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.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

“…1A). The 9 candidates are listed in Table 1 and include (besides lysostaphin) the constructs CHAPK_CWT-LST, a fusion protein consisting of the CHAP endopeptidase domain of the phage endolysin LysK (23,24) and the SH3b-type cell wall targeting domain (CWT) of lysostaphin; Ami2638A, a truncation construct harboring the amidase domain of the 2638A endolysin (25); CHAPK_CBD2638A, a fusion of the LysK CHAP domain with the SH3b CBD of the 2638A endolysin; M23LST_CBD2638A, a fusion of the M23 endopeptidase domain of lysostaphin and the CBD of 2638A; CHAPK_CHAPK_CWT-LST, a construct containing the duplicated CHAP domain of LysK fused to the CWT of lysostaphin; M23LST_CHAPH5_CWT-LST, a fusion protein featuring an N-terminal M23 domain of lysostaphin, a centrally located CHAP domain from LysH5 (an endolysin reportedly active in cow's milk) (26), and a C-terminal CWT of lysostaphin; CHAPH5_LST, which includes the CHAP domain of LysH5 fused to lysostaphin; and CHAPTwort, a truncation construct consisting of the CHAP domain of the phage Twort endolysin (27). Of note, this selection of 9 PGH constructs comprises enzymes with diverse domain architectures, ranging from single EADs without a cell wall binding module to chimeric multidomain fusion proteins, and includes EADs targeting multiple PG cleavage sites (amidase, CHAP endopeptidases, and lysostaphin M23 endopeptidase).…”

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

“…1A). The eight candidates are listed in Table 1 and include (besides lysostaphin) the constructs CHAPK_CWT-LST, a fusion protein consisting of the CHAP endopeptidase domain of the phage endolysin LysK (23,24) and the SH3b-type cell wall-targeting domain (CWT) of lysostaphin; CHAPK_CBD2638A, a fusion of the LysK CHAP domain with the SH3b CBD of the 2638A endolysin (25); M23LST_CBD2638A, a fusion of the M23 endopeptidase domain of lysostaphin and the CBD of 2638A; CHAPK_CHAPK_CWT-LST, a construct containing the duplicated CHAP domain of LysK fused to the CWT of lysostaphin; M23LST_CHAPH5_CWT-LST, a fusion protein featuring an N-terminal M23 domain of lysostaphin, a centrally located CHAP domain from LysH5 (an endolysin reportedly active in cow's milk) (26), and a C-terminal CWT of lysostaphin; CHAPH5_LST, which includes the CHAP domain of LysH5 fused to lysostaphin; and CHAPTwort, a truncation construct consisting of the CHAP domain of the phage Twort endolysin (27).…”

Recombinant Endolysins as Potential Therapeutics against Antibiotic-Resistant Staphylococcus aureus: Current Status of Research and Novel Delivery Strategies

“…A few staphylococcal endolysins containing a CBD unrelated to the common SH3b domain have been reported as well (121). For most of the dual-EAD staphylococcal endolysins, lytic activity relies mostly on the N-terminal CHAP endopeptidase domain (116,122,123), whereas the amidase domain is virtually inactive when applied externally. One exception is the 2638A endolysin, which features a highly active amidase domain (119).…”