LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain

Abstract

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure. IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

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

“…Endolysins encoded by phages infecting the B. cereus group show differences regarding the targeted bacteria, ranging from a narrow to a broad activity spectrum. For instance, LysPBC5 is only able to lyse members of the B. cereus group whereas LysPBC2 is also active against other Bacillus spp., and even L. monocytogenes and Clostridium perfringens [11,33]. 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.…”

“…B. cereus sensu stricto (s.s), Bacillus thuringiensis, Bacillus anthracis, Bacillus weihenstephanensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus cytotoxicus are the seven species most commonly recognized as members of this group, but other species have been suggested as new members [15,16]. In the last few years, several endolysins of phages infecting diverse B. cereus group members have been identified and characterized [11,[17][18][19][20][21]. Deep-Blue [22,23] and Deep-Purple [24], two phages infecting the B. cereus group, belong to the families Herelleviridae and Siphoviridae, respectively.…”

“…Endolysins can be used as antimicrobial compounds in the food and medical sectors instead of their parental phages [ 10 ]. Indeed, phages sometimes have such a narrow host spectrum that they cannot target all the pathogenic strains of a certain group whereas their corresponding endolysins usually display a broader spectrum [ 11 ]. Moreover, no bacterial resistance to endolysins has been identified so far.…”

Phages in Therapy and Prophylaxis of American Foulbrood – Recent Implications From Practical Applications

“…It is also possible that the already described P. larvae phages may encode previously unidentified lytic enzymes. It has been demonstrated that lysins may specifically bind spores, e.g., lysin LysPBC2 encoded by Bacillus cereus ( Kong et al, 2019 ). These findings suggest the probability of isolating an endolysin with activity against spores produced by Gram-positive bacteria similar to P. larvae .…”

“…The modular structure of these enzymes creates the possibility of engineering proteins and constructing endolysins with new or improved properties. Enhanced lysin activity can be achieved by manipulating their functional domains, e.g., random or directed mutagenesis in the cell-binding domain ( São-José, 2018 ; Kong et al, 2019 ), shuffling and fusion of catalytic domains with cell-wall binding domains of different origin and properties to obtain chimeric enzymes (chimeolysins), fusion of full-length lytic enzymes, domain deletion, addition or duplication, fusion to peptides, and combination of these methods ( São-José, 2018 ). With respect to AFB sporicidal activity, it would be desirable for tailored enzymes that would penetrate the spore coat and then facilitate bond cleavage in peptidoglycan layers both in the core wall and spore cortex ( Todar, 2009 ).…”