Preferences help
enabled [disable] Abstract
Number of results
2008 | 55 | 3 | 525-536
Article title

A novel member of the thermolysin family, cloning and biochemical characterization of metalloprotease from Staphylococcus pseudintermedius

Title variants
Languages of publication
Thermolysins constitute a family of secreted bacterial metalloproteases expressed, among others, by several pathogens. Strains of Staphylococcus pseudintermedius isolated from diseased dogs and judged as protease-positive, by skim milk agar plate culture, were investigated for protease content. No proteolytic activity was detected when the bacteria were grown in regular liquid media. Unexpectedly, supplementation of the medium with calcium ions resulted in expression of a metalloprotease and profound changes in the profile of extracellular proteins. On the basis of homology to other staphylococcal metalloproteases, the nucleotide sequence of the gene encoding this protease (Pst) and its flanking regions was determined. The full-length pst codes for a protein with an open reading frame of 505 amino acids. The internal region contains the HEXXH catalytic domain that is conserved in members of the thermolysin family. Regardless of the presence of calcium in the medium, the expression of the protease gene was of the same intensity. This suggests that regulation of the metalloprotease production by calcium ions is at a post-transcriptional level. Isolates of S. pseudintermedius exhibit a proteolytic phenotype due to the metalloprotease expression, however only in presence of calcium ions, which most probably stabilize the structure of the protease.
Physical description
  • Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Epidemiology and Clinical Microbiology, National Institute of Medicines, Warszawa, Poland
  • Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Department of Epidemiology and Clinical Microbiology, National Institute of Medicines, Warszawa, Poland
  • Department of Epidemiology and Clinical Microbiology, National Institute of Medicines, Warszawa, Poland
  • Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
  • Armstrong PB (2006) Proteases and protease inhibitors: a balance of activities in host-pathogen interaction. Immunobiology 211: 263-281.
  • Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22: 195-201.
  • Arvidson S (1973) Studies on extracellular proteolytic enzymes from Staphylococcus aureus. II. Isolation and characterization of an EDTA-sensitive protease. Biochim Biophys Acta 302: 149-157.
  • Auld D.S (2004) Catalytic mechanisms of metallopeptidases. In Handbook of Proteolytic Enzymes. Barrett AJ, Rawlings ND, Woessner JF, ed, pp 268-289. Elsevier, London.
  • Banbula A, Potempa J, Travis J, Fernandez-Catalan C, Mann K, Huber R, Bode W, Medrano F (1998) Amino-acid sequence and three-dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 A resolution. Structure 6: 1185-1193.
  • Becker K, Harmsen D, Mellmann A, Meier C, Schumann P, Peters G, von Eiff C (2004) Development and evaluation of a quality-controlled ribosomal sequence database for 16S ribosomal DNA-based identification of Staphylococcus species. J Clin Microbiol 42: 4988-4995.
  • Bonar E, Dubin A, Bierczyska-Krzysik A, Noga M, Silberring J, Staliska K, Koj A (2006) Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells. Cytokine 33: 111-117.
  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  • Buchanan JD, Corbett RJ, Roche RS (1986) The thermodynamics of calcium binding to thermolysin. Biophys Chem 23: 183-199.
  • Colman PM, Jansonius JN, Matthews BW (1972) The structure of thermolysin: an electron density map at 2-3 Å resolution. J Mol Biol 70: 701-724.
  • Corbett RJ, Roche RS (1983) The unfolding mechanism of thermolysin. Biopolymers 22: 101-105.
  • Descloux S, Rossano A, Perreten V (2008) Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius. J Clin Microbiol 46: 1818-1823.
  • Devriese LA, Vancanneyt M, Baele M, Vaneechoutte M, De Graef E, Snauwaert C, Cleenwerck I, Dawyndt P, Swings J, Decostere A, Haesebrouck F (2005) Staphylococcus pseudintermedius sp. nov, a coagulase-positive species from animals. Int J Syst Evol Microbiol 55: 1569-1573.
  • Drancourt M, Raoult D (2002) rpoB gene sequence-based identification of Staphylococcus species. J Clin Microbiol 40: 1333-1338.
  • Drapeau GR (1978) Role of metalloprotease in activation of the precursor of staphylococcal protease. J Bacteriol 136: 607-613.
  • Dubin A, Koj A, Chudzik J (1976) Isolation and some molecular parameters of elastase-like normal proteinases from horse blood leucocytes. Biochem J 153: 389-396.
  • Dubin G (2002) Extracellular proteases of Staphylococcus spp. Biol Chem 383: 1075-1086.
  • Eleaume H, Jabbouri S (2004) Comparison of two standardisation methods in real-time quantitative RT-PCR to follow Staphylococcus aureus genes expression during in vitro growth. J Microbiol Methods 59: 363-370.
  • Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A (2003) ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 31: 3784-3788.
  • Ghebremedhin B, Layer F, Konig W, Konig B (2008) Genetic classification and distinguishing of Staphylococcus species based on different partial gap 16S rRNA hsp60 rpoB sodA and tuf gene sequences. J Clin Microbiol 46: 1019-1025.
  • Ghorbel-Frikha B, Sellami-Kamoun A, Fakhfakh N, Haddar A, Manni L, Nasri M (2005) Production and purification of a calcium-dependent protease from Bacillus cereus BG1. J Ind Microbiol Biotechnol 32: 186-194.
  • Guex N, Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723.
  • Imamura T, Tanase S, Szmyd G, Kozik A, Travis J, Potempa J (2005) Induction of vascular leakage through release of bradykinin and a novel kinin by cysteine proteinases from Staphylococcus aureus. J Exp Med 201: 1669-1676.
  • Karlsson A, Saravia-Otten P, Tegmark K, Morfeldt E, Arvidson S (2001) Decreased amounts of cell wall-associated protein A and fibronectin-binding proteins in Staphylococcus aureus sarA mutants due to up-regulation of extracellular proteases. Infect Immun 69: 4742-4748.
  • Kawano Y, Kawagishi M, Nakano M, Mase K, Yamashino T, Hasegawa T, Ohta M (2001) Proteolytic cleavage of staphylococcal exoproteins analyzed by two-dimensional gel electrophoresis. Microbiol Immunol 45: 285-290.
  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
  • Lautz S, Kanbar T, Alber J, Lammler C, Weiss R, Prenger-Berninghoff E, Zschock M (2006) Dissemination of the gene encoding exfoliative toxin of Staphylococcus intermedius among strains isolated from dogs during routine microbiological diagnostics. J Vet Med B Infect Dis Vet Public Health 53: 434-438.
  • Massimi I, Park E, Rice K, Muller-Esterl W, Sauder D, McGavin MJ (2002) Identification of a novel maturation mechanism and restricted substrate specificity for the SspB cysteine protease of Staphylococcus aureus. J Biol Chem 277: 41770-41777.
  • Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262: 10035-10038.
  • McGavin MJ, Zahradka C, Rice K, Scott JE (1997) Modification of the Staphylococcus aureus fibronectin binding phenotype by V8 protease. Infect Immun 65: 2621-2628.
  • Miedzobrodzki J, Naidu AS, Watts JL, Ciborowski P, Palm K, Wadstrom T (1989) Effect of milk on fibronectin and collagen type I binding to Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine mastitis. J Clin Microbiol 27: 540-544.
  • Miedzobrodzki J, Kaszycki P, Bialecka A, Kasprowicz A (2002) Proteolytic activity of Staphylococcus aureus strains isolated from the colonized skin of patients with acute-phase atopic dermatitis. Eur J Clin Microbiol Infect Dis 21: 269-276.
  • Moore PC, Lindsay JA (2001) Genetic variation among hospital isolates of methicillin-sensitive Staphylococcus aureus: evidence for horizontal transfer of virulence genes. J Clin Microbiol 39: 2760-2767.
  • Nakano M, Kawano Y, Kawagish M, Hasegawa T, Iinuma Y, Oht M (2002) Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological applications. Microbiol Immunol 46: 11-22.
  • Nicholas KB, Nicholas HB, Deerfield DW (1997) GeneDoc: analysis and visualization of genetic variation. In EMBNEW.NEWS, vol 4, pp 14.
  • Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, Moghazeh S (1993) Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12: 3967-3975.
  • Popowicz GM, Dubin G, Stec-Niemczyk J, Czarny A, Dubin A, Potempa J, Holak TA (2006) Functional and structural characterization of Spl proteases from Staphylococcus aureus. J Mol Biol 358: 270-279.
  • Potempa J, Fedak D, Dubin A, Mast A, Travis J (1991) Proteolytic inactivation of α-1-anti-chymotrypsin. Sites of cleavage and generation of chemotactic activity. [erratum appears in J Biol Chem 1992 Mar 5, 267(7): 5016] J Biol Chem 266: 21482-21487.
  • Prokesova L, Porwit-Bobr Z, Baran K, Potempa J, Pospisil M, John C (1991) Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes. Immunol Lett 27: 225-230.
  • Sabat A, Kosowska K, Poulsen K, Kasprowicz A, Sekowska A, van Den Burg B, Travis J, Potempa J (2000) Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus. Infect Immun 68: 973-976.
  • Sabat A, Krzyszton-Russjan J, Strzalka W, Filipek R, Kosowska K, Hryniewicz W, Travis J, Potempa J (2003) New method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. J Clin Microbiol 41: 1801-1804.
  • Sabersheikh S, Saunders NA (2004) Quantification of virulence-associated gene transcripts in epidemic methicillin resistant Staphylococcus aureus by real-time PCR. Mol Cell Probes 18: 23-31.
  • Sarkisova S, Patrauchan MA, Berglund D, Nivens DE, Franklin MJ (2005) Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. J Bacteriol 187: 4327-4337.
  • Sasaki T, Kikuchi K, Tanaka Y, Takahashi N, Kamata S, Hiramatsu K (2007) Reclassification of phenotypically identified staphylococcus intermedius strains. J Clin Microbiol 45: 2770-2778.
  • Schagger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166: 368-379.
  • Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27: 157-162.
  • Schwarz S, Kadlec K, Strommenger B (2008) Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius detected in the BfT-GermVet monitoring programme 2004-2006 in Germany. J Antimicrob Chemother 61: 282-285.
  • Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31: 3381-3385.
  • Secades P, Alvarez B, Guijarro JA (2001) Purification and characterization of a psychrophilic calcium-induced growth-phase-dependent metalloprotease from the fish pathogen Flavobacterium psychrophilum. Appl Environ Microbiol 67: 2436-2444.
  • Shaw L, Golonka E, Potempa J, Foster SJ (2004) The role and regulation of the extracellular proteases of Staphylococcus aureus. Microbiology 150: 217-228.
  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850-858.
  • Sieprawska-Lupa M, Mydel P, Krawczyk K, Wojcik K, Puklo M, Lupa B, Suder P, Silberring J, Reed M, Pohl J, Shafer W, McAleese F, Foster T, Travis J, Potempa J (2004) Degradation of human antimicrobial peptide LL-37 by Staphylococcus aureus-derived proteinases. Antimicrob Agents Chemother 48: 4673-4679.
  • Teufel P, Gotz F (1993) Characterization of an extracellular metalloprotease with elastase activity from Staphylococcus epidermidis. J Bacteriol 175: 4218-4224.
  • Trulzsch K, Grabein B, Schumann P, Mellmann A, Antonenka U, Heesemann J, Becker K (2007) Staphylococcus pettenkoferi sp. nov., a novel coagulase-negative staphylococcal species isolated from human clinical specimens. Int J Syst Evol Microbiol 57: 1543-1548.
  • Van Hoovels L, Vankeerberghen A, Boel A, Van Vaerenbergh K, De Beenhouwer H (2006) First case of Staphylococcus pseudintermedius infection in a human. J Clin Microbiol 44: 4609-4612.
  • Wegrzynowicz Z, Heczko PB, Drapeau GR, Jeljaszewicz J, Pulverer G (1980) Prothrombin activation by a metalloprotease from Staphylococcus aureus. J Clin Microbiol 12: 138-139.
  • Wladyka B, Pustelny K (2008) Regulation of bacterial protease activity. Cell Mol Biol Lett 13: 212-229.
  • Yugueros J, Temprano A, Berzal B, Sanchez M, Hernanz C, Luengo JM, Naharro G (2000) Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp. J Clin Microbiol 38: 4351-4355.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.