Development Of Assay For Identification AI-2 Receptor And Identification Of AI-2 Target Proteins In Avian Pathogenic Escherichia Coli

Avian pathogenic Escherichia coli(APEC)can causes a variety of extra-intestinal diseases in chickens,turkeys,and other avian species,which result in avian colibacillosis with airsacculitis,cellulitis,salpingitis and peritonitis.APEC causes major economic losses through high mortality rates and low feed conversion in avian industry in worldwide.LuxS/AI-2 type quorum sensing system is a bacterial cell–cell communication process,which is widely distributed in Gram-positive and Gram-negative bacteria.LuxS/AI-2 type quorum sensing play important roles in controlling bacterial bioluminescence,sporulation,competence,antibiotic production,biofilm formation,and virulence factor secretion.AI-2 can be uptaked by the specific membrane receptor transport system.So far,two types of AI-2 receptor have been identified: LuxP type and LsrB type of receptors characterized first for Vibrio harveyi and Salmonella Typhimurium,respectively.Previous studies showed that APEC have been shown to respond to AI-2.However,no lsrB or luxP homolog was found in APEC genome sequences by bioinformatics analysis.The presnt study will develop the assay for AI-2 receptors identification,screen the AI-2 target proteins,which will be of benefit for future studies on the regulatory pathway of LuxS/AI-2 type quorum sensing system in APEC.(1)Development of assay for identification AI-2 receptor.To further verify LsrB binding capacity of AI-2,and develop the assay for AI-2 receptor identification,a luxS deletion strain of BL21(DE3)was constructed and named BL21(DE3)ΔluxS,which did not product AI-2.The LsrB protein was expressed using the BL21(DE3)ΔluxS strain and BL21(DE3)strain.The purification LsrB protein was used in AI-2 binding and releasing experiments.The result showed that LsrB have the binding activity of AI-2,which could be used to screen AI-2 receptor in APEC.(2)Screening AI-2 target proteins by proteomicsThe results of AI-2 uptake showed that AI-2 was internalized(about 72%)occurred at 3 h after the AI-2 was added.In addition,the transcription levels of four virulence genes(luxS,pfs,iss and tsh)were measured by real-time PCR.The most significant(p <0.001)change of transcript levels of the four genes occurred at 5 h after AI-2 uptake.These results indicate that APEC internalizing AI-2 and AI-2 regulating levels of gene transcription are not synchronized in APEC.In order to screen AI-2 target proteins in APEC,the bacterial samples were collected after adding AI-2 5 h based on the aboved results of the internalization kinetics and the RealTime-PCR.The sample of APEC without AI-2 was as a control group.Then the differences protein were screened by using non-labeled quantitative proteomics method to detect AI-2 regulation.In total 479 proteins show significant differences in protein expression level after internalizing AI-2 by unlabeled quantitative proteomics.And the protein bioinformatics analysis showed that,these proteins are mainly involved in the regulation of metabolism and signal transduction in APEC.(3)Suspicious AI-2 receptor gene screening in APECBased on the aboved results of proteomics,five AI-2 target genes(3630,tufB,phoH,kgtP and yjaE)were selected for construction mutants by Red homologous recombination technology.The mutant strains of the aboved five genes were successfully obtained.The kinetic study of AI-2 uptake by the mutant strains showed that the five mutant strains have no effects on AI-2 uptake.Furthermore,the results of LD50 showed that the pathogenic of these mutant strains do not show significant change.These results indicates that the selected five AI-2 target genes are not involved in the regulation of virulence of APEC.The five selected AI-2 target gene(3630,tufB,phoH,kgtP and yjaE)may be involved in the regulation of some other physiological processes which regulated by LuxS/AI-2 type quorum sensing system in APEC,and there specific mechanisms remains to be further study in the future.In summary,this study develop the assay for AI-2 receptors identification,screen the AI-2 target proteins,which will be of benefit for future studies on the regulatory pathway of LuxS/AI-2 type quorum sensing system in APEC.