title: Lytic activity of the staphylolytic Twort phage endolysin CHAP domain is enhanced by the SH3b cell wall binding domain.
abstract: Increases in the prevalence of antibiotic-resistant strains of Staphylococcus aureus have elicited efforts to develop novel antimicrobials to treat these drug-resistant pathogens. One potential treatment repurposes the lytic enzymes produced by bacteriophages as antimicrobials. The phage Twort endolysin (PlyTW) harbors three domains, a cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), an amidase-2 domain and a SH3b-5 cell wall binding domain (CBD). Our results indicate that the CHAP domain alone is necessary and sufficient for lysis of live S. aureus, while the amidase-2 domain is insufficient for cell lysis when provided alone. Loss of the CBD results in ∼10X reduction of enzymatic activity in both turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW Δ172-373) with lytic activity that exceeded the activity of the full length construct in both the turbidity reduction and plate lysis assays. Addition of Ca(2+) enhanced the turbidity reduction activity of both the full length protein and truncation constructs harboring the CHAP domain. Chelation by addition of EDTA or the addition of zinc inhibited the activity of all PlyTW constructs.
authors: Animal Biosciences and Biotechnology Laboratory, BARC, NEA, ARS, USDA, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA.
authors: Stephen C
authors: SC
authors: Becker
authors: Animal Biosciences and Biotechnology Laboratory, BARC, NEA, ARS, USDA, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA.
authors: Steven
authors: S
authors: Swift
authors: Federal Budget Institution of Science, State Research Center for Applied Microbiology and Biotechnology, 142279, Obolensk, Serpukhov district, Moscow Region, Russia.
authors: Olga
authors: O
authors: Korobova
authors: Federal Budget Institution of Science, State Research Center for Applied Microbiology and Biotechnology, 142279, Obolensk, Serpukhov district, Moscow Region, Russia.
authors: Nina
authors: N
authors: Schischkova
authors: Federal Budget Institution of Science, State Research Center for Applied Microbiology and Biotechnology, 142279, Obolensk, Serpukhov district, Moscow Region, Russia.
authors: Pavel
authors: P
authors: Kopylov
authors: Animal Biosciences and Biotechnology Laboratory, BARC, NEA, ARS, USDA, 10300 Baltimore Avenue, Beltsville, MD 20705-2350, USA david.donovan@ars.usda.gov.
authors: David M
authors: DM
authors: Donovan
authors: Federal Budget Institution of Science, State Research Center for Applied Microbiology and Biotechnology, 142279, Obolensk, Serpukhov district, Moscow Region, Russia.
authors: Igor
authors: I
authors: Abaev
mesh_terms: D001433:Bacteriolysis; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D020536:Enzyme Activators / Q000378:metabolism; D006868:Hydrolysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013204:Staphylococcus Phages / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000821:virology*
chemical_list: D002413:Cations, Divalent; D020536:Enzyme Activators; D010450:Endopeptidases; C001143:endolysin; D002118:Calcium
keywords: Staphylococcus aureus; autolysin; bacteriophage endolysin; peptidoglycan hydrolase