Sxt1, Isolated from a Therapeutic Phage Cocktail, Is a Broader Host Range Relative of the Phage T3

Abstract

Using Escherichia coli BW25113 as a host, we isolated a novel lytic phage from the commercial poly-specific therapeutic phage cocktail Sextaphage^® (Microgen, Russia). We provide genetic and phenotypic characterization of the phage and describe its host range on the ECOR collection of reference E. coli strains. The phage, hereafter named Sxt1, is a close relative of classical coliphage T3 and belongs to the Teetrevirus genus, yet its internal virion proteins, forming an ejectosome, differ from those of T3. In addition, the Sxt1 lateral tail fiber (LTF) protein clusters with those of the phages from the Berlinvirus genus. A comparison of T7, T3, and Sxt1 LTFs reveals the presence of insertions leading to the elongation of Sxt1 tail fibers, which, together with the difference in the HRDRs (host range-determining regions), might explain the expanded host specificity for the Sxt1.

Keywords: Sextaphage; bacteriophage T3; bacteriophage T7; ejectosome; lateral tail fibers; phage therapy.

Figure 1

(A) Morphology of Sxt1 plaques on the E. coli BW25113 cell lawn. (B) Infection of E. coli BW25113 liquid cultures with Sxt1 or phage T7 at low (0.001) or high (1) MOI. (C) One-step growth curve of Sxt1 and T7 phages in liquid LB media at 37 °C. (D) Representative TEM images of the Sxt1 virions.

Figure 2

Genomic comparison of Sxt1, T3, and T7 phage genomes. Colors represent functional annotations according to the PHROG database. Predicted HNH endonucleases genes are marked in blue. Sequence identity was calculated using fastANI with a 150 bp window. Genetic maps were prepared using gggenomes library in R.

Figure 3

The position of Sxt1 on the phylogenetic tree of bacteriophages from the Studiervirinae subfamily. (A) Phylogenetic tree of indicated bacteriophages based on protein orthogroups, except those analyzed in panels (B,C). Phylogenetic trees of internal virion proteins gp15 (sxt1_p36) (B), internal virion proteins with endolysin domains gp16 (sxt1_p37) (C), and lateral tail fiber proteins gp17 (sxt1_p38) (D). Bacteriophage genera are indicated with color. Bootstrap support is provided at each node. Branch lengths reflect the numbers of substitutions per site. Sxt1 position is indicated with a star.

Figure 4

(A) C-terminal receptor-sensing domains of lateral tail fiber protein homotrimer from phage T7 (PDB: 4A0U, left) and Sxt1 (AF2 model, right). (B) Reconstruction of the Sxt1 virion, revealing the structure of the tail and positions of the six lateral tail fiber attachment sites. The model was obtained using a C6 symmetry axis.

Figure 5

(A) Efficiency of Sxt1, T3, and T7 plaquing on common E. coli strains. (B) Efficiency of Sxt1, T3, and T7 plaquing on a collection of natural E. coli isolates producing O-antigens. (C) An adsorption assay with E. coli ECOR50 and Sxt1, T3, and T7. % of phage particles adsorbed on the surface of bacterial cells 7 min after infection is shown.