The Colibactin Toxicity Related Genes In Escherichia Coli Contributes To Meningitis Development

Avian pathogenic Escherichia coli(APEC)is an important member of extraintestinal pathogenic Escherichia coli(ExPEC),which causes local or systemic infectious bacterial diseases in fowl and threatens the development of the poultry industry.APEC and neonatal meningitis E.coli(NMEC)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.APEC XM(O2:K1)strain was isolated from the brain tissue of a duckling with sepsis/meningitis in Xinmin,Shandong in 2007.The whole-genome sequencing revealed that APEC XM carried pks island and multiple ExPEC characteristic virulence genes.In the previous study,APEC XM produced meningitis in newborn rats,newborn mice and chicks.The whole-genome sequencing and animal infection test have proven that APEC XM is a high virulent strain,which might be harmful to the poultry industry.At present,the role of genes related to colibactin genotoxicity in the pathogenicity of meningitis and molecular mechanisms involved are still unclear.In this study,the transcriptomes of APEC strain and bEnd.3 cells were measured by dual RNA-seq after the interaction of 3 h,and the mRNA levels of all 19 genes in pks island had significantly changed in APEC XM during infection.Then,the ?-Red homologous recombination system and sequence and ligation independent cloning technology were used to construct clbH,clbK,clbF,clbI or clbG deletion mutants and complemented mutants,respectively.Comparison of the genotoxic effects of different strains on bEnd.3 cells by methylene blue staining,immunofluorescence and flow cytometry.Western blot,clinical observation,CBC test,MRI,pathological sections and immunohistochemistry were used to detect changes in meningitis-related indicators in bEnd.3 cells and 4-week ICR mice to explore clbG and clbH in pathogenicity of meningitis and molecular mechanisms involved in.The main research contents are as follows:1.The transcriptomes of APEC strain and bEnd.3 cells were measured by dual RNA-seq after the interaction of 3 h.When APEC infected the bEnd.3 cells,several significant changes in the expression of genes related to cell junctional complexes,extracellular matrix degradation,actin cytoskeleton rearrangement,immune activation and the inflammatory response in bEnd.3 cells were observed as compared to the mock infection group.Thus,the immune activation of bEnd.3 cells indicated that APEC infection activated host defenses.Furthermore,APEC may exploit cell junction degradation to invade the BBB.In addition,amino acid metabolism and energy-metabolism related genes were downregulated,and the protein export pathway related genes were upregulated in APEC cultured with bEnd.3 cells,compared to that in control.Thus,APEC may encounter starvation and express virulence factors during incubation with bEnd.3 cells.Importantly,the mRNA levels of all 19 genes in pks island had significantly changed(p<0.01)in APEC XM during infection,which indicated that genes related to colibactin genotoxicity might be new virulence factors for APEC XM meningitis.2.The effect of clbH,clbK,clbF,clbI or clbG deletion on the genotoxicity of colibactin.ClbH and ClbI are essential for cyclopropane(C3H3)formation,which is responsible for colibactin-induced DNA alkylation.ClbG recognizes AM and transfers it to ClbK,which incorporates AM into colibactin.ClbF is a biosynthesis dehydrogenase for AM synthesis.ClbK generates heterocyclic structures containing both sulphur and nitrogen(C3H3NS)to the thiazole rings.C3H3,AM and C3H3NS are important structures suspected to be important for colibactin biological function.In this paper,the ?-Red homologous recombination system and sequence and ligation independent cloning technology were used to construct clbH,clbK,clbF,clbI or clbG deletion mutants and complemented mutants,respectively.To confirm the role of clbH,clbK,clbF,clbI or clbG during the colibactin biosynthesis process,we detected the yH2AX expression,megalocytosis level and cell cycle phases in bEnd.3 cells to quantify the colibactin production of deletion mutants.Compared with the APEC XM group,the expression of yH2AX in 5 deletion strain groups decreased significantly at 0 hpi(hour post-infection,hpi)and 72 hpi.The phenomenon of megalocytosis and the cell-cycle distribution(G1 phase,S phase,and G2 phase)of bEnd.3 cells in 5 deletion strain groups were similar to those in the control group.The results indicated that the absence of clbH,clbK,clbF,clbI or clbG significantly reduced the genotoxicity effect of colibactin,and they were the key genes for the colibactin toxic effect.3.Genes related to colibactin genotoxicity(clbG and clbH)contributes to the occurrence of APEC meningitis.Western blot,clinical observation,CBC test,MRI,pathological sections and immunohistochemistry were used to detect changes in meningitis-related indicators in bEnd.3 cells and 4-week ICR mice.The results of vitro infection of bEnd.3 cells showed that,compared with the APEC XM group,APEC XM ?clbG and APEC XM ?clbH significantly reduced the relative expression of cytokines(IL-1,IL-6 and TNF-?;p<0.01)in bEnd.3 cells.The protein expressions of Claudin-5,Occludin and ZO-1in the APEC XM ?clbG group or APEC XM ?clbH group were higher than those in APEC group(p<0.01 and p<0.05).None of the 4-week ICR mice,infected with the APEC XM ?clbG or APEC XM ?clbH,contracted meningitis and displayed weakened clinical symptoms.Fewer radiological and histopathological lesions were observed in the APEC XM ?clbG group and APEC XM ?clbH group.The deletion of clbG or clbH reduced the bacterial colonization of tissues and the relative expression of cytokines(IL-1,IL-6,and TNF-?)in the brains in the APEC XM ?clbG group or APEC XM ?clbH group,compared to those in the APEC XM group.The tight junction proteins(claudin-5,occludin and ZO-1;p<0.01 and p<0.05)were not significantly destructed in vivo after APEC XM ?clbG and APEC XM ?clbH infection Therefore,colibactin contributes to the occurrence of APEC meningitis.Therefore,clbG and clbH contribute to the inflammation response and tight junction protein disruption during APEC XM infection in vitro and in vivo.In conclusion,the absence of clbH,clbK,clbF,clbI or clbG significantly reduced the genotoxicity effect of colibactin,and they are the key genes for the colibactin toxic effect.clbG and clbH contribute to activation of innate immune system and destruction of the blood-brain barrier during infection.Therefore,clbG and clbH may be important virulence factors for APEC XM.