Transcriptional Regulator YqeI, Locating at ETT2 Locus, Affects the Pathogenicity of Avian Pathogenic Escherichia coli

Abstract

Avian pathogenic Escherichia coli (APEC) is the leading cause of systemic infections in poultry worldwide and has a hidden threat to public health. Escherichia coli type three secretion system 2 (ETT2), similar to the Salmonella pathogenicity island SPI1, is widely distributed in APEC and associated with virulence. The function of YqeI, which is one of the hypothetical transcriptional regulators locating at the ETT2 locus of APEC, is unknown. In this study, we successfully obtained the mutant strain AE81ΔyqeI of the wild type strain AE81 and performed the transcriptional profiling assays. Additionally, the transcriptional sequencing results revealed that YqeI influenced localization, locomotion and biological adhesion and so on. The transmission electron microscope observation showed that the wild type strain AE81 possessed long curved flagella, whereas the mutant strain AE81ΔyqeI hardly had any. The strain AE81ΔyqeI exhibited lower motility than AE81 after culturing the dilute bacterial suspension on a semisolid medium. It was also found that the survival ability of AE81ΔyqeI weakened significantly when AE81ΔyqeI was cultured with 0%, 10%, 20%, 30%, 40% and 50% SPF serum in PBS, and AE81ΔyqeI had decreased adherence to DF-1 cells compared with AE81 in the bacterial adhesion assay. The bacterial colonization assay indicated that the virulence of AE81ΔyqeI was reduced in the heart, liver, spleen, and lung. These results confirmed that the transcription regulator YqeI is involved in APEC's pathogenicity, and this study provides clues for future research.

Keywords: YqeI; avian pathogenic Escherichia coli; pathogenesis; transcription regulator.

Figure 1

(A) The schematic diagram of the strategy for deleting the yqeI gene in AE81: (B) confirmation of the mutant strain AE81ΔyqeI and the complemented strain AE81ΔyqeI-pCmΔyqeI. M: 5000 DNA marker; Lane 1: PCR product (1309 bp) amplified from AE81 with primers yqeI-out; Lane 2: PCR product (577 bp) amplified from AE81ΔyqeI with primers yqeI-out; Lane 3: negative control with primers yqeI-out; Lane 4: PCR product (870 bp) amplified from AE81 with primers C-yqeI; Lane 5: PCR product (870 bp) amplified from AE81ΔyqeI-pCmΔyqeI with primers C-yqeI; Lane 6: negative control with primers C-yqeI; (C) the growth curves of AE81, AE81ΔyqeI and AE81ΔyqeI-pCmΔyqeI in lysogeny broth.

Figure 2

The relative expression of some flagella-related genes detected by qRT-PCR, * p < 0.05, ** p < 0.01, ns (no significant difference).

Figure 3

(A). The flagella of AE81, AE81ΔyqeI and AE81ΔyqeI-pCmyqeI in the transmission electron micrographs views (×10,000). (a) The morphological observation of AE81 (×10,000); (b) the morphological observation of AE81ΔyqeI (×10,000); (c) the morphological observation of AE81ΔyqeI-pCmyqeI (×10,000) (B). The observation of motility ability. (d) The motility circle of the wild strain AE81; (e) the motility circle of AE81ΔyqeI; (f) the motility circle of AE81ΔyqeI-pCmyqeI.

Figure 4

Bacterial resistance to chicken serum. Growth was determined at OD_600nm. (A) SPF chicken serum was added at concentrations of 0%; (B) SPF chicken serum was added at concentrations of 10%; (C) SPF chicken serum was added at concentrations of 20%; (D) SPF chicken serum was added at concentrations of 30%; (E) SPF chicken serum was added at concentrations of 40%; (F) SPF chicken serum was added at concentrations of 50%.

Figure 5

The cell adhesion number was calculated by plate counting. The cell number of AE81ΔyqeI was significantly decreased compared with AE81. The cell numbers of AE81 and AE81ΔyqeI-pCmyqeI were similar, * p < 0.05.

Figure 6

Bacterial colonization during systemic infection in chickens at 24 h post infection (**, p < 0.01). (A) The number of AE81, AE81∆yqeI and AE81∆yqeI-pCmyqeI was counted from the heart; (B) The number of AE81, AE81∆yqeI and AE81∆yqeI-pCmyqeI was counted from the heart liver; (C) The number of AE81, AE81∆yqeI and AE81∆yqeI-pCmyqeI was counted from the heart spleen; (D) The number of AE81, AE81∆yqeI and AE81∆yqeI-pCmyqeI was counted from the heart lung.