Uncovering the determinants of model Escherichia coli strain C600 susceptibility and resistance to lytic T4-like and T7-like phage

Abstract

As antimicrobial resistance (AMR) continues to increase, the therapeutic use of phages has re-emerged as an attractive alternative. However, knowledge of phage resistance development and bacterium-phage interaction complexity are still not fully interpreted. In this study, two lytic T4-like and T7-like phage infecting model Escherichia coli strain C600 are selected, and host genetic determinants involved in phage susceptibility and resistance are also identified using TraDIS strategy. Isolation and identification of the lytic T7-like show that though it belongs to the phage T7 family, genes encoding replication and transcription protein exhibit high differences. The TraDIS results identify a huge number of previously unidentified genes involved in phage infection, and a subset (six in susceptibility and nine in resistance) are shared under pressure of the two kinds of lytic phage. Susceptible gene wbbL has the highest value and implies the important role in phage susceptibility. Importantly, two susceptible genes QseE (QseE/QseF) and RstB (RstB/RstA), encoding the similar two-component system sensor histidine kinase (HKs), also identified. Conversely and strangely, outer membrane protein gene ompW, unlike the gene ompC encoding receptor protein of T4 phage, was shown to provide phage resistance. Overall, this study exploited a genome-wide fitness assay to uncover susceptibility and resistant genes, even the shared genes, important for the E. coli strain of both most popular high lytic T4-like and T7-like phages. This knowledge of the genetic determinants can be further used to analysis the behind function signatures to screen the potential agents to aid phage killing of MDR pathogens, which will greatly be valuable in improving the phage therapy outcome in fighting with microbial resistance.

Keywords: Antimicrobial resistance (AMR); Lytic T4-like phage; Lytic T7-like phage; Tn5 transposons, Phage therapy.

Graphical abstract

Fig. 1

Morphological characteristics of lytic phage AH67C600_Q5. (A) Culture map of phage AH67C600_Q5 plaques in double-layer agar. (B) TEM images of phage AH67C600_Q5 in 100 nm vision. (C) TEM images of phage AH67C600_Q5 in 50 nm vision.

Fig. 2

Physiological characteristics of T7-like lytic phage AH67C600_Q5. (A) One step curve of phage AH67C600_Q5. 10 to 30 min is the latent time and 30 to 80 min is the burst time. (B) Optimal MOI test. The phage AH67C600_Q5 achieved the highest titer after cultured while the MOI is 0.1:1 (0.96 × 10⁹ PFU per mL) (C) pH stability tests. The phage AH67C600_Q5 is stable at the pH of 6 to 8, then sharply decreased with the pH increase and decrease. (D) Thermal stability tests. The phage AH67C600_Q5 had stable biological activity at temperatures of 4 to 45 °C, then sharply decreased with the temperatures increase.

Fig. 3

Bioinformatics analysis of T7-like lytic Phage AH67C600_Q5. (A) Annotation of the predicted genes of phage AH67C600_Q5. The genes located at upper right are replication and transcription -related and the structural proteins are concentrated in the lower left corner. (B) Phylogenetic analysis based on the large subunit of terminase for Enterobacteriaceae phage, demonstrating an independent branch for phage AH67C600_Q5. (C) Genome sequence alignment of phage T7, phage AH67C600_Q5, and phage CICC 80,001. The functional modules indicated by color, and similarities were shown in gray according to the scale on the bottom right corner, with green representing replication and transcription -related, red representing lytic-related gene, blue representing structure-related genes and black representing hypothetical genes.

Fig. 4

Volcano plot showing changes in prevalence of mutants in the mutant pool compared to the control during the addition of T4-like phage AH67C600_Q9 (A) and T7-like phage AH67C600_Q5 (B) selections, as shown by the relationship of log2 fold change in selection condition compared to the control (x axis), with the q value (y axis) indicating the false-discovery rate. Colored points show those shared genes in T4-like phage and T7-like phage group that a log2FC of greater than 2 (red) or less than 2 (green). Those shared genes are labeled with annotated gene name.

Fig. 5

Gene ontology (Go) enrichment analysis of genes involved in phage resistance(A) and susceptibility (B) during the addition of T4-like phage AH67C600_Q9 (A) and T7-like phage AH67C600_Q5 (B) selections. The X-axis represents the number of genes involved in a GO terms, and the Y-axis represents the GO terms. Colored column shows genes number in different groups, with light green and green representing resistant and susceptible gene in T7-like group respectively, and light red and red representing resistant and susceptible gene in T4-like group respectively. Background color shows the GO terms involved in different process, with cyan representing cellular component, purple representing molecular functions and light yellow representing biological processes.

Fig. 6

Changes of phage host range and efficiency of plating. (A) Minimum phage titer of three stains (E. coli strain C600, E. coli strain C600ΔqseE and E. coli strain C600ΔrstB) for two test phages (AH67C600_Q5 and AH67C600_Q9). (B) Spot assay phenotypes.