Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria.

Phage-encoded endolysins are emerging antibacterial agents based on their ability to efficiently degrade peptidoglycan on Gram-positive bacteria, but the envelope characteristics of Gram-negative bacteria limit their application. Engineering modifications of endolysins can improve the optimization of their penetrative and antibacterial properties. This study constructed a screening platform to screen for engineered Artificial-Bp7e (Art-Bp7e) endolysins with extracellular antibacterial activity against Escherichia coli. An oligonucleotide of 20 repeated NNK codons was inserted upstream of the endolysin gene Bp7e to construct a chimeric endolysin library in the pColdTF vector. The chimeric Art-Bp7e proteins were expressed by transforming the plasmid library into E. coli BL21 and released by chloroform fumigation, and the protein activities were evaluated by the spotting method and the colony-counting method to screen for promising proteins. Sequence analysis showed that all screened proteins with extracellular activities had a chimeric peptide with a positive charge and an α-helical structure. Also, a representative protein, Art-Bp7e6, was further characterized. It exhibited broad antibacterial activity against E. coli (7/21), Salmonella enterica serovar Enteritidis (4/10), Pseudomonas aeruginosa (3/10), and even Staphylococcus aureus (1/10). In the transmembrane process, the chimeric peptide of Art-Bp7e6 depolarized the host cell envelope, increased the permeability of the cell, and facilitated the movement of Art-Bp7e6 across the envelope to hydrolyze the peptidoglycan. In conclusion, the screening platform successfully screened for chimeric endolysins with extracellular antibacterial activities against Gram-negative bacteria, which provides methodological support for the further screening of engineered endolysins with high extracellular activities against Gram-negative bacteria. Also, the established platform showed broad application prospects and can be used to screen various proteins. IMPORTANCE The presence of the envelope in Gram-negative bacteria limits the use of phage endolysins, and engineering endolysins is an efficient way to optimize their penetrative and antibacterial properties. We built a platform for endolysin engineering and screening. A random peptide was fused with the phage endolysin Bp7e to construct a chimeric endolysin library, and engineered Artificial-Bp7e (Art-Bp7e) endolysins with extracellular activity against Gram-negative bacteria were successfully screened from the library. The purposeful Art-Bp7e had a chimeric peptide with an abundant positive charge and an α-helical structure, which led Bp7e to acquire the ability for the extracellular lysis of Gram-negative bacteria and showed a broad lysis spectrum. The platform provides a huge library capacity without the limitations of reported proteins or peptides. It can be utilized for the further screening of optimal endolysins against Gram-negative bacteria as well as for the screening of additional proteins with specific modifications.

A Novel Polyvalent Bacteriophage vB_EcoM_swi3 Infects Pathogenic Escherichia coli and Salmonella enteritidis.

A novel virulent bacteriophage vB_EcoM_swi3 (swi3), isolated from swine feces, lyzed 9% (6/65) of Escherichia coli and isolates 54% (39/72) of Salmonella enteritidis isolates, which were all clinically pathogenic multidrug-resistant strains. Morphological observation showed that phage swi3 belonged to the Myoviridae family with an icosahedral head (80 nm in diameter) and a contractile sheathed tail (120 nm in length). At the optimal multiplicity of infection of 1, the one-step growth analysis of swi3 showed a 25-min latent period with a burst size of 25-plaque-forming units (PFU)/infected cell. Phage swi3 remained stable both at pH 6.0-8.0 and at less than 50°C for at least 1 h. Genomic sequencing and bioinformatics analysis based on genomic sequences and the terminase large subunit showed that phage swi3 was a novel member that was most closely related to Salmonella phages and belonged to the Rosemountvirus genus. Phage swi3 harbored a 52-kb double-stranded DNA genome with 46.02% GC content. Seventy-two potential open reading frames were identified and annotated, only 15 of which had been assigned to functional genes. No gene associated with pathogenicity and virulence was identified. The effects of phage swi3 in treating pathologic E. coli infections in vivo were evaluated using a mouse model. The administration of a single intraperitoneal injection of swi3 (106 PFU) at 2 h after challenge with the E. coli strain (serotype K88) (108 colony-forming units) sufficiently protected all mice without toxic side effects. This finding highlighted that phage swi3 might be used as an effective antibacterial agent to prevent E. coli and S. enteritidis infection.

Characterization of a Novel Bacteriophage swi2 Harboring Two Lysins Can Naturally Lyse Escherichia coli.

The novel virulent Siphoviridae bacteriophage swi2 was isolated from a pig farm, and its biological characteristics, genome architecture, and infection-related properties were characterized. Phage swi2 has a high titer of 1.01 × 1012 PFU/mL with good tolerance to UV rays and remains stable in the pH range of 6-10 and at temperatures less than 50°C. One-step growth analysis revealed that phage swi2 had a 25 min latent period with a large burst size (1,000 PFU/cell). The biological characteristics indicated that swi2 had good host infectivity and effective lytic activities. The genome of phage swi2 is composed of 47,611 bp with a G + C content of 46.50%. Eighty-nine orfs were predicted, and only 18 of them have known functions. No virulence genes or drug resistance genes were found in the genome. Genome sequence comparison of phage swi2 showed that there were a total of 10 homologous phages in the database with low similarity (less than 92.51% nucleotide identity and 66% query coverage). The predicted host lysis-related genes of phage swi2 consist of one holin, two endolysins, and Rz/Rz1 equivalents. Antibacterial activity assays showed that both endolysins could naturally reduce the host Escherichia coli 51 titers by -1 log unit both in vitro and in vivo, EDTA showed no obvious synergistic action, and holin had no lytic effects on the host cell. These results provide necessary information for the development of antibiotic alternatives for the treatment of multidrug-resistant Escherichia coli infection.