|Don, Shengli - UNIV OF AL BIRMINGHAM|
|Baker, John - UNIV OF AL BIRMINGHAM|
|Foster Frey, Juli|
|Pritchard, David - UNIV OF AL BIRMINGHAM|
Submitted to: FEMS Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 29, 2009
Publication Date: March 10, 2009
Citation: Becker, S.C., Don, S., Baker, J.R., Foster Frey, J.A., Pritchard, D.G., Donovan, D.M. 2009. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiology Letter. 294(1):52-60. Interpretive Summary: Problem: Staphylococci are gram positive pathogens that have large negative impacts on both agriculture and human health. S. aureus and Coagulase-negative staphylococcus (CoNS) are leading causes of bovine mastitis, a 2 billion dollar a year loss, accounting for more than 40% of all cases in one study. Traditional treatments for mastitis, most commonly antibiotics, have been shown to be only marginally effective. Bacteriophage endolysins are a potential source of narrow spectrum antibiotics for treatment of antibiotic resistant pathogens. Accomplishments: We have determined the cut sites of each active domain and further dissected LysK to determine the CHAP domain of LysK appears to be the crucial element in promoting exolysis, but the presence of a SH3b cell wall binding domain greatly increases the efficiency of lysis. The Amidase domain, while active, does not appear to contribute to exolysis, and the loss of the amidase domain does not decrease the activity of the enzyme when tested against live S. aureus and CoNS. Contribution of Accomplishment to Solving the Problem: The findings of this work identify the necessary components of this phage endolysin for its application as an antimicrobial, and will aid scientists in the development of similar molecules as antimicrobials.
Technical Abstract: LysK is a staphylococcal bacteriophage endolysin composed of three domains, an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain (cleaves between D-alanine of the stem peptide and glycine of the cross-bridge peptide) a mid-protein amidase 2 domain (N-acetylmuramoyl-L-alanine amidase activity), and a C-terminal SH3b_5 (SH3b) cell-wall binding domain, . Both catalytic domains were shown to be active on purified peptidoglycan by positive-ion electrospray ionization mass spectrometry, from which the cut sites were determined to be identical to LytA (phi11 endolysin). Truncations of the LysK protein which contain the CHAP domain are demonstrated to lyse S. aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays. The amidase domain, with the SH3b domain shows only faint activity in either zymogram or turbidity reduction assays but has no detectable activity in either the plate lysis or MIC assays. When the amidase domain is deleted, resulting in a fusion of the CHAP domain to the SH3b domain, the resulting fusion protein has near full length LysK lytic activity in all of the lytic assays including MICs, suggesting the need for a c-terminal binding domain for full activity. Both the wild type molecule and the CHAP-SH3b fusion were shown to lyse untreated staphylococci S. aureus Newman, S. aureus Newbould 305, and the coagulase-negative strains S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri and, S. xylocus in turbidity reduction assays and act equally synergisticwith the bacterocin Lysostaphin.