Identification and Characterization of Dpo42, a Novel Depolymerase Derived from the Escherichia coli Phage vB_EcoM_ECOO78

Abstract

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 vB_EcoM_ECOO78 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 vB_EcoM_ECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vB_EcoM_ECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vB_EcoM_ECOO78 was found to be 41,289 bp in size, with a GC content of 53.07%. Additionally, vB_EcoM_ECOO78 has 56 predicted open reading frames, 51 (91.07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vB_EcoM_ECOO78 (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.

Overexpression of AmpC Promotes Bacteriophage Lysis of Ampicillin-Resistant Escherichia coli

“…In previous studies, we have identified and characterized two related phage, vB_EcoM-ep3 and vB_EcoM_ECOO78, both Myoviridae capable of infecting a number of multiple-drug resistant E. coli clinical isolates (Lv et al, 2015;Guo et al, 2017). In this study, we show that hyper-production of AmpC is directly related to decreased OmpF, and increased OmpA, expression in E. coli and that these changes are associated with increased adsorption and lysis by these phages.…”

“…That might be the reason why the difference between the two groups didn't seem that significant (Figure 1). Furthermore, strain CVCC1418 (also serotype O78, but with a low MIC of ampicillin) hosted fewer of the O78-derived phages than the other E. coli of this serotype and was less susceptible to lysis by the previously characterized Myoviridae phages, vB_EcoM-ep3 and vB_EcoM_ECOO78 (Figure 2) (Lv et al, 2015;Guo et al, 2017). These results suggested that differences in the level of ampicillin resistance amongst O78 serotype strains may affect susceptibility to their specific phage.…”

“…Here, we have shown that the increased production of OmpA associated with increased expression of AmpC promotes the adsorption of, and lysis by, tested Myoviridae phages (Figures 4, 6D). We demonstrated that OmpA functions as a receptor for the tail fiber protein, Dpo41, of phage vB_EcoM-ep3, which is homologous to the tail fiber protein, Dpo42, of phage vB_EcoM_ECOO78 (Guo et al, 2017).…”

“…Escherichia coli strains and their sources are listed in Table 1. Phage vB_EcoM-ep3 and vB_EcoM_ECOO78 were isolated previously in our laboratory (Lv et al, 2015;Guo et al, 2017); all other phages were isolated from hospital or domestic sewage, or water from rivers and lakes around Changchun, either as part of this or a previous study in our laboratory. Except for phage vB_EcoM-ep3 and vB_EcoM_ECOO78, the remaining phages were not characterized in depth.…”

“…Four of the clinical isolates from chicken farms with high MICs of ampicillin (>800 mg/L) belong to serotype O78 and were used to isolate a total of 17 of the tested phage, including previously characterized members of the Myoviridae family, vB_EcoM-ep3 and vB_EcoM_ECOO78 (Lv et al, 2015;Guo et al, 2017). Full host range determination further revealed that these four strains were able to host a total of 27 phages amongst them, with individual strains hosting up to 13 different phages ( Supplementary Table S1).…”