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Avian pathogenic Escherichia coli (APEC) is an important pathogen that causes avian colibacillosis in poultry. APEC infection can lead to pathological changes in chicken trachea. The type VI secretion system (T6SS) of APEC contribute to the pathogenicity of APEC. However, whether T6SS plays a role in infection of the trachea remains unclear. We constructed mutant strain Δhcp2a by the Red recombination method system. The role of hcp2a (the structural secretion components and secretory protein of the T6SS) in the infection of trachea was investigated. The mutation strain displayed a significant increase in biofilm formation and a decrease in resistance to chicken serum. Moreover, RNA sequencing analyses showed that infection of chicken tracheal epithelium by the mutant strain Δhcp2a induced differential expression of genes. The result also showed that 14 genes (13 genes were downregulated) were enriched in cytokine-cytokine receptor interaction signalling pathway at 12 and 24 h post infection. The mutation Δhcp2a resulted in significant decreases in the bacterial loads in trachea at 6 and 12 h post infection. Real-time PCR analyses showed that the hcp2a mutation downregulated the expression of IL8 and IL1β at mRNA level in chicken tracheal epithelium. Our results indicate that mutation of hcp2a influenced genes expression of the cytokine-cytokine receptor interaction pathway by decreasing APEC colonization in the trachea.

ybjX gene mutation decreased the pathogenicity of the avian pathogenic Escherichia coli strain, AE17. However, the associated regulatory mechanism of ybjX remains unknown. In this study, we examined the bactericidal activity of chicken serum and blood, as well as bacterial survival in HD11 macrophages. We compared the transcriptome of ybjX mutations with those of the wild strain and studied the effects of ybjX on miRNA expression in the spleen. Our findings revealed that the mutant strain, ΔybjX, had a lower resistance to chicken serum and blood, as well as lower bacterial survival in HD11 macrophages than AE17. RNA sequencing analyses showed that the ybjX mutation reduced stress resistance by down-regulating mRNAs in metabolic pathways. Infection with the ybjX mutant strain caused changes in the splenic miRNA profile. We verified Kelch repeat and BTB domain-containing protein 11 to be the target of miR-133b. Together, these findings suggest that the ybjX mutation reduces serum, blood, and environmental stress resistance by down-regulating the mRNA in metabolic pathways.

Mutation of the PhoP/Q two-component system decreases the expression of ybjX and pagP encoding outer membrane proteins, and mutation of ybjX or pagP attenuates avian pathogenic Escherichia coli (APEC) pathogenicity. However, whether ybjX/pagP mutation (double-deletion mutant) has a synergistic effect on pathogenicity remains unknown. Herein, electrophoresis mobility shift assay (EMSA) experiments showed that the PhoP/Q system regulated ybjX and pagP transcription indirectly. The APECΔybjX/pagP mutant strain, constructed using the Red recombination method, exhibited reduced invasion of chicken embryo fibroblast (DF-1) cells, but had no effect on virulence in a chicken model. Using RNA sequencing to identify differential mRNAs in APECΔybjXΔpagP and native strains, we revealed up-regulation of genes involved in the bacterial chemotaxis pathway. The ybjX/pagP mutant strain displayed significantly increased motility, suggesting that double deletion of ybjX and pagP enhances motility via the bacterial chemotaxis pathway.