EspE3 plays a role in the pathogenicity of avian pathogenic Escherichia coli

Abstract

APEC encodes multiple virulence factors that have complex pathogenic mechanisms. In this study, we report a virulence factor named EspE3, which can be secreted from APEC. This protein was predicted to have a leucine-rich repeat domain (LRR) and may have a similar function to IpaH class effectors of the type III secretion system (T3SS). For further exploration, the regulatory correlation between the espE3 and ETT2 genes in APEC was analysed. We then assessed the pathogenicity of EspE3, detected it in APEC secretion proteins and screened the proteins of EspE3 that interact with chicken trachea epithelial cells. This study provides data on a new virulence factor for further exploring the pathogenic mechanism of APEC.

Keywords: APEC; ETT2; EspE3; effector; pathogenicity.

Figure 1

espE3 gene locus map. Blue represents some other functional genes in the APEC81 genome; Purple represents a predicted gene encoding a hypothetical protein related to espE3; Yellow represents espE3 gene, and green represents genes encoded by ETT2 gene cluster.

Figure 2

Transcription level of espE3 in different growth phases of each gene mutant. We used APEC16S gene as an internal reference to detect the transcription level of espE3 of different strains. A In the lag phase, the transcription level of espE3 in the deletion strains of yqeH, ygeG and ETT2 were decreased compared with that of the wild strains (AE81), and the complementary strains had obvious recovery. B, C In the logarithmic phase and stationary phase, there was no significant difference in the transcription level of espE3 between the three gene deletion strains and the wild strains, and the same was true for the complementary strains.

Figure 3

Adhesion and invasion. We diluted the samples after bacterial infection, cultured them in LB solid medium and counted the number of colonies. The results showed that the adhesion and invasion ability of the espE3 deletion strain were significantly decreased, and the complementary strain had a significant recovery.

Figure 4

Transcription levels of inflammatory cytokines. We carried out RT-qPCR on bacterial infected cell samples, and the transcription level of IL-1β and TNF-α decreased significantly after the cells were infected by the espE3 deletion strain, and the transcription level of the complementary strain recovered. The transcription levels of IL-6 and IL-8 were significantly increased after the infection of the cells with the espE3 deletion strain, and the transcription levels of the complementary strain were restored.

Figure 5

Survival rate of chickens infected with AE81, ΔespE3 and CespE3. After infecting chicks with the espE3 deletion strain, the complementary strain and the wild strain, we calculated the number of chicks surviving every day thereafter. On the first day, only 60% of chicks infected with wild strain survived, while 85% of chicks infected with espE3 deletion strain survived, and 85% of chicks infected with complementary strain survived. There was a difference in the survival rate between chicks infected with the complementary strain and chicks infected with the espE3 deletion strain on the second day. The survival rate of chicks infected with the espE3 deletion strain within 7 days after infection were significantly higher than the other two strains.

Figure 6

Effect of EspE3 on the pathogenicity of APEC. A In the heart, the loss of espE3 alleviates the necrosis, roughness and deformation of myocardial fibers. B In the liver, the loss of espE3 alleviated the degeneration of hepatocytes and blurred cell edges. C In the trachea, the loss of espE3 alleviates the degeneration and proliferation of tracheal epithelial cells, the congestion of tracheal mucosa and the exudation of mucus. D In the lung, the loss of espE3 leads to a significant reduction in inflammatory and proliferative lesions of cells.

Figure 7

Screening of proteins that interact with EspE3 in CTE. A Interaction experiment between EspE3 and CTE proteins. 1: GST-EspE3 was used as bait, and PBS was used as prey. The interaction combined with eluent was used as a negative control. 2. GST-EspE3 was used as bait, and CTE protein was used as prey. 3: PBS was used as bait, and CTE protein was used as prey. B The total number of interacting proteins and the number of differential proteins were identified by mass spectrometry and sequencing.