Alterations in gp37 Expand the Host Range of a T4-Like Phage

Abstract

The use of phages as antibacterial agents is limited by their generally narrow host ranges. The aim of this study was to make a T4-like phage, WG01, obtain the host range of another T4-like phage, QL01, by replacing its host-determinant gene region with that of QL01. This process triggered a direct expansion of the WG01 host range. The offspring of WG01 obtained the host ranges of both QL01 and WG01, as well as the ability to infect eight additional host bacteria in comparison to the wild-type strains. WQD had the widest host range; therefore, the corresponding fragments, named QD, could be used for constructing a homologous sequence library. Moreover, after a sequencing analysis of gene 37, we identified two different mechanisms responsible for the expanded host range: (i) the first generation of WG01 formed chimeras without mutations, and (ii) the second generation of WG01 mutants formed from the chimeras. The expansion of the host range indicated that regions other than the C-terminal region may indirectly change the receptor specificity by altering the supportive capacity of the binding site. Additionally, we also found the novel means by which subsequent generations expanded their host ranges, namely, by exchanging gene 37 to acquire a wider temperature range for lysis. The method developed in this work offers a quick way to change or expand the host range of a phage. Future clinical applications for screening phages against a given clinical isolate could be achieved after acquiring more suitable homologous sequences.IMPORTANCE T4-like phages have been established as safe in numerous phage therapy applications. The primary drawbacks to the use of phages as therapeutic agents include their highly specific host ranges. Thus, changing or expanding the host range of T4-like phages is beneficial for selecting phages for phage therapy. In this study, the host range of the T4-like phage WG01 was expanded using genetic manipulation. The WG01 derivatives acquired a novel means of expanding their host ranges by acquiring a wider temperature range for lysis. A region was located that had the potential to be used as a sequence region for homologous sequence recombination.

Keywords: Escherichia coli; T4-like phages; gp37; homologous recombination; host range expansion.

FIG 1

Comparisons of the whole-genome and amino acid sequences of gp37. (A1) Neighbor-joining tree based on the alignment of the entire genomes of five T4-like phages derived from the “distance tree results” in the BLASTN search of the NCBI database. The results revealed that WG01 shared close phylogenic relationships with JS98, QL01, and JS10 but exhibited a distant phylogenic relationship with wV7. (A2) Comparisons of the entire genome sequences were carried out using the Artemis Comparison Tool (ACT). Different functional modules are represented by different colors, and the color legend is shown at the bottom. The different colored lines represent different similarities of the genes. The red lines represent the highest similarity and the blue lines represent the lowest similarity. (B1) Neighbor-joining tree analysis and bootstrap analysis (1,000 bootstrap replicates) based on the alignment of the gp37 amino acid sequences of four T4-like phages were carried out using ClustalX and MEGA. WG01 shared a closer phylogenic relationship with wV7 than with QL01 and JS10. (B2) The different amino acids domains (1 to 456, 457 to 713, 714 to 910, and 911 to 1,100) of WG01 gp37 were compared with the corresponding protein sequences of QL01, JS10, and wV7. The color keys representing the different similarities are presented at the bottom. In A1 and B1, the scales at the bottom represent the evolutionary distances.

FIG 2

Rationale for the homologous recombination of WG01. The different parts from gene 37 of the QL01 phage were digested and inserted into pUC118 to produce different recombinant plasmids. DE017 was transformed with the recombinant plasmid. Then, the transformant cells were infected by WG01. When the WG01 genome was injected into the bacteria and nucleic acid was synthesized, homologous recombination of gene 37 between QL01 and WG01 occurred.

FIG 3

Sketch map of gene 37 positions of QL01 for WG01 recombination, results of the isolated chimeras, and mutation sites of the mutants derived from the same WQT chimera. (A) Sketch map of gene 37 positions of QL01 for WG01 recombination and results of the chimeras isolated with the DE205B host. Different positions from the end indicate the DNA fragments for WG01 recombination; these fragments were named QT, QC, QD, QE, QF, QB, and QA. Four types of recombinant phages, namely, WQT1, WQC1, WQD1, and WQE1, were isolated from DE205B, the host of QL01. The Xs indicate that no chimeric phages were isolated with DE205B by infecting the plasmid-containing cell with phage WG01. (B) The mutation sites of mutants derived from the same WQT chimera. There are 12 types of mutants that originate from DE017 cells carrying plasmids containing the QT inserts and that were infected with WG01, including single point mutations, multipoint mutations, and DNA fragment deletions. The sequences of the 12 types of mutants were compared with that of WQT. The mutation sites are marked with red lines. The numbering scale at the bottom represents the nucleotide positions.

FIG 4

Host range results of WG01, QL01, and WG01 derivatives. The heat map of host range results was produced using the OmicShare platform. The red color represents that the bacteria could be infected by the phages. The light green color represents that the bacteria could not be infected by the phages. QL01, WG01, WQC1, WQD1, and WQE1 lysed 50, 22, 47, 63, and 28 of the 113 E. coli strains, respectively. The mutants derived from the same chimeras of WQT, WQT2, WQT3, WQT4, WQT5, WQT6, WQT8, WQT9, WQT10, WQT11, and WQT12 had the same host ranges, each of which could lyse 49 strains. WQT7 was able to lyse the largest number of strains (52 strains).

FIG 5

Summary of the final experimental host range results. (Top) The experimental results of host range analysis showed that in 113 E. coli strains used for the host spectrums testing, WG01 could lyse 22 E. coli strains and QL01 could lyse 50 E. coli strains. There were 14 strains that WG01 and QL01 could lyse. (Bottom) There were 22 strains that WG01 and WG01 derivatives could lyse, 47 strains that QL01 and WG01 derivatives could lyse, and 3 strains that only QL01 could lyse. All the WG01 derivatives could lyse 63 E. coli strains, including 41 strains that WG01 could not lyse, 16 strains that QL01 could not lyse, 33 strains that QL01 could lyse and WG01 could not, and 8 strains that WG01 could lyse and QL01 could not. Moreover, the derivative phages lysed 8 bacterial strains that neither WG01 nor QL01 lysed.

FIG 6

Growth characteristics of the WG01, QL01, and WG01 derivatives. The heat map of the growth characteristics was produced using the OmicShare platform. The growth characteristics of WG01, QL01, WQT1, WQC1, WQD1, and WQE1 are displayed. On a lawn of DE205B cells, QL01 and WQT1 formed large clear plaques at 28°C, 16°C, and 4°C; WQC1 formed small clear plaques at 28°C and turbid and small plaques at 16°C and 4°C; WQD1 formed large clear plaques at 28°C and 4°C and small clear plaques at 16°C; WQE1 formed small clear plaques at 28°C. WG01 could not infect DE205B but could form large clear plaques at 28°C, small clear plaques at 37°C, and small turbid plaques at 16°C and 4°C with the DE017 host.