title: Peptidoglycan degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin.
abstract: The use of bacteriophage endolysins as specific antibacterial agents is a prospective strategy to treat bacterial infections caused by antibiotic-resistant pathogens. In case of Gram-negative species this strategy has limited applications since outer membrane shields the enzyme target and prevents bacteria lysis. We aimed to obtain and characterize the endolysin of the newly discovered anti-Salmonella bacteriophage S-394 (Lys394) and to choose an appropriate permeabilizing agent to disrupt Escherichia coli cells suspended in buffer solution and grown on agar surface. Lys394 synthesized in E. coli C41(DE3) was obtained as an electrophoretically homogenous protein. The protein of 18 kDa molecular weight shows high muralytic activity against various genera of chloroform treated Gram-negatives. Maximum of enzyme activity was observed at pH 8.5 and low ionic strength. In silico analysis of amino acid sequence identified Lys394 as an endopeptidase. Various outer membrane permeabilizers were analyzed in combination with Lys394 to degrade laboratory strain of E. coli CR63. Permeabilizing activity was evaluated using a periplasmic β-lactamase leakage test with untreated E. coli cells as a substrate. The highest rate of planktonic E. coli lysis was reached for Lys394 applied together with 25 μg/ml of poly-l-arginine with molecular weight distribution from 5 to 15 kDa or 20 μg/ml PGLa peptide. Lawn E. coli colony forming ability was decreased by 4 orders of magnitude after 30 min treatment with 25 μg of Lys394, 1 mM EDTA and 50 μg/ml of PGLa peptide at a room temperature.
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-11, Moscow 119991, Russia.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Legotsky
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-11, Moscow 119991, Russia.
authors.forename: Ksenia Yu
authors.initials: KY
authors.lastname: Vlasova
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-11, Moscow 119991, Russia.
authors.forename: Anastasia D
authors.initials: AD
authors.lastname: Priyma
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 ul. Miklukho-Maklaya, Moscow 117997, Russia.
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Shneider
authors.affiliation: State Research Center of Virology and Biotechnology "VECTOR", Koltsovo, Novosibirsk Region, 630559, Russia.
authors.forename: Vladimir G
authors.initials: VG
authors.lastname: Pugachev
authors.affiliation: State Research Center of Virology and Biotechnology "VECTOR", Koltsovo, Novosibirsk Region, 630559, Russia.
authors.forename: Olga D
authors.initials: OD
authors.lastname: Totmenina
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-11, Moscow 119991, Russia; Center for Nanotechnology in Drug Delivery, University of North Carolina, 120 Mason Farm Rd., Chapel Hill, NC 27599, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Kabanov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, 16/10 ul. Miklukho-Maklaya, Moscow 117997, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Miroshnikov
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-11, Moscow 119991, Russia. Electronic address: klyachko@enzyme.chem.msu.ru.
authors.forename: Natalia L
authors.initials: NL
authors.lastname: Klyachko
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D006088:Gram-Negative Aerobic Bacteria / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009994:Osmolar Concentration; D010457:Peptidoglycan / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D012483:Salmonella Phages / Q000737:chemistry* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D010457:Peptidoglycan; D011994:Recombinant Proteins; D010450:Endopeptidases; C001143:endolysin
keywords: Anti-Salmonella; Antimicrobials; Bacteriophage endolysin; Enterobacteriaceae; Outer membrane permeabilization