Applied Microbiology and Biotechnology

, Volume 99, Issue 15, pp 6315–6326 | Cite as

Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity

  • Daniel C. Osipovitch
  • Sophie Therrien
  • Karl E. Griswold
Biotechnologically relevant enzymes and proteins


Staphylococcus aureus is a dangerous bacterial pathogen whose clinical impact has been amplified by the emergence and rapid spread of antibiotic resistance. In the search for more effective therapeutic strategies, great effort has been placed on the study and development of staphylolytic enzymes, which benefit from high potency activity toward drug-resistant strains, and a low inherent susceptibility to emergence of new resistance phenotypes. To date, the majority of therapeutic candidates have derived from either bacteriophage or environmental competitors of S. aureus. Little to no consideration has been given to cis-acting autolysins that represent key elements in the bacterium’s endogenous cell wall maintenance and recycling machinery. In this study, five putative autolysins were cloned from the S. aureus genome, and their activities were evaluated. Four of these novel enzymes, or component domains thereof, demonstrated lytic activity toward live S. aureus cells, but their potencies were 10s to 1000s of times lower than that of the well-characterized therapeutic candidate lysostaphin. We hypothesized that their poor activities were due in part to suboptimal cell wall targeting associated with their native cell wall binding domains, and we sought to enhance their antibacterial potential via chimeragenesis with the peptidoglycan binding domain of lysostaphin. The most potent chimera exhibited a 140-fold increase in lytic rate, bringing it within 8-fold of lysostaphin. While this enzyme was sensitive to certain biologically relevant environmental factors and failed to exhibit a measurable minimal inhibitory concentration, it was able to kill lysostaphin-resistant S. aureus and ultimately proved active in lung surfactant. We conclude that the S. aureus proteome represents a rich and untapped reservoir of novel antibacterial enzymes, and we demonstrate enhanced bacteriolytic activity via improved cell wall targeting of autolysin catalytic domains.


MRSA Lysins Lytic enzyme CHAP Lysostaphin Antibiotic 



We would like to thank Dr. Gary Sloan at the University of Alabama for kindly providing the RN4220 strains used in this paper. We would also like to thank Ony, Inc. for supplying the Infasurf used in this study. This work was supported in part by R21 grant 1R21AI098122 from the National Institutes of Health NIAID to KEG.

Conflict of interest

The authors claim no conflict of interest.

Supplementary material

253_2015_6443_MOESM1_ESM.pdf (931 kb)
ESM 1 (PDF 930 kb)


  1. Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM (2009) LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol Lett 294(1):52–60. doi: 10.1111/j.1574-6968.2009.01541.x PubMedCrossRefGoogle Scholar
  2. Bose JL, Lehman MK, Fey PD, Bayles KW (2012) Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation. PLoS One 7(7):e42244. doi: 10.1371/journal.pone.0042244 PubMedCentralPubMedCrossRefGoogle Scholar
  3. Brunskill EW, Bayles KW (1996) Identification of LytSR-regulated genes from Staphylococcus aureus. J Bacteriol 178(19):5810–5812PubMedCentralPubMedGoogle Scholar
  4. Buist G, Steen A, Kok J, Kuipers OP (2008) LysM, a widely distributed protein motif for binding to (peptido)glycans. Mol Microbiol 68(4):838–847. doi: 10.1111/j.1365-2958.2008.06211.x PubMedCrossRefGoogle Scholar
  5. Centers for Disease Control and Prevention (2013) Antibiotic resistance threats in the United States, 2013 Threats ReportGoogle Scholar
  6. Cheung AL, Bayer AS, Zhang G, Gresham H, Xiong YQ (2004) Regulation of virulence determinants in vitro and in vivo in Staphylococcus aureus. FEMS Immunol Med Microbiol 40(1):1–9PubMedCrossRefGoogle Scholar
  7. Chu X, Xia R, He N, Fang Y (2013) Role of Rot in bacterial autolysis regulation of Staphylococcus aureus NCTC8325. Res Microbiol 164(7):695–700. doi: 10.1016/j.resmic.2013.06.001 PubMedCrossRefGoogle Scholar
  8. Cole C, Barber JD, Barton GJ (2008) The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36(Web Server issue):W197–W201. doi: 10.1093/nar/gkn238 PubMedCentralPubMedCrossRefGoogle Scholar
  9. Cosgrove SE, Qi Y, Kaye KS, Harbarth S, Karchmer AW, Carmeli Y (2005) The impact of methicillin resistance in Staphylococcus aureus bacteremia on patient outcomes: mortality, length of stay, and hospital charges. Infect Control Hosp Epidemiol 26(2):166–174. doi: 10.1086/502522 PubMedCrossRefGoogle Scholar
  10. Dehart HP, Heath HE, Heath LS, Leblanc PA, Sloan GL (1995) The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus. Appl Environ Microbiol 61(7):2811PubMedCentralPubMedGoogle Scholar
  11. Donovan DM, Dong S, Garrett W, Rousseau GM, Moineau S, Pritchard DG (2006) Peptidoglycan hydrolase fusions maintain their parental specificities. Appl Environ Microbiol 72(4):2988–2996. doi: 10.1128/AEM.72.4.2988-2996.2006 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Dziarski R, Gupta D (2006) The peptidoglycan recognition proteins (PGRPs). Genome Biol 7(8):232. doi: 10.1186/gb-2006-7-8-232 PubMedCentralPubMedCrossRefGoogle Scholar
  13. Fernandes S, Proença D, Cantante C, Silva FA, Leandro C, Lourenço S, Milheiriço C, de Lencastre H, Cavaco-Silva P, Pimentel M, São-José C (2012) Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus. Microb Drug Resist (Larchmont, NY) 18(3):333–343. doi: 10.1089/mdr.2012.0025 CrossRefGoogle Scholar
  14. Frankel MB, Schneewind O (2012) Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan. J Biol ChemGoogle Scholar
  15. Frankel MB, Hendrickx AP, Missiakas DM, Schneewind O (2011) LytN, a murein hydrolase in the cross-wall compartment of Staphylococcus aureus, is involved in proper bacterial growth and envelope assembly. J Biol Chem 286(37):32593–32605. doi: 10.1074/jbc.M111.258863 PubMedCentralPubMedCrossRefGoogle Scholar
  16. Gargis SR, Heath HE, LeBlanc PA, Dekker L, Simmonds RS, Sloan GL (2010) Inhibition of the activity of both domains of lysostaphin through peptidoglycan modification by the lysostaphin immunity protein. Appl Environ Microbiol 76(20):6944–6946. doi: 10.1128/AEM. 01066-10 PubMedCentralPubMedCrossRefGoogle Scholar
  17. Gotz F, Heilmann C, Stehle T (2014) Functional and structural analysis of the major amidase (Atl) in Staphylococcus. Int J Med Microbiol 304(2):156–163. doi: 10.1016/j.ijmm.2013.11.006 PubMedCrossRefGoogle Scholar
  18. Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Molecular cloning: a laboratory manualGoogle Scholar
  19. Grundling A, Schneewind O (2006) Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus. J Bacteriol 188(7):2463–2472. doi: 10.1128/JB.188.7.2463-2472.2006 PubMedCentralPubMedCrossRefGoogle Scholar
  20. Kokai-Kun JF (2012) Lysostaphin: a silver bullet for staph. Antimicrobial Drug Discovery: Emerging StrategiesGoogle Scholar
  21. Kusuma C, Jadanova A, Chanturiya T, Kokai-Kun JF (2007) Lysostaphin-resistant variants of Staphylococcus aureus demonstrate reduced fitness in vitro and in vivo. Antimicrob Agents Chemother 51(2):475–482. doi: 10.1128/AAC. 00786-06 PubMedCentralPubMedCrossRefGoogle Scholar
  22. Letunic I, Doerks T, Bork P (2014) SMART: recent updates, new developments and status in 2015. Nucleic Acids Res. doi: 10.1093/nar/gku949 Google Scholar
  23. Ling B, Berger-Bachi B (1998) Increased overall antibiotic susceptibility in Staphylococcus aureus femAB null mutants. Antimicrob Agents Chemother 42(4):936–938PubMedCentralPubMedGoogle Scholar
  24. Mao J, Schmelcher M, Harty WJ, Foster-Frey J, Donovan DM (2013) Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme. FEMS Microbiol Lett 342(1):30–36. doi: 10.1111/1574-6968.12104 PubMedCentralPubMedCrossRefGoogle Scholar
  25. Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH (2011) CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 39(Database issue):D225–D229. doi: 10.1093/nar/gkq1189 PubMedCentralPubMedCrossRefGoogle Scholar
  26. Martin PK, Bao Y, Boyer E, Winterberg KM, McDowell L, Schmid MB, Buysse JM (2002) Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus. J Bacteriol 184(20):5810–5813PubMedCentralPubMedCrossRefGoogle Scholar
  27. Mellroth P, Sandalova T, Kikhney A, Vilaplana F, Hesek D, Lee M, Mobashery S, Normark S, Svergun D, Henriques-Normark B, Achour A (2014) Structural and functional insights into peptidoglycan access for the lytic amidase LytA of Streptococcus pneumoniae. MBio 5(1):e01120-13. doi: 10.1128/mBio.01120-13 PubMedCentralPubMedCrossRefGoogle Scholar
  28. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2(9):2212–2221. doi: 10.1038/nprot.2007.321 PubMedCrossRefGoogle Scholar
  29. Osipovitch DC, Griswold KE (2015) Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent. FEMS Microbiol Lett 362(2):1-7. doi: 10.1093/femsle/fnu035 PubMedCrossRefGoogle Scholar
  30. Osipovitch DC, Parker AS, Makokha CD, Desrosiers J, Kett WC, Moise L, Bailey-Kellogg C, Griswold KE (2012) Design and analysis of immune-evading enzymes for ADEPT therapy. Protein Eng Des Sel 25(10):613–623. doi: 10.1093/protein/gzs044 PubMedCentralPubMedCrossRefGoogle Scholar
  31. Pastagia M, Schuch R, Fischetti VA, Huang DB (2013) Lysins: the arrival of pathogen-directed anti-infectives. J Med Microbiol 62(Pt 10):1506–1516. doi: 10.1099/jmm.0.061028-0 PubMedCrossRefGoogle Scholar
  32. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786. doi: 10.1038/nmeth.1701 PubMedCrossRefGoogle Scholar
  33. Resch A, Rosenstein R, Nerz C, Gotz F (2005) Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. Appl Environ Microbiol 71(5):2663–2676. doi: 10.1128/AEM.71.5.2663-2676.2005 PubMedCentralPubMedCrossRefGoogle Scholar
  34. Rice KC, Bayles KW (2003) Death's toolbox: examining the molecular components of bacterial programmed cell death. Mol Microbiol 50(3):729–738. doi: 10.1046/j.1365-2958.2003.t01-1-03720.x PubMedCrossRefGoogle Scholar
  35. Rodriguez-Rubio L, Martinez B, Rodriguez A, Donovan DM, Garcia P (2012) Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5. Appl Environ Microbiol 78(7):2241–2248. doi: 10.1128/AEM.07621-11 PubMedCentralPubMedCrossRefGoogle Scholar
  36. Rodríguez-Rubio L, Martínez B, Donovan DM, Rodríguez A, García P (2013) Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics. Crit Rev Microbiol 39(4):427–434. doi: 10.3109/1040841X.2012.723675 PubMedCrossRefGoogle Scholar
  37. Sabala I, Jonsson I-MM, Tarkowski A, Bochtler M (2012) Anti-staphylococcal activities of lysostaphin and LytM catalytic domain. BMC Microbiol 12:97. doi: 10.1186/1471-2180-12-97 PubMedCentralPubMedCrossRefGoogle Scholar
  38. Sabala I, Jagielska E, Bardelang PT, Czapinska H, Dahms SO, Sharpe JA, James R, Than ME, Thomas NR, Bochtler M (2014) Crystal structure of the antimicrobial peptidase lysostaphin from Staphylococcus simulans. FEBS J 281(18):4112–4122. doi: 10.1111/febs.12929 PubMedCentralPubMedCrossRefGoogle Scholar
  39. Scanlon TC, Teneback CC, Gill A, Bement JL, Weiner JA, Lamppa JW, Leclair LW, Griswold KE (2010) Enhanced antimicrobial activity of engineered human lysozyme. ACS Chem Biol 5(9):809–818. doi: 10.1021/cb1001119 PubMedCentralPubMedCrossRefGoogle Scholar
  40. Schlag M, Biswas R, Krismer B, Kohler T, Zoll S, Yu W, Schwarz H, Peschel A, Götz F (2010) Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl. Mol Microbiol 75(4):864–873. doi: 10.1111/j.1365-2958.2009.07007.x PubMedCrossRefGoogle Scholar
  41. Schmelcher M, Powell AM, Becker SC, Camp MJ, Donovan DM (2012) Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands. Appl Environ Microbiol 78(7):2297–2305. doi: 10.1128/AEM.07050-11 PubMedCentralPubMedCrossRefGoogle Scholar
  42. Schuch R, Lee HM, Schneider BC, Sauve KL, Law C, Khan BK, Rotolo JA, Horiuchi Y, Couto DE, Raz A, Fischetti VA, Huang DB, Nowinski RC, Wittekind M (2014) Combination therapy with lysin CF-301 and antibiotic is superior to antibiotic alone for treating methicillin-resistant Staphylococcus aureus-induced murine bacteremia. J Infect Dis 209(9):1469–1478. doi: 10.1093/infdis/jit637 PubMedCentralPubMedCrossRefGoogle Scholar
  43. Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc Natl Acad Sci U S A 95(11):5857–5864PubMedCentralPubMedCrossRefGoogle Scholar
  44. Shurland S, Zhan M, Bradham DD, Roghmann MC (2007) Comparison of mortality risk associated with bacteremia due to methicillin-resistant and methicillin-susceptible Staphylococcus aureus. Infect Control Hosp Epidemiol 28(3):273–279. doi: 10.1086/512627 PubMedCrossRefGoogle Scholar
  45. Singh PK, Donovan DM, Kumar A (2014) Intravitreal injection of the chimeric phage endolysin Ply187 protects mice from Staphylococcus aureus endophthalmitis. Antimicrob Agents Chemother 58(8):4621–4629. doi: 10.1128/AAC.00126-14 PubMedCentralPubMedCrossRefGoogle Scholar
  46. Sundarrajan S, Raghupatil J, Vipra A, Narasimhaswamy N, Saravanan S, Appaiah C, Poonacha N, Desai S, Nair S, Bhatt RN, Roy P, Chikkamadaiah R, Durgaiah M, Sriram B, Padmanabhan S, Sharma U (2014) Bacteriophage-derived CHAP domain protein, P128, kills Staphylococcus cells by cleaving interpeptide cross-bridge of peptidoglycan. Microbiology 160(Pt 10):2157–2169. doi: 10.1099/mic.0.079111-0 PubMedCrossRefGoogle Scholar
  47. Surovtsev VI, Fedorov TV, Borozdina MA (2004) Michaelis-menten kinetics for determining enzymatic activity of lysostaphin. Biochemistry (Mosc) 69(7):754–756CrossRefGoogle Scholar
  48. Szweda P, Schielmann M, Kotlowski R, Gorczyca G, Zalewska M, Milewski S (2012) Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus. Appl Microbiol Biotechnol 96(5):1157–1174. doi: 10.1007/s00253-012-4484-3 PubMedCentralPubMedCrossRefGoogle Scholar
  49. Takahashi J, Komatsuzawa H, Yamada S, Nishida T, Labischinski H, Fujiwara T, Ohara M, Yamagishi J-i, Sugai M (2001) Molecular characterization of an atl null mutant of Staphylococcus aureus. Microbiol Immunol 46(9):601–612CrossRefGoogle Scholar
  50. Taubes G (2008) The bacteria fight back. Science 321(5887):356–361. doi: 10.1126/science.321.5887.356 PubMedCrossRefGoogle Scholar
  51. Thomas VC, Hancock LE (2009) Suicide and fratricide in bacterial biofilms. Int J Artif Organs 32(9):537–544PubMedGoogle Scholar
  52. Tillman GE, Simmons M, Garrish JK, Seal BS (2013) Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium. Arch Microbiol 195(10–11):675–681. doi: 10.1007/s00203-013-0916-4 PubMedCentralPubMedCrossRefGoogle Scholar
  53. Vollmer W, Joris B, Charlier P, Foster S (2008) Bacterial peptidoglycan (murein) hydrolases. FEMS Microbiol Rev 32(2):259–286. doi: 10.1111/j.1574-6976.2007.00099.x PubMedCrossRefGoogle Scholar
  54. Wang X, Wilkinson BJ, Jayaswal RK (1991) Sequence analysis of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity. Gene 102(1):105–109PubMedCrossRefGoogle Scholar
  55. Whisstock JC, Lesk AM (1999) SH3 domains in prokaryotes. Trends Biochem Sci 24(4):132–133PubMedCrossRefGoogle Scholar
  56. Willson D (2001) Calfactant. Expert Opin Pharmacother 2(9):1479–1493. doi: 10.1517/14656566.2.9.1479 PubMedCrossRefGoogle Scholar
  57. World Health Organization (2014) Antimicrobial resistance: global report on surveillance. p 257Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Daniel C. Osipovitch
    • 1
  • Sophie Therrien
    • 2
  • Karl E. Griswold
    • 3
    • 4
  1. 1.Program in Experimental and Molecular MedicineDartmouth CollegeHanoverUSA
  2. 2.New England CollegeHennikerUSA
  3. 3.Thayer School of EngineeringDartmouth CollegeHanoverUSA
  4. 4.Program in Molecular and Cellular BiologyDartmouth CollegeHanoverUSA

Personalised recommendations