Evolutionary responses of Escherichia coli to phage pressure: insights into mucoidy and colanic acid overexpression

Abstract

Background: Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC.

Results: In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance.

Conclusions: This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.

Keywords: E. coli; yrfF; APEC; Bacteriophage; Colanic acid; Mucoidy; Phage resistance.

Fig. 1

Morphology of phage 66. Transmission electron micrograph of phage 66. Sample was stained with 2% uranyl acetate. 60,000 × magnification, 200 kV accelerating voltage. Scale bar represents 200 nm

Fig. 2

Spot tests of different dilutions of phage 66 on (A) its host strain E. coli APEC17 and (B) E. coli K-12. C Growth kinetics of strains APEC17 and K-12 with or without phage 66 at an MOI of 1. Error bars represent the standard error of the mean for three replicates

Fig. 3

Mutations identified in BIMs isolated from K-12 (A) and APEC17 (B) strains. Non-synonymous, synonymous, and structural mutations (nucleotide additions or deletions) are represented by green, blue, and red bars, respectively. A mutation in the traG gene (T899 K) was also identified in the 138,781 bp plasmid of APEC17-BIM1-M, APEC17-BIM1a-NM, and APEC17-BIM2-M

Fig. 4

Structural analysis of the L643R mutation on the YrfF protein in E. coli K-12. A YrfF-WT (cyan, left) and YrfF-L643R (green, right) in the presence of the membrane. The mutated residue (Leu643 to Arg643) is highlighted in orange spheres in both structures. Red arrows represent the average angle of the periplasmic domain according to the normal of the bilayer (z-axis, black arrow). B Residues in YrfF forming hydrogen bonds with L643 (left) or R643 (right) are shown in orange. Residues in red (G389, V390, V392, D394, K630, I631, and F632) are predicted to lose membrane contact in YrfF-L643R but not in YrfF-WT

Fig. 5

Grouping of proteins according to the STRING database with genes that were (A) up-regulated and (B) down-regulated. Some proteins are colored based on their membership in enriched functional categories. Only functional categories enriched for the main cluster-forming proteins are displayed. The cluster numbers, accompanied by their descriptions, the number of proteins in the network compared with the total number of proteins of this category found in the reference genome, the strength of clustering (log₁₀ observed/expected), and the False Discovery Rate value (FDR, corrected p-value using the Benjamini–Hochberg procedure) are shown