PMID:26167894

Citation	Oliveira, A, Leite, M, Kluskens, LD, Santos, SB, Melo, LD and Azeredo, J (2015) The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood. PLoS ONE 10:e0132095
Abstract	Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.
Links	PubMed PMC4500393 Online version:10.1371/journal.pone.0132095
Keywords	Animals; Anti-Infective Agents/therapeutic use; Bacteriophages/physiology; Bees/microbiology; Endopeptidases/isolation & purification; Endopeptidases/pharmacology; Gram-Positive Bacterial Infections/drug therapy; Gram-Positive Bacterial Infections/veterinary; Larva/microbiology; Microbial Sensitivity Tests; Paenibacillus/drug effects; Paenibacillus/virology; Polymerase Chain Reaction; Spores, Bacterial/drug effects
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:R9VY83		GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000314:		F		The authors claim that 'BlastP, Pfam and HHpred identified an N-acetylmuramoyl-L-alanine amidase belonging to the Amidase_2 family (Pfam 01510) at the N-terminus encompassing two third of the protein. CDD database identified 5 putative amidase catalytic residues: two histidines (position 29 and 129), a phenylalanine (position 53), a lysine (position 135) and a cysteine (position 137)' (First paragraph of the results section). Table 2 however confirmed that PlyPl23 has lytic activity and also shows the lytic spectrum of PlyPl23 when assessed against a panel of 20 P. larvae isolates and 4 additional strains belonging to other related bacterial species (Bacillus cereus, Lactobacillus paracasei, Lactobacillus pentosus, Bacillus subtilis), and compared with that of the respective phage phiIBB_Pl23. Results showed that while the phage phiIBB_Pl23 was able to lyse 16 of the 20 strains (80%), its endolysin (PlyPl23) was active against all the tested Paenibacillus larvae strains. The Bacillus and Lactobacillus strains were not lysed by the phage or by the enzyme	complete CACAO 12773

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