Applied Microbiology and Biotechnology

, Volume 89, Issue 6, pp 1783–1795 | Cite as

Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

  • Jonathan E. Schmitz
  • Maria Cristina Ossiprandi
  • Kareem R. Rumah
  • Vincent A. Fischetti
Biotechnologically Relevant Enzymes and Proteins

Abstract

With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low μg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or non-clostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.

Keywords

Lysin Prophage Enzybiotic Muramidase Clostridium perfringens 

Notes

Acknowledgments

This research was funded by NIH/NIAID grants AI057472 and AI11822 to VAF. JES acknowledges the kind support of the NIH MSTP program (Weill Cornell/Rockefeller/Sloan-Kettering grant GM 07739). The authors would like to thank Dr. Davise Larone of New York Hospital for providing clostridial strains. We also acknowledge Prof. Ezio Bottarelli of the University of Parma for his valued insights, as well as Ms. Cinzia Reverberi and Mr. Roberto Lurisi for technical assistance. Electron micrographs were acquired by Ms. Eleana Sphicas of the Rockefeller University Electron Microscopy Core Facility.

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410Google Scholar
  2. Baker JR, Liu C, Dong S, Pritchard DG (2006) Endopeptidase and glycosidase activities of the bacteriophage B30 lysin. Appl Environ Microbiol 72:6825–6828Google Scholar
  3. Bendtsen JD, Nielsen H, von Heijne G, Brunak S (2005) Improved prediction of signal peptides: signalP 3.0. J Mol Biol 340:783–795Google Scholar
  4. Borysowksi J, Weber-Dabrowska B, Gorski A (2005) Bacteriophage endolysins as a novel class of antibacterial reagents. Exp Biol Med 231:366–377Google Scholar
  5. Briers Y, Volckaert G, Cornelissen LS, Michiels CW, Hertveldt K, Lavigne R (2007) Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages ϕKZ and EL. Mol Microbiol 65:1334–1344CrossRefGoogle Scholar
  6. Bryant AE, Stevens DL (1997) The pathogenesis of gas gangrene. In: Rood JI, McClane BA, Songer JG, Titball RW (eds) The clostridia: molecular biology and pathogenesis, 1st edn. Academic, London, pp 185–196Google Scholar
  7. Camiade E, Peltier J, Bourgeois I, Couture-Tosi E, Courtin P, Antunes A, Chapot-Chartier MP, Dupuy B, Pons JL (2010) Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N-acetylglucosaminidase activity that is implicated in cell separation and stress-induced autolysis. J Bacteriol 192:2373–2384CrossRefGoogle Scholar
  8. Carman RJ, Sayeed S, Li J, Genheimer CW, Hiltonsmith MF, Wilkins TD, McClane BA (2008) Clostridium perfringens toxin genotypes in the feces of healthy North Americans. Anaerobe 14:102–108CrossRefGoogle Scholar
  9. Cheng Q, Nelson D, Zhu S, Fischetti VA (2005) Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Antimicrob Agents Chemother 49:111–117CrossRefGoogle Scholar
  10. Daniel A, Euler C, Collin M, Chahales P, Gorelick KJ, Fischetti VA (2010) Synergism between a novel chimeric lysin and oxacillin protects against infection by methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 54:1603–1612CrossRefGoogle Scholar
  11. Deutsch S, Guezenec S, Piot M, Foster S, Lortal S (2004) Mur-LH, the broad-spectrum endolysin of Lactobacillus helveticus temperate bacteriophage phi-0303. Appl Environ Microbiol 70:96–103CrossRefGoogle Scholar
  12. Diaz E, López R, Garcia JL (1991) Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits. J Biol Chem 266:5464–5471Google Scholar
  13. Felsenstein J (1989) PHYLIP—Phylogeny inference package (version 3.2). Cladistics 5:164–166Google Scholar
  14. Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR, Bateman A (2010) The Pfam protein families database. Nucleic Acid Res 38:D211–D222CrossRefGoogle Scholar
  15. Fischetti VA (2010) Bacteriophage endolysins: a novel anti-infective to control Gram-positive pathogens. Int J Med Microbiol 300:357–362CrossRefGoogle Scholar
  16. Fischetti VA (2008) Bacteriophage lysins as effective antibacterials. Curr Opin Microbiol 11:393–400CrossRefGoogle Scholar
  17. García P, García JL, García E, Sánchez-Puelles JM, López R (1990) Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 86:81–88CrossRefGoogle Scholar
  18. Garnier, Cole ST (1986) Characterization of a bacteriocinogenic plasmid from Clostridium perfringens and molecular genetic analysis of the bacteriocin-encoding gene. J Bacteriol 168:1189–1196Google Scholar
  19. Grandgirard D, Loeffler JM, Fischetti VA, Leib SL (2008) Phage lytic enzyme Cpl-1 for antibacterial therapy in experimental pneumococcal meningitis. J Infect Dis 197:1519–1522CrossRefGoogle Scholar
  20. Hamada H, Arakawa T, Shiraki K (2009) Effect of additives on protein aggregation. Curr Pharm Biotechnol 10:400–407CrossRefGoogle Scholar
  21. Hermoso JA, Monterroso B, Albert A, Galán B, Ahrazem O, García P, Martínez-Ripoll M, García JL, Menéndez M (2003) Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 11:1239–1249CrossRefGoogle Scholar
  22. Jay JM, Loessner MJ, Golden DA (2005) Food protection with chemicals, and by biocontrol. In: Modern Food Microbiology, 7th edn. Springer, New York, pp 301–350Google Scholar
  23. Kim W, Salm H, Geider K (2004) Expression of bacteriophage phiEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora. Microbiology 150:2702–2714Google Scholar
  24. Korndörfer IP, Kanitz A, Danzer J, Zimmer M, Loessner MJ, Skerra A (2008) Structural analysis of the l-alanoyl-d-glutamate endopeptidase domain of Listeria bacteriophage endolysin Ply500 reveals a new member of the LAS peptidase family. Acta Cryst 64:644–650CrossRefGoogle Scholar
  25. Lawrence GW (1997) The pathogenesis of enteritis necroticans. In: Rood JI, McClane BA, Songer JG, Titball RW (eds) The clostridia: molecular biology and pathogenesis, 1st edn. Academic, London, pp 197–210Google Scholar
  26. Lima-Mendez G, Van Helden J, Toussaint A, Leplae R (2008) Prophinder: a computational tool for prophage prediction in prokaryotic genomes. Bioinformatics 24:863–865CrossRefGoogle Scholar
  27. Loeffler JM, Nelson D, Fischetti VA (2001) Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 294:2170–2172CrossRefGoogle Scholar
  28. Loessner MJ (2005) Bacteriophage endolysins—current state of research and applications. Curr Opin Microbiol 8:480–487CrossRefGoogle Scholar
  29. Matches JR, Liston J, Curran D (1974) Clostridium perfringens in the environment. Appl Microbiol 28:655–660Google Scholar
  30. 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, Watkins 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 16:1031–1040CrossRefGoogle Scholar
  31. 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 98:4107–4112CrossRefGoogle Scholar
  32. Nelson D, Schuch R, Chahales P, Zhu S, Fischetti VA (2006) PlyC: a multimeric bacteriophage lysin. Proc Natl Acad Sci USA 103:10765–10770CrossRefGoogle Scholar
  33. Porter CJ, Schuch R, Pelzek AJ, Buckle AM, McGowan S, Wilce MC, Rossjohn J, Russell R, Nelson D, Fischetti VA, Whisstock JC (2007) The 1.6-A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis. J Mol Biol 366:540–550CrossRefGoogle Scholar
  34. Pritchard DG, Dong S, Kirk MC, Cartee RT, Baker JR (2007) LambdaSa1 and lambdaSa2 prophage lysins of Streptococcus agalactiae. Appl Environ Microbiol 73:7150–7154CrossRefGoogle Scholar
  35. Pichoff S, Lutkenhaus J (2007) Overview of cell shape: cytoskeletons shape bacterial cells. Curr Opin Microbiol 10:601–605CrossRefGoogle Scholar
  36. Rashel M, Uchiyama J, Takemura I, Hoshiba H, Ujihara T, Takatsuji H, Honke K, Matsuzaki S (2008) Tail-associated structural protein gp61 of Staphylococcus aureus phage phi MR11 has bifunctional lytic activity. FEMS Microbiol Lett 284:9–16CrossRefGoogle Scholar
  37. Schmitz JE, Schuch R, Fischetti VA (2010) Identifying phage lytic enzymes: past, present, and future. In: Villa TG, Veiga-Crespo P (eds) Enzybiotics: antibiotic enzymes as drugs and therapeutics, 1st edn. Wiley, Hoboken, pp 219–251Google Scholar
  38. Schuch R, Nelson D, Fischetti VA (2002) A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 418:884–889CrossRefGoogle Scholar
  39. Seal BS, Fouts DE, Simmons M, Garrish JK, Kuntz RL, Woolsey R, Schegg KM, Kropinski AM, Ackermann HW, Siragusa GR (2010) Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions. Arch Virol. doi: 10.1007/s00705-010-0812-z Google Scholar
  40. 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 99:996–1001CrossRefGoogle Scholar
  41. Simmons M, Donovan DM, Siragusa GR, Seal BS (2010) Recombinant expression of two bacteriophage proteins that lyse Clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains. J Agric Food Chem 58:10330–10337CrossRefGoogle Scholar
  42. Smedly JG III, Fisher DJ, Sayeed S, Chakrabarti G, McClane BA (2004) The enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 152:183–204CrossRefGoogle Scholar
  43. United States Food and Drug Administration (2009) The bad bug book: foodborne pathogenic microorganisms and natural toxins handbook. http://www.fda.gov/Food/FoodSafety/FoodborneIllness/default.htm. Accessed 22 October 2010
  44. Uzal FA, Songer JG (2008) Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J Vet Diagn Invest 20:253–265Google Scholar
  45. Van Immerseel F, Rood JI, Moore RJ, Titball RW (2009) Rethinking our understanding of the pathogenesis of necrotic enteritis in chickens. Trends Microbiol 17:32–36CrossRefGoogle Scholar
  46. Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32:259–286CrossRefGoogle Scholar
  47. Wang S, Kong J, Zhang X (2008) Identification and characterization of the two-component cell lysis cassette encoded by temperate bacteriophage phiPYB5 of Lactobacillus fermentum. J Appl Microbiol 105:1939–1944CrossRefGoogle Scholar
  48. Xu Q, Sudek S, McMullen D, Miller MD, Geierstanger B, Jones DH, Krishna SS, Spraggon G, Bursalay B, Abdubek P, Acosta C, Ambing E, Astakhova T, Axelrod HL, Carlton D, Caruthers J, Chiu HJ, Clayton T, Deller MC, Duan L, Elias Y, Elsliger MA, Feuerhelm J, Grzechnik SK, Hale J, Han GW, Haugen J, Jaroszewski L, Jin KK, Klock HE, Knuth MW, Kozbial P, Kumar A, Marciano D, Morse AT, Nigoghossian E, Okach L, Oommachen S, Paulsen J, Reyes R, Rife CL, Trout CV, van den Bedem H, Weekes D, White A, Wolf G, Zubieta C, Hodgson KO, Wooley J, Deacon AM, Godzik A, Lesley SA, Wilson IA (2009) Structural basis of murein peptide specificity of a γ-d-glutamyl-l-diamino acid endopeptidase. Structure 17:303–313CrossRefGoogle Scholar
  49. Ye T, Zhang X (2008) Characterization of a lysin from deep-sea thermophilic bacteriophage GVE2. Appl Microbiol Biotechnol 78:635–641CrossRefGoogle Scholar
  50. Yoong P, Schuch R, Nelson D, Fischetti VA (2006) PlyPH, a bacteriolytic enzyme with a broad pH range of activity and lytic action against Bacillus anthracis. J Bacteriol 188:2711–2714CrossRefGoogle Scholar
  51. Zimmer M, Loessner M, Morgan AJ (2008) US Patent 7,371,375Google Scholar
  52. Zimmer M, Scherer S, Loessner MJ (2002a) Genomic analysis of Clostridium perfringens bacteriophage phi3626, which integrates into guaA and possibly affects sporulation. J Bacteriol 184:4359–4368CrossRefGoogle Scholar
  53. Zimmer M, Vukov N, Scherer S, Loessner MJ (2002b) The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 68:5311–5317CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Jonathan E. Schmitz
    • 1
  • Maria Cristina Ossiprandi
    • 2
  • Kareem R. Rumah
    • 1
  • Vincent A. Fischetti
    • 1
  1. 1.Laboratory of Bacterial Pathogenesis and ImmunologyThe Rockefeller UniversityNew YorkUSA
  2. 2.Department of Animal Health, Microbiology and Immunology SectionUniversity of Parma, Faculty of Veterinary MedicineParmaItaly

Personalised recommendations