Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis

Abstract

BackgroundEndolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33).ResultsBioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50°C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60°C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins.ConclusionsPlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.

Bacteriophages and bacteriophage-derived endolysins as potential therapeutics to combat Gram-positive spore forming bacteria

“…), while lysins of Gram‐positive bacteria are usually 25–40 kDa large (Fischetti ), modular in structure and exhibit variety in their architecture that goes beyond the classical two domain structure of endolysins (Yuan et al . ).…”

“…The number of binding domains varies between endolysins (Yuan et al . ). The affinity is so strong that it may be compared to antigen‐antibody binding (Loessner et al .…”

Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against Bacillus cereus

“…Although PBC1 has a virulent lifestyle and rapidly lyses the host B. cereus strain, its extremely narrow host range may limit its efficiency as a biocontrol agent unless it is used in a phage cocktail. It has been reported that the endolysin, a phage-encoded peptidoglycan hydrolase, generally shows a broader lytic spectrum than the phage (39)(40)(41)(42)(43). Moreover, the development of resistance against endolysins has not been reported (44)(45)(46)(47).…”

“…LysPBC1 has three Zn 2ϩ -coordinating residues (Glu23, His79, and Glu140) in the catalytic domain, and these residues are conserved in all four endolysins. Considering that the C-terminal amidase02_C domain of LysPBC1 did not show any homology to PlyPSA and CD27L and that several endolysins have an amidase02_C domain (PF12123) as a cell wall binding domain (42,51), the C-terminal domain is presumed to function in binding the B. cereus cell wall.…”

Preclinical Safety Evaluation of Intravenously Administered SAL200 Containing the Recombinant Phage Endolysin SAL-1 as a Pharmaceutical Ingredient

“…Since their discovery, the use of phage endolysins as antibacterial agents has been proposed due to their distinct mode of action and highly specific antibacterial activity, which is independent of the antibiotic susceptibility pattern of the bacterium (5). A number of studies have applied phage endolysins to target pathogens such as Bacillus anthracis (7), Streptococcus pneumoniae (8), S. aureus (9), and Bacillus thuringiensis (10), and promising results have been observed in animal models of human disease (8,(11)(12)(13)(14)(15)(16)(17)(18). Thus, phage endolysins represent a promising line of research for the discovery and development of novel antibacterial therapeutic agents (19).…”