The transcriptional regulator PhoP mediates the <i>tolC</i> molecular mechanism on APEC biofilm formation and pathogenicity

Abstract

As a transcriptional regulator of the classical binary regulatory system, PhoP plays an important role in the life activities of avian pathogenic <i>Escherichia coli</i> (APEC). In previous experiments, we found that the absence of <i>phoP</i> affects APEC biofilm formation and pathogenicity. To further explore the molecular mechanism of <i>phoP</i> regulation of these phenomena, the differentially expressed gene <i>tolC</i> was screened based on <i>phoP</i>-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 <i>tolC</i> promoter. On this basis, <i>tolC</i> 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 <i>tolC</i> reduced biofilm formation. We also examined pathological changes in organ paraffin sections by challenging chicks with the strains. After loss of <i>tolC</i>, 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 <i>tolC</i> deletion downregulated <i>secA</i>/<i>secB</i>/<i>secE</i>/<i>secY</i> transcript levels, which are part of the type II secretion system secreting virulence effector element. These results indicate that <i>tolC</i> contributes to the <i>phoP</i>-mediated effect on APEC biofilm formation and pathogenicity.