As a transcriptional regulator of the classical binary regulatory system, PhoP plays an important role in the life activities of avian pathogenic Escherichia coli (APEC). In previous experiments, we found that the absence of phoP affects APEC biofilm formation and pathogenicity. To further explore the molecular mechanism of phoP regulation of these phenomena, the differentially expressed gene tolC was screened based on phoP-derived transcriptional data, and the specific sequence identity of the PhoP binding sequence was predicted by bioinformatics and verified by electrophoretic mobility shift assay (EMSA). The results showed that PhoP can directly bind to the tolC promoter. On this basis, tolC deletion and complementary strains were constructed. Biofilm formation was quantified by crystal violet staining and rdar morphology change was observed in these strains. Loss of tolC reduced biofilm formation. We also examined pathological changes in organ paraffin sections by challenging chicks with the strains. After loss of tolC, the clinical signs of pericarditis and liver and spleen enlargement in chicks were alleviated, and pathogenicity to the host cells was decreased. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that tolC deletion downregulated secA/secB/secE/secY transcript levels, which are part of the type II secretion system secreting virulence effector element. These results indicate that tolC contributes to the phoP-mediated effect on APEC biofilm formation and pathogenicity.
Keywords: Avian pathogenic Escherichia coli; PhoP; biofilm; pathogenicity; tolC.