Fur/Ryh B Regulates The Biological Phenotype And Pathogenic Mechanism Of Avian Pathogenic E.coli

APEC(Avian Pathogenic Escherichia coli)is one of the important Pathogenic bacteria caused birds colon bacillus disease,can cause the local or systemic infection of poultry,serious damage to the development of the industry.Because the urinary tract Pathogenic Escherichia coli(UPEC),neonatal meningitis Escherichia coli(NMEC)are highly gene homology with APEC,which is considered Pathogenic Escherichia coli important repository of gene,has potential risks to public health.Ferric uptake regulator(Fur),as an important protein regulating iron ion metabolism in bacteria,is encoded by Fur gene,which is the most widely and deeply studied protein related to iron metabolism.Ryh B is a size of 90 nucleotides bacterial non-coding small RNA molecules(s RNA),is the expression by negative regulation factors of the Fur,there are reports: Ryh B as known control bacteria in the present study targets the highest number of s RNA,with APEC of pathogenic bacteria,including a certain relationship,thus expounds Fur/Ryh B whether in the aspect of control bacteria biological phenotype and pathogenic have synergy and determine the downstream control targets,to reveal the pathogenic mechanism of APEC has certain reference value.In this study,Fur,Ryh B and Fur/Ryh B as a whole were taken as research objects,and the effects of Fur,Ryh B and Fur/Ryh B on the biological phenotype and transcription level of key virulence genes of APEC were investigated by constructing single/dual gene deletion strains.The enrichment of different genes of the three strains was analyzed by transcriptome technology,and the significantly different virulence genes were screened for EMSA verification.The specific results are as follows:1.Construction and biological characteristics comparison of single and double gene deletion strains Fur/Ryh B of APECIn this experiment,Red homologous recombination technology was used to successfully construct Fur isolated and Fur/Ryh B isolated strains(Ryh B isolated strains were kept in the laboratory).Comparing the biological characteristics of the three strains,the results showed that the absence of Fur and Ryh B did not affect the growth performance of APEC.?Fur and ?Fur/Ryh B significantly reduced the athletic ability of APEC as well as the number and length of flagella,and ?Ryh B had little effect on this phenotype.?Fur and ?Fur/Ryh B significantly enhanced the ability of APEC biofilm formation,while?Ryh B slightly reduced the ability of APEC biofilm formation;?Fur and ?Ryh B had no significant effect on common antibiotic resistance of bacteria,and ?Fur/Ryh B could significantly increase the sensitivity of APEC to cotrimoxazole.?Fur and ?Fur/Ryh Bsignificantly reduced the oxidative stress tolerance of APEC,and ?Ryh B had little effect on it.2.Differentially expressed genes of Fur/Ryh B strains of APECTranscriptometric analysis of Fur and Ryh B isolated and Fur/Ryh B isolated strains showed that there were 1,286 differentially expressed genes in APEC before and after Fur gene deletion,of which 649 genes were up-regulated and 637 genes were down-regulated.Before and after the deletion of the Ryh B gene,there were 1,001 differentially expressed genes in APEC,of which 459 genes were up-regulated and 542 genes were down-regulated.Before and after the Fur/Ryh B gene deletion,there were 1266 differentially expressed genes in APEC,of which 668 were up-regulated and 598 were down-regulated.GO analysis and functional classification of these differentially expressed genes showed that:?Fur-influenced differentially expressed genes were mainly enriched in the REDOX process,REDOX enzyme activity and cofactor binding,but genes with significant differences were enriched in genes related to chemotaxis such as cilia or flagella-dependent cell movement.?Ryh B-affected differentially expressed genes were mainly enriched in organic biosynthesis process,cell biosynthesis process and biosynthesis process.The differentially expressed genes influenced by ?Fur/Ryh B were mainly enriched in the REDOX process,and again,the genes related to cell motility were the most varied.In the flagella-related pathway with the greatest difference,94% of genes(34/36)were down-regulated,and the largest factor was Fli C,which was down-regulated by about 12 times.These results are basically consistent with the results of fluorescence quantitative PCR.3.Anchor the downstream targets for Fur regulation of flagellate biological phenotypes by APECThe recombinant vector BL21-pet28a-Fur was successfully constructed and expressed.Fur protein had the best expression effect and protein activity at 16 ?.The protein concentration of the target protein was 0.3 g/ L.The key flagellate regulatory genes Flh D and Fli A were screened,and the interaction between Fur protein and Flh D and Fli A genes was detected by EMSA assay with biotin probe.The results showed that Fur protein did not bind to Fli A's marker probe,but could bind to Flh D's marker probe,but the concentration dependence was not observed,suggesting that Fur may directly regulate Flh D and down-regulate flagella-related genes.