The spontaneously produced lysogenic prophage phi456 promotes bacterial resistance to adverse environments and enhances the colonization ability of avian pathogenic Escherichia coli strain DE456

Abstract

In the last decade, prophages that possess the ability of lysogenic transformation have become increasingly significant. Their transfer and subsequent activity in the host have a significant impact on the evolution of bacteria. Here, we investigate the role of prophage phi456 with high spontaneous induction in the bacterial genome of Avian pathogenic Escherichia coli (APEC) DE456. The phage particles, phi456, that were released from DE456 were isolated, purified, and sequenced. Additionally, phage particles were no longer observed either during normal growth or induced by nalidixic acid in DE456Δphi456. This indicated that the released phage particles from DE456 were only phi456. We demonstrated that phi456 contributed to biofilm formation through spontaneous induction of the accompanying increase in the eDNA content. The survival ability of DE456Δphi456 was decreased in avian macrophage HD11 under oxidative stress and acidic conditions. This is likely due to a decrease in the transcription levels of three crucial genes-rpoS, katE, and oxyR-which are needed to help the bacteria adapt to and survive in adverse environments. It has been observed through animal experiments that the presence of phi456 in the DE456 genome enhances colonization ability in vivo. Additionally, the number of type I fimbriae in DE456Δphi456 was observed to be reduced under transmission electron microscopy when compared to the wild-type strain. The qRT-PCR results indicated that the expression levels of the subunit of I fimbriae (fimA) and its apical adhesin (fimH) were significantly lower in DE456Δphi456. Therefore, it can be concluded that phi456 plays a crucial role in helping bacterial hosts survive in unfavorable conditions and enhancing the colonization ability in DE456.

Keywords: Prophage; avian pathogenic E. coli; biofilm; colonization; environmental stress.

Figure 1

Morphological characteristics and comparative genomic analysis of phi456. A phi456 forms tiny and blurry plaques on MC1061 in the plaque assay, which are indicated by black arrows. B Transmission electron microscopy images of purified phage phi456. C The structure of prophage phi456. Phi456 showed a 70% nucleotide sequence identity with Escherichia phage RCS47. The unbroken line indicates the length of the sequence, while ORFs classified in the same functional categories are color-coded accordingly.

Figure 2

Induction rate of prophage in different environments. The number of bacterial genomes without phi456 was quantified by using primers flanking of phi456. The quantification of the target sequence was performed by normalizing to the reference gene purA. The error bars indicate standard deviations. Student’s t-test was used to analyse the data.

Figure 3

Presence of prophage phi456 in bacterial genome increases biofilm formation. A Biofilm formation of DE456 and DE456Δphi456 with or without DNase for 36 h. B The amount of phage particles phi456 during biofilm formation. The data represent the mean values obtained from six replicate wells in 96-well plates across three independent experiments. Error bars indicate standard deviations. Student’s t-test was used to analyse the data.

Figure 4

The effect of prophage on stress-related phenotypes. Survival for DE456 and DE456Δphi456 after challenging with A osmotic stress (2.4 M NaCl for 30 min), B oxidative stress (30 mM H₂O₂ for 15 min), C acid stress (pH 3 for 30 min) and D heat stress (65 °C for 10 min). Data are the mean of 3 replicates ± SD. Error bars indicate standard deviations. Student’s t-test was used to analyse the data.

Figure 5

Bacterial counts during infection in vivo. Chickens were inoculated by the air sac route with strains or a bacteria-phage suspension, respectively. At 24 hpi, chickens were sacrificed and the bacterial load in A lung, B heart, and C liver was quantified. The data was subjected to analysis using the Mann–Whitney U test.

Figure 6

Cytotoxicity of phage particles phi456. After being exposed to phi456 in DMEM for 24 h, the viability of DF-1 chicken embryo fibroblast cells was assessed using the trypan blue exclusion assay. The data represented the average of three independent assays. Error bars indicate standard deviations. Student’s t-test was used to analyze the data.

Figure 7

Deletion of phi456 leads to decreased type I fimbriae and adhesion ability. Transmission electron microscopy images of DE456 (A) and DE456Δphi456 (B). The red arrow points to the I fimbria, while the blue arrow points to the flagella. C Quantification of genes related to type I fimbriae by qRT-PCR. The relative expression level of fimA and fimH in the DE456Δphi456 was compared to WT. The quantification of the target gene was performed by normalizing to the reference gene dnaE. D Adhesion assay. Values are the average of three independent experiments. The error bars indicate standard deviations. Student’s t-test was used to analyse the data.

Figure 8

Prophage phi456 genes influence survival ability in macrophage cells HD-11. A The bacterial survival in HD-11 infected with DE456 or DE456Δphi456 at a MOI of 20 bacteria per cell. At 3 h or 6 hpi, the cells were lysed and bacterial counts were measured. B Quantification of genes related to toll-like receptors and tumor necrosis factor by qRT-PCR. The quantification of the target gene was performed by normalizing to the reference gene β-Actin. C Quantification of genes related to oxidative stress response by qRT-PCR. The relative expression level of rpoS, katE and oxyR in the DE456Δphi456 was compared to WT. The quantification of the target gene was performed by normalizing to the reference gene dnaE. The error bars indicate standard deviations. Student’s t-test was used to analyze the data.