LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells.
LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.
Becker
Stephen C
SC
Animal Biosciences and Biotechnology Laboratory, Animal and Natural Resources Institute, BARC, ARS, USDA, Beltsville, MD 20705-2350, USA.
Dong
Shengli
S
Baker
John R
JR
Foster-Frey
Juli
J
Pritchard
David G
DG
Donovan
David M
DM
D000900:Anti-Bacterial Agents / Q000378:metabolism; D001433:Bacteriolysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008248:Lysostaphin / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability; D017384:Sequence Deletion; D013204:Staphylococcus Phages / Q000201:enzymology*; D013211:Staphylococcus aureus / Q000187:drug effects / Q000821:virology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D014764:Viral Proteins; D010450:Endopeptidases; D008248:Lysostaphin; C001143:endolysin