YqeH contributes to avian pathogenic Escherichia coli pathogenicity by regulating motility, biofilm formation, and virulence

Abstract

Avian pathogenic Escherichia coli (APEC) is a pathotype of extraintestinal pathogenic E. coli and one of the most serious infectious diseases of poultry. It not only causes great economic losses to the poultry industry, but also poses a serious threat to public health worldwide. Here, we examined the role of YqeH, a transcriptional regulator located at E. coli type III secretion system 2 (ETT2), in APEC pathogenesis. To investigate the effects of YqeH on APEC phenotype and virulence, we constructed a yqeH deletion mutant (APEC40-ΔyqeH) and a complemented strain (APEC40-CΔyqeH) of APEC40. Compared with the wild type (WT), the motility and biofilm formation of APEC40-ΔyqeH were significantly reduced. The yqeH mutant was highly attenuated in a chick infection model compared with WT, and showed severe defects in its adherence to and invasion of chicken embryo fibroblast DF-1 cells. However, the mechanisms underlying these phenomena were unclear. Therefore, we analyzed the transcriptional effects of the yqeH deletion to clarify the regulatory mechanisms of YqeH, and the role of YqeH in APEC virulence. The deletion of yqeH downregulated the transcript levels of several flagellum-, biofilm-, and virulence-related genes. Our results demonstrate that YqeH is involved in APEC pathogenesis, and the reduced virulence of APEC40-ΔyqeH may be related to its reduced motility and biofilm formation.

Keywords: Avian pathogenic Escherichia coli; biofilm formation; motility; pathogenicity; virulence; yqeH.

Figure 1

PCR analysis of mutant strain APEC40-ΔyqeH and complemented strain APEC40-CΔyqeH. A Schematic diagram of the strategy used to construct the APEC40 yqeH deletion mutant. The yqeH gene was deleted by replacing the partial gene sequence with a chloramphenicol-resistance cassette. The primers used for confirmation of the yqeH deletion are indicated. B Confirmation of the yqeH mutant strain APEC40-ΔyqeH. Lane identities in (B)—M: 2000-bp DNA marker; 1–2: identification of wild-type APEC40-yqeH strain (lane 1, 252 bp; lane 2, 2256 bp); 3–4: identification of APEC40-ΔyqeH-Cm (lane 3, 0 bp; lane 4, 2637 bp); 5–6: identification of APEC40-ΔyqeH (lane 5, 0 bp; lane 6, 1623 bp). C Confirmation of the complemented strain APEC40-CΔyqeH. Lane identities in (C)—M: 2000-bp DNA marker; 1: Recombinant plasmid pSTV28 blank control identification, 193 bp; 2: APEC40-CΔyqeH identification, 826 bp.

Figure 2

Characteristics of strains APEC40, APEC40-ΔyqeH, and APEC40-CΔyqeH. A Growth curves of APEC40, APEC40-ΔyqeH, and APEC40-CΔyqeH in LB broth. Growth rates of APEC40, APEC40-ΔyqeH, and APEC40-CΔyqeH were monitored by measuring their OD₆₀₀ values in LB medium at 2 h intervals for 24 h. During culture for 24 h, there was no significant difference in the growth rates of APEC40, APEC40-ΔyqeH, and APEC40-CΔyqeH grown in LB broth (P > 0.05). Each value is the average of three independent experiments. B Bacterial motility. Swimming motility of strain APEC40-ΔyqeH was significantly lower than that of the wild-type APEC40 strain, whereas the swimming motility of complemented strain APEC40-CΔyqeH was restored. The experiment was repeated three times and all samples were measured in triplicate. C Biofilm formation by APEC strains as determined with crystal violet (CV) staining. APEC40-ΔyqeH showed significantly lower biofilm formation than wild-type strain APEC40 after crystal violet staining (**P < 0.01 for the WT vs. mutant).

Figure 3

Deletion of yqeH reduced the adherence and invasion abilities of APEC. Adherence to and invasion of DF-1 cells was significantly lower in mutant strain APEC40-ΔyqeH than in WT strain APEC40. (**P < 0.01 for WT vs. mutant).

Figure 4

Animal systemic infection in vivo. A Determination of bacterial virulence. Seven-day-old chicks were inoculated intratracheally with 10⁸ colony-forming units (CFUs) of APEC40, APEC40-ΔyqeH, or APEC40-CΔyqeH. Chicks that were administered PBS were used as negative control. Survival was monitored for 7 days post-infection. B Bacterial colonization and proliferation in chicks. Groups of eight 7-day-old chicks were intratracheally infected with bacteria (10⁸ CFUs). Bacteria were recovered from the lung, blood, liver, and spleen at 24 h post-infection. The nonparametric Mann–Whitney U-test was used to determine statistical significance (**P < 0.01, ***P < 0.001 for WT vs. mutant).

Figure 5

Analysis of differentially expressed genes by RNA-seq. A Volcano plots were used to visualize the differentially expressed genes in APEC40 and APEC40-ΔyqeH. Genes with significantly upregulated expression in APEC40-ΔyqeH relative to their expression in APEC40 are indicated with red dots; those downregulated are indicated with blue dots; and grey dots represent genes with no significant difference in expression between the strains. The results showed that 112 genes were upregulated and 101 genes were downregulated in APEC40-ΔyqeH relative to their expression in wild-type strain APEC40 (differentially expressed genes were selected at fold change > 2 and q < 0.005). B KEGG enrichment analysis of differentially expressed genes. Top 20 pathways enriched in differentially expressed genes are shown in the figure. C Quantitative differences in the expression levels of 19 differentially expressed virulence-related genes between wild-type strain APEC40 and mutant strain APEC40-ΔyqeH are shown with a heatmap.

Figure 6

Verification of RNA-seq results with reverse transcription–quantitative real-time PCR (RT–qPCR). RT–qPCR was used to determine the expression profiles of several genes differentially expressed in the yqeH mutant strain. The x-axis shows the annotations of the selected genes. The dnaE gene was used for standardization.