Genomic and proteomic characterization of four novel Schitoviridae family phages targeting uropathogenic Escherichia coli strain

Abstract

Background: Escherichia coli-associated urinary tract infections (UTIs) are among the most prevalent bacterial infections in humans. Typically, antibiotic medication is used to treat UTIs, but over the time, growth of multidrug resistance among these bacteria has created a global public health issue that necessitates other treatment modalities, such as phage therapy.

Methods: The UPEC strain PSU-5266 (UE-17) was isolated from human urine samples, while phages were obtained from wastewater. These phages were characterized through host range analysis, stability studies, adsorption assays, and electron microscopy. Additionally, genomic, phylogenetic, and proteomic analyses were conducted to provide further insights.

Results: The current study describes the isolation and characterization of four Escherichia coli phages designated as UE-S5a, UE-S5b, UE-M3 and UE-M6. Bactericidal assays depicted that all bacteriophages exhibited a strong lytic ability against uropathogenic E. coli (UPEC) strain PSU-5266 (UE-17). The phages displayed a broad host range (31-41%) among 104 tested isolates and adsorption rate of 15-20 min. They were stable within pH range of 5-11 and temperature range of 4 to 55 °C. Electron microscopy showed that all phages have icosahedral heads (70-74 nm) and short non-contractile tails, thus exhibiting a podovirus morphology. Sequencing results showed that they have linear double stranded DNA, genome of 73 to 76 kb in length, with GC content of 42% and short direct terminal repeats. Their genomes contain 84-88 predicted genes with putative functions predicted to 42-48% of gene products. The phylogenetic and comparative genomic analysis results depicted that these phages, sharing > 98% sequence similarity, are new members of genus Gamaleyavirus of subfamily Enquatrovirinae, in the Schitoviridae family. Mass spectrometric analysis of purified phage particles identified 44-56 phage particle-associated proteins (PPAPs) belonging to various functional groups such as lysis proteins, structural proteins, DNA packaging related proteins, and proteins involved in replication, metabolism and regulation. In addition, no genes encoding virulence factors, antibiotic resistance or lysogeny factors were identified.

Conclusion: The overall findings suggest that these bacteriophages are potential candidates for phage therapy in treating UTIs caused by UPEC strains.

Keywords: Schitoviridae; Phage particle-associated proteins (PPAPs); Phage therapy; Urinary tract infections; Uropathogenic E. coli.

Fig. 1

Plaques formed by phages (A) UE-S5a (B) UE-S5b (C) UE-M3 (D) UE-M6, on their UPEC host strain after an overnight incubation at 37°C

Fig. 2

Transmission electron micrograph of (A) UE-S5a (B) UE-S5b (C) UE-M3 (D) UE-M6

Fig. 3

Adsorption curve of phages UE-S5a, UE-S5b, UE-M3, and UE-M6 against their UPEC host strain. The percentage of unabsorbed/free phages was calculated by T/T₀×100. The results presented here are the mean values with SD indicated by error bars from three independent experiments

Fig. 4

The stability of phages UE-S5a, UE-S5b, UE-M3, and UE-M6 under various (A) temperature and (B) pH conditions. Statistical differences were determined by comparing values (a) at 4°C and (b) at pH 7. Results are the mean values with SD indicated by error bars from three independent experiments. The asterisks **, *** and **** denotes the significant values P<0.01, P<0.001 and P<0.0001, respectively, ns not significant

Fig. 5

In-vitro time killing curve of bacteriophages (A) UE-S5a, (B) UE-S5b, (C) UE-M3, and (D) UE-M6 at various MOIs of 0.01, 0.1, 1, 10 and 100. Control represent UPEC strains PSU-5266 with 20 µl of LB broth. The OD₆₀₀ of the culture was measured for 7 hours. Results are the mean values with SD indicated by error bars from three independent experiments

Fig. 6

This heatmap visualizes pairwise comparisons of intergenomic similarity between four isolated and sixteen closely related phages using VIRDIC software. The upper right half shows percentage intergenomic similarity, darker colours indicate higher percentage similarity between genomes. The lower left half shows, three values for each genome pair (top to bottom); top value: Proportion of Genome 1 aligned with its partner, middle value: ratio of the two genomes' lengths, bottom value proportion of Genome 2 aligned with its partner. Darker colours represent lower values, potentially indicating less aligned sequence or significant length differences. Horizontal and vertical axes list corresponding phage GenBank accession numbers. Bacteriophages of current study are marked with a red asterisk (*) next to their accession numbers.

Fig. 7

The phylogenetic tree analysis based on the large terminase subunit sequence, illustrating relationship between current study phages and thirty other Caudoviricetes phages (supplementary table S4). UPEC Phages are highlighted in red, while different groups are indicated by different colours

Fig. 8

Comparison of the genome sequences of UE-S5a, UE-S5b, UE-M3, and UE-M6 with closely related members of Escherichia_phage-PGN829.1 (NC_070871.1) and Enterobacteria_phageBp4 (NC_024142.2) and phage_U1G (NC_070872.1). The grey colour between the genome maps indicates level of homology with the scales representing the percentage genome identity between the regions obtained through BLASTn