The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates

Abstract

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereus sensu lato group species, including B. cereus sensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereus sensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereus sensu lato isolates tested (n ϭ 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium. IMPORTANCE Organisms of the Bacillus cereus sensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereus sensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereus sensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis. Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

Characterization of PlyB221 and PlyP32, Two Novel Endolysins Encoded by Phages Preying on the Bacillus cereus Group

“…PlyB221 showed high activity (>50%) from pH 6 to 9 whereas PlyP32 activity rapidly dropped outside the range of pH 6 and 7. Endolysins from phages targeting B. cereus that are effective at pH 6 have already been described (i.e., PlyPH and PlyB) but they were also highly active up to pH 9, contrary to PlyP32 [47,49]. The addition of salt did not improve the activity of PlyP32 and PB221, contrary to what was observed for PlyB, and they were able to withstand up to 100 mM and 200 mM of NaCl, respectively, without any loss of activity.…”

“…It was previously shown for LysB4 and PlyP56, that the activity is lost in the presence of EDTA and then restored with the addition of metal ions-such as zinc, calcium, and manganese-thus reinforcing the fact that PlyB221 also relies on divalent cations for its activity [18,20]. PlyP32 possesses a GH25_muramidase EAD that is similar to that of PlyB, the endolysin encoded by the Bacillus phage Bcp1, and PlyBt33 from phage BtCS33 [47,48]. Both PlyB221 and PlyP32 displayed high lytic activity within the first 10 min of incubation with the most sensitive bacteria when 100 µg/mL of protein was tested, which corresponds to 2.6 and 2.9 µM, respectively.…”

“…PlyB221 and PlyP32 both have intermediate activity spectra, as they are active against strains of the B. cereus group but also B. megaterium, B. pumilus, and B. licheniformis. Noteworthy, PlyB221 and PlyP32 were not tested against B. anthracis, but based on their close relationships to PlyP56 and PlyB whose activity against B. anthracis was demonstrated [20,47], their activity spectrum is expected to include this pathogenic bacterium. Also, PlyB221 and PlyP32 are active on a much wider range of bacteria than that of their parental phages.…”

“…In both endolysins, the CBD is represented by SH3 domains, a type of CBD widespread among endolysins of phages infecting B. cereus along with the Amidase02_C domain (PF12123) [17,21,47,52]. Contrary to the endolysins activity range that extended to several species outside the B. cereus group, binding of both CBD was limited to strains of the B. cereus group and to B. megaterium (Table 3).…”

The endolysin of the Acinetobacter baumannii phage vB_AbaP_D2 shows broad antibacterial activity

“…Endolysins are phage‐encoded enzymes that degrade the bacterial cell walls at the end of a phage replication cycle (Schmelcher et al ., 2012) and have antibacterial activity against Gram‐positive bacteria. In the last decade, endolysins have been investigated as therapeutic agents, applied to a wide range of Gram‐positive bacteria, such as streptococci (Nelson et al ., 2001), Enterococcus faecalis (Swift et al ., 2016), Bacillus cereus (Schuch et al ., 2019), Staphylococcus aureus (Gilmer et al ., 2013) and Streptococcus pneumoniae (Jado et al ., 2003). Several Gram‐positive phage endolysins have been shown to possess anti‐biofilm abilities including PlyC (Shen et al ., 2013), LySMP (Meng et al ., 2011) and ClyR (Yang et al ., 2016).…”