AI-2 Affects Biofilm Formation And Motility Of Plesiomonas Shigelloides By Regulating The C-di-GMP-metabolizing Enzyme DosC

Autoinducer-2(AI-2),a Quorum Sensing(QS)signal molecule,is widely distributed in bacteria and is one of the molecules that initiate the population density response of bacteria.Unlike most self-inducers,gram-negative and gram-positive bacteria that produce AI-2 signals can not only regulate their own population behavior,but also affect bacteria that do not produce AI-2.When cells reach high density,their activated gene expression is involved in a series of life activities,including bioluminescence,motility,biofilm formation,resistance to stress,and production of pathogenic factors(Alencar et al.2021).However,the regulation and molecular mechanism of this signaling molecule on physiological functions of many bacteria have not been reported.Plesiomonas shigelloides is a common pathogenic bacteria,which not only poses a serious threat to human health,but also poses a great risk to aquaculture production.In this study,P.shigelloides NCTC 10360 was used as the research object to reveal the internal mechanism by which AI-2 regulates the formation and motility of the biofilm of P.Shigelloides NCTC 10360 by affecting the level of its intracellular cyclodiguan-monophosphate(c-di-GMP).It provides a new idea for the prevention and treatment of Plesiomonas shigelloides infection.The main results are as follows:1.We constructed the lux S gene knockout strain(Δlux S)by homologous recombination method.Soft agar plate assays and crystal violet staining assays were used to detect the differences of swimming motility and biofilm formation between Δlux S and the wild type.The results showed that the deletion of lux S gene would weaken the biofilm formation and swimming ability.2.The potential receptor protein DosC of AI-2 was identified by sequence alignment(SAMEA2665130＿2180).The ligand-binding domain(LBD)of Dos C and AI-2 were analyzed by bioluminescence experiment of Vibrio harveyi and isothermal titration thermal experiment(ITC).The results showed that AI-2 showed a high affinity with Dos C-LBD(Kd = 2.83±0.17 μmol/L).3.In addition to the periplasmic ligand binding domain(LBD),the intracellular part of the membrane protein Dos C also contains GGDEF and EAL tandem domains related to c-di-GMP metabolism.Through the expression and purification of full-length transmembrane protein Dos C,enzyme activity experiments were carried out in vitro with or without DPD/AI-2,reaction substrates and appropriate reaction conditions,and the reaction products were analyzed by HPLC.The results showed that AI-2 enhanced the phosphodiesterase activity of receptor protein Dos C and promoted the degradation of c-di-GMP.4.Finally,the AI-2 receptor DosC coding gene knockout strain(Δdos C)was constructed,extracted and the intracellular c-di-GMP content of the wild type,Δlux S andΔdos C strains was determined by liquid chromatography-tandem mass spectrometry(LC-MS).It was found that compared with the wild type,the intracellular c-di-GMP levels of Δlux S and Δdos C strains were significantly increased.Finally,Δdos C and wild-type biofilm formation ability were measured,and it was found that compared with wild-type,Δdos C strain biofilm formation level and biofilm formation ability were significantly decreased.These results suggest that AI-2 regulates the level of intracellular c-di-GMP through Dos C,thereby affecting the biofilm formation and swimming ability of P.shigelloides.These results indicate that the accumulation of intracellular c-di-GMP level in p.shigelomonas NCTC 10360 inhibits the biofilm formation and the ability of biofilm movement,and the c-di-GMP metabolic enzyme Dos C enhances its phosphodiesterase activity by responding to the quorum sensing signal molecule AI-2,promoting the degradation of c-di-GMP.It affects the content of intracellular c-di-GMP,and then regulates the biofilm formation and swimming ability of bacteria.