Abstract
Clostridium perfringens produces potent toxins and histolytic enzymes, causing various diseases including life-threatening fulminant diseases in humans and other animals. Aiming at utilizing a phage endolysin as a therapeutic alternative to antibiotics, we surveyed the genome and bacteriophage sequences of C. perfringens. A phiSM101 muramidase gene (psm) revealed by this study can be assumed to encode an N-acetylmuramidase, since the N-terminal catalytic domain deduced from the gene shows high homology of those of N-acetylmuramidases. The psm gene is characteristic in that it is present in phiSM101, an episomal phage of enterotoxigenic C. perfringens type A strain, SM101, and also in that homologous genes are present in the genomes of all five C. perfringens toxin types. The psm gene was cloned and expressed in Escherichia coli as a protein histidine-tagged at the N-terminus (Psm-his). Psm-his was purified to homogeneity by nickel-charged immobilized metal affinity chromatography and anion-exchange chromatography. The purified enzyme lysed cells of all C. perfringens toxin types but not other clostridial species tested, as was shown by a turbidity reduction assay. These results indicate the Psm-his is useful as a cell-wall lytic enzyme and also suggest that it is potentially useful for biocontrol of this organism.
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References
Adak GK, Long SM, O'Brien SJ (2002) Trends in indigenous foodborne disease and deaths, England and Wales: 1992 to 2000. Gut 51:832–841
Bryant AE, Bayer CR, Aldape MJ, Wallace RJ, Titball RW, Stevens DL (2006) Clostridium perfringens phospholipase C-induced platelet/leukocyte interactions impede neutrophil diapedesis. J Med Microbiol 55:495–504
Chen Y, Miyata S, Makino S, Moriyama R (1997) Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene. J Bacteriol 179:3181–3187
Cooper KK, Songer JG (2009) Necrotic enteritis in chickens: a paradigm of enteric infection by Clostridium perfringens type A. Anaerobe 15:55–60
Courchesne NM, Parisien A, Lan CQ (2009) Production and application of bacteriophage and bacteriophage-encoded lysins. Recent Pat Biotechnol 3:37–45
Croux C, Canard B, Goma G, Soucaille P (1992) Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC824 that acts on non-N-acetylated peptidoglycan. Appl Environ Microbiol 58:1075–1081
Dhalluin A, Bourgeois I, Pestel-Caron M, Camiade E, Raux G, Courtin P, Chapot-Chartier MP, Pons JL (2005) Acd, a peptidoglycan hydrolase of Clostridium difficile with N-acetylglucosaminidase activity. Microbiology 151:2343–2351
Fischetti VA (2008) Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol 11:393–400
Fischetti VA (2010) Bacteriophage endolysins: a novel anti-infective to control Gram-positive pathogens. Int J Med Microbiol 300:357–362
Harbarth S, Samore MH (2005) Antimicrobial resistance determinants and future control. Emerg Infect Dis 11:794–801
Huard C, Miranda G, Wessner F, Bolotin A, Hansen J, Foster SJ, Chapot-Chartier MP (2003) Characterization of AcmB, an N-acetylglucosaminidase autolysin from Lactococcus lactis. Microbiology 149:695–705
Layec S, Decaris B, Leblond-Bourget N (2008) Diversity of Firmicutes peptidoglycan hydrolases and specificities of those involved in daughter cell separation. Res Microbiol 159:507–515
Loeffler JM, Fischetti VA (2003) Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrob Agents Chemother 47:375–377
Loeffler JM, Nelson D, Fischetti VA (2001) Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294:2170–2172
Loeffler JM, Djurkovic S, Fischetti VA (2003) Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect Immun 71:6199–6204
Lynch J, Painter J, Woodruff R, Braden C (2006) Surveillance for foodborne-disease outbreaks—United States, 1998–2002. Morbidity and Mortality Weekly Report, CDC 55:1–42
McReynolds JL, Byrd JA, Anderson RC, Moore RW, Edrington TS, Genovese KJ, Poole TL, Kubena LF, Nisbet DJ (2004) Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis. Poult Sci 83:1948–1952
Myers GS, Rasko DA, Cheung JK, Ravel J, Seshadri R, DeBoy RT, Ren Q, Varga J, Awad MM, Brinkac LM, Daugherty SC, Haft DH, Dodson RJ, Madupu R, Nelson WC, Rosovitz MJ, Sullivan SA, Khouri H, Dimitrov GI, Watkin KL, Mulligan S, Benton J, Radune D, Fisher DJ, Atkins HS, Hiscox T, Jost BH, Billington SJ, Songer JG, McClane BA, Titball RW, Rood JI, Melville SB, Paulsen IT (2006) Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16:1031–1040
Nariya H, Inouye S (2005) Identification of a protein Ser/Thr kinase cascade that regulates essential transcriptional activators in Myxococcus xanthus development. Mol Microbiol 58:367–379
Nariya H, Miyata S, Suzuki M, Tamai E, Okabe A (2011) Development and application of a method for counterselectable in-frame deletion in Clostridium perfringens. Appl Environ Microbiol 77:1375–1382
Nelson D, Loomis L, Fischetti VA (2001) Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Acad Sci USA 98:4107–4112
Norrby SR, Nord CE, Finch R, European Society of Clinical Microbiology and Infectious Diseases (2005) Lack of development of new antimicrobial drugs: a potential serious threat to public health. Lancet Infect Dis 5:115–119
O'Flaherty S, Ross RP, Coffey A (2009) Bacteriophage and their lysins for elimination of infectious bacteria. FEMS Microbiol Rev 33:801–881
Petit L, Gibert M, Popoff MR (1999) Clostridium perfringens: toxino-type and genotype. Trends Microbiol 7:104–710
Present DA, Meislin R, Shaffer B (1990) Gas gangrene. A review. Orthop Rev 19:333–341
Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M (2004) Cold-shock induced high-yield protein production in Escherichia coli. Nat Biotechnol 22:877–882
Rau A, Hogg T, Marquardt R, Hilgenfeld R (2001) A new lysozyme fold. Crystal structure of the muramidase from Streptomyces coelicolor at 1.65 Å resolution. J Biol Chem 276:31994–31999
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Sheng S, Cherniak R (1998) Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 10 determined by NMR spectroscopy. Carbohydr Res 305:65–72
Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, Shiba T, Ogasawara N, Hattori M, Kuhara S, Hayashi H (2002) Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci USA 99:996–1001
Tamai E, Miyata S, Tanaka H, Nariya H, Suzuki M, Matsushita O, Hatano N, Okabe A (2008) High-level expression of his-tagged clostridial collagenase in Clostridium perfringens. Appl Microbiol Biotechnol 80:627–635
Tanaka H, Tamai E, Miyata S, Taniguchi Y, Nariya H, Hatano N, Houchi H, Okabe A (2008) Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression. Appl Microbiol Biotechnol 277:1063–1071
Tanaka H, Nariya H, Suzuki M, Houchi H, Tamai E, Miyata S, Okabe A (2011) High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens. Protein Expr Purif 76:83–89
Tsutsui K, Minami J, Matsushita O, Katayama S, Taniguchi Y, Nakamura S, Nishioka M, Okabe A (1995) Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi. J Bacteriol 177:7164–7170
Wang IN, Smith DL, Young R (2000) Holins: the protein clocks of bacteriophage infections. Annu Rev Microbiol 54:799–825
Zimmer M, Vukov N, Scherer S, Loessner MJ (2002) The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 68:5311–5317
Acknowledgments
We are grateful to Dr. M. R. Sarker, Oregon State University, for providing us with the C. perfringens SM101. We also wish to thank Mr. N. J. Halewood for his assistance in preparing the manuscript. This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (Grant for Scientific Research C 21590482). It was also partly supported by the Kagawa University Characteristic Prior Research Funds 2010.
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Nariya, H., Miyata, S., Tamai, E. et al. Identification and characterization of a putative endolysin encoded by episomal phage phiSM101 of Clostridium perfringens . Appl Microbiol Biotechnol 90, 1973–1979 (2011). https://doi.org/10.1007/s00253-011-3253-z
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DOI: https://doi.org/10.1007/s00253-011-3253-z