Identification and Characterization of Dpo42, a Novel Depolymerase Derived from the Escherichia coli Phage vBEcoMECOO78

Biofilm formation, one of the most important virulence factors of pathogenic bacteria, protects bacteria against desiccation, antibiotics, phages and host immune responses. However, phage-derived depolymerases show antibiofilm activity and demonstrate great potential to treat infections caused by biofilm-forming bacteria. In this study, the Escherichia coli phage vBEcoMECOO78 was isolated and characterised, and we observed its ability to lyse five out of 34 tested E. coli clinical isolates. The highest phage titre was observed at a multiplicity of infection of 10-5 and a burst size of approximately 74 plaque forming units (PFU) /infection. Electron micrographs indicated that vBEcoMECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vBEcoMECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vBEcoMECOO78 was found to be 41, 289 bp in size, with a GC content of 53. 07%. Additionally, vBEcoMECOO78 has 56 predicted open reading frames, 51 (91. 07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vBEcoMECOO78 (Dpo42) has low identity with other reported phage-associated depolymerases. Dpo42 was expressed and purified as a soluble protein using E. coli BL21. The biofilm formation ability of E. coli isolates and the antibiofilm activity of Dpo42 were tested by performing spot assays and using a 96-well micro-titre plate method. Dpo42 degraded the capsular polysaccharides surrounding E. coli and exhibited dose-dependent biofilm-formation prevention activity. Based on these results, Dpo42 appears to be a novel phage-derived depolymerase that represents a new potential strategy for preventing E. coli biofilm formation.