hg2⫹
DPP IV-specific Metallo Cysteine Serine Serine Cysteine NAc NA NA
1 mM 10 mM 10 M 100 M 1 mM 10 M 35 mM 16 mM 1 mM
29 ⫾ 1 66 ⫾ 8 81 ⫾ 2 82 ⫾ 1 118 ⫾ 1 125 ⫾ 1 0⫾0 78 ⫾ 1 30 ⫾ 1
a
AEBSF, aminoethyl-benzene sulfonyl fluoride hydrochloride; TLCK, N-␣-pE-64, N-(trans-epoxysuccinyl)-L-leucine-4guanidinobutylamide; EDTA, ethylenediaminetetraacetic acid; IAN, iodoacetamide; 3,4-DIC, 3,4-dichloroisocoumarin. b The activity obtained with 324 U of enzyme in the absence of inhibitor was given a value of 100%. Means of three assays ⫾ standard deviations. c Not applicable. L-tosyl-lysine-chloromethylketone;
The DPP IV activity of the purified preparation was measured at different pH values using the following buffers at 50 mM: citrate (pH 3 to 6), phosphate (pH 7), Tris (pH 8 and 9), and carbonate (pH 10 and 11) (Table 4). The assay mixture contained 324 U of the recombinant DPP IV in 90 l of buffer with 10 l of Gly-Pro-pNa (20 mM). The A415 was recorded following a 45-min incubation at 37°C. The activity of DPP IV was optimal at pH 8, although more than 50% of activity was obtained at pH values of 6 and 9. Several incubation temperatures were tested (4, 10, 24, 37, 45, and 55°C) using PBS as the buffer and the optimal temperature was found to be 37°C (Table 4). These optimal conditions observed for the S. suis recombinant DPP IV were similar or close to those reported for other bacterial DPP IVs (2, 7, 17, 25, 27). To evaluate the stability of the recombinant DPP IV, the enzyme was diluted to the working concentration and kept for 24 h at ⫺20, 4, 10, or 24°C prior to assaying the activity as described above (Table 4). The percent residual activity was established by comparison with a fresh dilution of recombinant DPP IV. The diluted preparation of recombinant DPP IV was unstable, with approximately 14% of the original activity remaining after 24-h incubation at 4°C. On the other hand, the stock preparation of recombinant DPP IV was not affected by prolonged storage at ⫺20°C. A basal medium (10) was used to investigate the effect of growth conditions on DPP IV production by S. suis S735. This medium was supplemented with glucose at concentrations ranging from 0.1 to 2% and casein hydrolysate (peptone; Becton Dickinson, Ontario, Canada) at concentrations ranging from 0.2 to 10%. S. suis S735 was grown overnight in ToddHewitt broth and inoculated (10% inoculum) into prewarmed basal medium. Samples were taken every 30 min and subjected to centrifugation (16,000 ⫻ g, 5 min), and residual bacteria in the supernatant were removed by filtration through a 0.22-mpore-size filter. The pellets were washed once, resuspended in PBS (50 mM phosphate, 150 mM NaCl, pH 7.2), and stored at ⫺20°C until used. The DPP IV activity of the culture supernatants and bacterial cells was determined as follows. The reaction mixtures contained 90 l of cells (optical density at 660 nm of 0.5 in PBS) or culture supernatant and 10 l of Gly-Pro-pNA (20 mg/ml in 10% dimethyl sulfoxide). They
were incubated at 37°C for 1.5 h and the cells were removed by centrifugation, if needed, prior to reading the A415. DPP IV production by S. suis S735 was modulated by the concentration of casein hydrolysate added to the basal culture medium. The highest cell-associated activity was obtained following growth in the presence of 10% casein hydrolysate (data not shown). This was twice the activity obtained with 0 and 1% peptone. No differences were noted for extracellular DPP IV activity. The modulation of DPP IV by casein hydrolysate was also observed for P. albensis (27). The glucose concentration had no effect on either the cell-associated or extracellular DPP IV activity of S. suis S735. The DPP IV produced by S. suis was both cell bound and extracellular, which differs from those produced by other bacteria. For instance, DPP IV activity is extracellular for S. gordonii (7), intracellular for S. thermophilus (25), and loosely bound to the cell membrane for L. lactis subsp. cremoris (29). DPP IV cleaves peptides and proteins with either X-Pro or X-Ala at their N termini. The action of the enzyme on proteins and polypeptides may modify the biological activity of the resulting truncated molecule. Many natural cytokines, including interleukin 1, interleukin 2, and RANTES (regulated on activation normal T cell expressed and secreted), have a proline in the second position in their polypeptide chains (16). If the S. suis DPP IV is active on these molecules, it may result in local changes to host responses during the course of certain S. suis-associated infections. On the other hand, the removal of dipeptides from the N termini of peptides can generate new biologically active peptides. DPP IV may thus contribute significantly to the deregulation of inflammatory processes during meningitis. The S. suis DPP IV may also contribute to tissue destruction and systemic bacterial dissemination. Indeed, Abiko et al. (1) reported that the DPP IV of P. gingivalis can further degrade peptides from partially digested type I collagen. The dipeptides produced may also promote bacterial
TABLE 4. Effect of different conditions on the activity the recombinant S. suis DPP IV Conditiona
% Activity
Assay pH 4 ............................................................................................... 5 ............................................................................................... 6 ............................................................................................... 7 ............................................................................................... 8 ............................................................................................... 9 ............................................................................................... 10 ...............................................................................................
0⫾0 13 ⫾ 0 61 ⫾ 2 68 ⫾ 1 100 ⫾ 2 54 ⫾ 2 2⫾2
Assay temperature (°C) 4 ............................................................................................... 10 ............................................................................................... 24 ............................................................................................... 37 ............................................................................................... 45 ............................................................................................... 55 ...............................................................................................
24 ⫾ 1 26 ⫾ 2 52 ⫾ 3 100 ⫾ 4 78 ⫾ 5 1⫾2
Storage temperature (°C) ⫺20............................................................................................ 100 ⫾ 2 4............................................................................................ 14 ⫾ 3 10............................................................................................ 8 ⫾ 1 24............................................................................................ 7 ⫾ 0 a The highest activity obtained with 324 U of enzyme was given a value of 100%. Means of three assays ⫾ standard deviations.
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growth. Inactivation of the Ss-xPDPP gene in S. suis using genetic tools should make it possible to determine the contribution of this protease to bacterial growth and pathogenicity. Nucleotide sequence accession number. The Ss-xPDPP (strain S735) sequence has been submitted to GenBank under accession no. AY533504. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Research Network on Bacterial Pathogens of Swine (NSERC). We would like to thank Sonia Lacouture for technical assistance as well as Katy Vaillancourt and Benjamin Pe´ant for their helpful advice.
12.
13. 14. 15. 16. 17.
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