The Regulation Of Quorum Sensing Of LuxS/AI-2in Avian Pathogenic Escherichia Coli

Avian Pathogenic Escherichia Coli (APEC) causes avian colibacillosis, the mostsignificant infectious bacterial disease of poultry worldwide. The quorum sensing systemshared by Gram-positive and Gram-negative bacteria involves the production ofautoinducer-2. It has been proposed that AI-2is a universal signaling molecule thatfunctions in interspecies cell-to-cell communication. Nowadays various APEC serogroupsand increasingly serious problem of bacterial antibiotic resistance have become thechallenge in controlling APEC. It has been reported to be a key player in regulation ofmany physiological functions.It has been no research about LuxS/AI-2quorum sensing system of APEC to date.This study aims to supply new methods to prevent the infection of APEC from the aspect ofbactria population through the research of LuxS/AI-2type quorum sensing system, whichwill be benefit to future studies of the role of quorum sensing in physilology regulation ofAPEC.1.Detection of autoinducer-2of avian pathogenic Escherichia coli and analysis ofregulation.There have been no reports concerning the AI-2in APEC. In this study, weinvestigated AI-2production in APEC by Vibrio harveyi BB170bioassay. The resμLtsshowed that APEC can produce AI-2.In this study, we analyzed the relationship between the transcription level of luxS andpfs and AI-2production in different growth phases and culture conditions. AI-2activity indifferent growth phases and culture conditions was measured using the V. harveyibioluminescence assay. The levels of luxS and pfs mRNA were analyzed using real-timePCR. The results showed that the level of AI-2is consistent with the level of luxS mRNA indifferent growth phases. AI-2production and luxS mRNA by APEC was increased bysupplementation with glucose, maltose, and Nacl, while the addition of sucrose wasdecreased. The transcription level of luxS is correlated to the level of AI-2production,white the transcription level of pfs did not correlate with the level of AI-2production.2. In vitro biosynthesis AI-2and the modulation of AI-2on the biofilm forming abilityAPEC luxS and pfs was cloned, expressed, purified and used to investigate theproduction of AI-2in vitro. LuxS and Pfs were incubated with S-ribosylhomocysteine, thereaction was detected for the production of luminescence of Vibrio harveyi BB170. Theresults showed that the reaction products can contain300μmol/LAI-2.The aim of the study was to investigate the modulation of AI-2on the biofilm forming ability in Avian Pathogenic Escherichia coli (APEC). The improved crystal violetseme-quantitative method and fluorescence staining method were used to study the effectsof AI-2on the biofilm forming ability in APEC. The results showed that the biofilmforming ability decreased at the concentration of0.185mmol/L AI-2.3.Construction and pathogenicity characterization of luxSLuxS, the product of the luxS gene, mediates the quorum sensing(QS) mechanism.This involves the production of autoinducer-2(AI-2), which regulates importantphysiological traits and a variety of adaptive processes in different bacteria. In this study,luxS-delated mutant strain, DE17, was constructed using strain DE17. Analysis ofbioluminescence indicated that deletion of the luxS gene abolished the production of the QSsignal AI-2in the bacteria. Further studies showed that deletion of the luxS gene in DE17reduced the bacterial virulence by31.5-fold in ducklings, based on the measurement of the50%lethal dose. The mutant strain reduced significantly the abilities of adherence andinvasion, by50.0%and40.7%respectively, compared with the wild strain DE17. Themutant strain also showed reduced survival in vivo: the bacterial loads of the mutant strainin infected liver, spleen and blood were46.4-fold,5.2-fold, and3.7-fold reduced,respectively, compared with the wild-type strain DE17. Real-time polymerase chainreaction (PCR) demonstrated further that the mRNA levels of the virulence-related genesiucD, fyuA, vat, ompA, iss, fimC and tsh were significantly decreased in the mutant strainDE17, when compared with DE17(p <0.05). In addition, the deletion of the luxS genereduced the motility of the bacterium.Above all, this study demonstrated APEC has luxS/AI-2QS system. The level oftranscription of luxS is highly correlated with the level of AI-2production, while the levelof transcription of pfs does not correlate with the level of AI-2production.The resultsdemonstrated that recombinant Pfs and LuxS synthesize AI-2in vitro from SAH.AI-2involves in regulating the biofilm formation of APEC. This study suggests that theluxS gene functions in the pathogenesis of diseases caused by APEC. Our study provide thereference for the further explore of how LuxS/AI-2quorum system regulates APEC.