Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity
- 677 Downloads
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.
KeywordsMRSA 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.
- Centers for Disease Control and Prevention (2013) Antibiotic resistance threats in the United States, 2013 Threats ReportGoogle Scholar
- 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
- 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
- Frankel MB, Schneewind O (2012) Determinants of murein hydrolase targeting to cross-wall of Staphylococcus aureus peptidoglycan. J Biol ChemGoogle Scholar
- 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
- 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
- Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual. Molecular cloning: a laboratory manualGoogle Scholar
- Kokai-Kun JF (2012) Lysostaphin: a silver bullet for staph. Antimicrobial Drug Discovery: Emerging StrategiesGoogle Scholar
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- World Health Organization (2014) Antimicrobial resistance: global report on surveillance. p 257Google Scholar