| Avian pathogenic Escherichia coli(APEC)is an important member of extraintestinal pathogenic Escherichia coli(ExPEC),which is an important pathogen that causes acute or chronic infectious bacterial diseases with local or systemic infection in poultry.It has high homology with the genotype of neonatal meningitis E.coli(NMEC).They have some similar virulence genes and both of them can cause host meningitis.The genotoxin Colibactin is a kind of non-ribosomal peptide/polyketide secondary metabolite produced by bacteria,which can cause chromosomal instability and DNA damage in eukaryotic cells.It is encoded by pks gene Island(19 genes in total from clbA to clbS).The pks gene island is closely related to the virulence factors(adhesin,hemolysin,toxins and siderophores)of B2 parenteral pathogenic E.coli,which are important for meningitis occurrence.But it is unclear whether Colibactin is directly related to the occurrence of APEC meningitis by now.The colibactin biosynthesis requires the enzymatic activity of the phosphopantetheinyl transferase(PPTase)ClbA.ClbC constitutes the most uncommon part of the PKS/NRPS cluster as it encodes protein with unusual domain architecture.In this paper,the Red homologous recombination system was used to construct clbA and clbC deletion mutants,and the effects of clbA and clbC genes on Colibactin production were explored.The result showed that the phenomenon of megalocytosis and H2AX phosphorylation was obviously reduced when the clbA and clbC gene deletion strain interacted with bEnd.3 cells.Therefore,the deletion of clbA and clbC genes reduced the DNA double-strand breaks in bEnd.3 cells and affected the normal synthesis of Colibactin.It indicated that the clbA and clbC genes are the key genes for Colibactin synthesis.As a key enzyme for Colibactin synthesis,ClbA can also promote the production of siderophores.RyhB is a non-coding small RNA that regulates bacterial iron homeostasis and is involved in the regulation of clbA transcription in E.coli.However,it is unclear whether RyhB regulates ClbA directly and whether RyhB participates in the regulation of other genes on the Colibactin assembly line.Therefore,this study constructed APEC ryhB-deletion mutant and complemented mutant,and detected the transcription of clbA and clbC in wild type and ryhB-deletion mutant using qRT-PCR.The result showed that the deletion of ryhB gene down-regulated both clbA and clbC expressions.The bioinformatics prediction and dual plasmids fluorescence reporting system were used to study the regulatory mechanism of RyhB on clbA and clbC.The results showed that RyhB directly regulated the expression of clbA,while indirectly regulated the expression of clbC.It indicates that the synthesis of Colibactin is regulated by RyhB.To investigate whether Colibactin contributes to the occurrence of APEC meningitis,and whether RyhB,as a regulator of clbA,affects the pathogenicity of APEC by regulating Colibactin synthesis,a series of meningitis-related indicators were detected by using APEC wild strains,clbA,ryhB deletion mutants,and complemented strains to infect mice.The results showed that compared with the wild type strain,the clinical symptoms such as opisthotonus of mice infected with clbA and ryhB deletion mutants were significantly reduced or disappeared.The blood-brain barrier permeability was not damaged significantly,the bacterial load of the brain and other organs and the expression of inflammatory factors in the brain tissue were significantly reduced in clbA and ryhB deletion mutants.The clbA complemented strain restored its virulence to a great extent,and the ryhB complemented strain also recovered to some extent.The above results indicate that clbA and Colibactin are directly related to the occurrence of APEC-induced meningitis.Although RyhB down-regulates the transcription of clbA,it is also related to meningitis occurrence.It is supposed that RyhB enhances the pathogenicity of APEC XM by regulating other virulence related genes expression but not only Colibactin. |
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