Establishment Of Exo-RPA Detection Method For Vibrio Alginolyticus And Its Molecular Mechanism Of NLRP3 Inflammasome Activation In Host Macrophages

Vibrio alginolyticus is a zoonotic Marine Vibrio,mainly causing serious infection of fish,shrimp,shellfish and other Marine organisms,as well as gastroenteritis,conjunctivitis,otitis media and otitis externally in humans,and severely infected people die from sepsis.In recent years,with the strong economic strength and the vigorous development of mariculture,seafood has gradually become a part of people’s daily diet in China.Subsequently,reports of food poisoning caused by Vibrio alginolyticus become common.Vibrio disease caused by Vibrio alginolyticus is distributed worldwide.Once the disease occurs,it often causes a pandemic,which is a huge damage to the economy of the aquaculture industry and potential threat to people’s health.In order to achieve early intervention and achieve the purpose of"prevention instead of treatment",the establishment of a rapid on-site detection method for the pathogenic bacteria of Vibrio alginolyticus has far-reaching significance for controlling the outbreak and spread of the disease.In addition,the current treatment of Vibrio alginolytes disease is the use of broad-spectrum antibiotic antibacterial therapy,and there is still a lack of special drugs or vaccines for the prevention and treatment of the disease.However,antimicrobial therapy with antibiotics can cause the emergence of resistant strains of Vibrio alginolytes which makes treatment more difficult.In order to achieve the purpose of reducing the use of antibiotics,the treatment idea is changed from the use of antibiotics"antibacterial"to the treatment of host inflammation"anti-inflammatory",expecting to find new therapeutic targets.At the same time,more and more attention has been paid to the natural immune response between Vibrio alginolyticus and its host.The rapid propagation of Vibrio alginolyticus into human body is the main cause of acute infection.The innate immune response system of the host plays a vital role in identifying and responding to Vibrio alginolyticus and mediating the secretion of inflammatory cytokines to promote pathogen clearance,among which the pathogen pattern recognition receptor（PRRs）expressed by innate immune cells is the first stop to play its role.Although it has been reported that Vibrio alginolyticus can induce the production of inflammatory cytokines in mice,how PRRs are involved in the recognition of Vibrio alginolyticus in host cells and regulate the host inflammatory response has not been reported.This topic takes Vibrio alginolyticus as the research object,established a rapid on-site detection method for Vibrio alginolyticus based on Recombinase Polymerase Amplification（RPA）and Exo fluorescence probe,and evaluated the method on the aspects of specificity,sensitivity,and application.The present study recognized the key pattern-like recognition receptors TLR2 and NLRP3 in response to host cells by establishing mouse macrophage infection model in vitro.NLRP3 and Caspase-1/11 gene deletion mice were used to confirm that Vibrio alginolyticus positively regulates the secretion of inflammatory cytokines by activating the host NLRP3 inflammasome.The roles of TLR2,MAPK,AKT and NF-κB signaling pathways in regulating host inflammation and NLRP3 inflammasome activation in Vibrio alginolyticus were determined.Finally,the molecular mechanism of activation of NLRP3 inflammasome by Vibrio alginolyticus was clarified,in order to provide theoretical basis for the development of potential anti-inflammatory treatment against Vibrio alginolyticus.Firstly,an Exo-RPA method was established for rapid detection of Vibrio alginolyticus using tox R gene as target sequence.Common Vibrio pathogenic bacteria strains and common food-borne pathogenic bacteria strains were used as templates to verify the interspecific specificity of Exo-RPA in the detection of Vibrio alginolyticus.The sensitivity of Exo-RPA was determined with the gradient concentration of 10² CFU/μL～10^-2 CFU/μL bacterial solution as template.Genomic DNA samples from 80 clinical samples were used as templates to verify the feasibility of Exo-RPA assay.The results showed that the method had good specificity,except Vibrio alginolyticus,no other common Vibrio pathogenic bacteria and common food-borne pathogenic bacteria were detected.The sensitivity of the method was excellent,and it could detect Vibrio alginolyticus at 10^-1 CFU/μL,and the lower limit of detection was 0.174 CFU/reaction,and it had good compatibility with crude samples.Among the 80 clinical samples tested,both the standard method of q PCR and the Exo-RPA method were able to detect the same 11 positive samples,with a 100%concordance rate.The successful establishment of this method has a wide development prospect in field detection.Secondly,an in vitro model of mouse peritoneal macrophages infected by Vibrio alginolyticus was established and the transcriptional levels of inflammatory-related cytokines in peritoneal macrophages detected by q PCR.Next,the related proteins NLRP3,Caspase-1 and IL-1βof NLRP3 inflammasome were determined by Western blotting.Enzyme-linked immunosorbent assay（ELISA）was used to further determine the expression levels of IL-1β,IL-12,IL-6 and TNF-α.The subcellular localization of NLRP3 protein was determined by immunofluorescence.In addition,to further identify the key role of NLRP3 inflammasome in Vibrio alginolyticus infection,related inhibitors,such as Glyburide,CA-074 Methyl ester,AC-YAD-CHO and Z-VAD-FMK,as well as NLRP3^-/-,Caspase-1/11^-/-mouse macrophages were used in this experiment.q PCR results showed that Vibrio alginolyticus could induce the transcriptional levels of various cytokines in macrophages,including IL-1β,IL-12,IL-18,TNF-α,IL-17,IL-6,IFN-γ,and IL-10,of which the secretion level of IL-1βis the most significant.Western blotting assay of NLRP3,Caspase-1,and IL-1βrevealed that Vibrio alginolyticus induced activation of NLRP3 inflammasome leading to maturation of IL-1βand activation of Caspase-1 in a time-and dose-dependent manner.ELISA confirmed this result.When Vibrio alginolyticus infected macrophages,the NLRP3 protein changed from scattered distribution to punctate aggregation in the cells,which indicated that the NLRP3 inflammasome could be activated by Vibrio alginolyticus.Macrophages pretreated with glyburide,CA-074 Methyl ester,AC-YVAD-CHO,and Z-VAD-FMK were significantly inhibited by the downstream effector molecule IL-1βof NLRP3 inflammasome.Moreover,the inhibitors and NLRP3^-/-,Caspase-1/11^-/-significantly reduced the release levels of inflammatory cytokines such as IL-12,IL-6 and TNF-α.This will lay the foundation for further exploration of the role and mechanism of NLRP3 inflammasome in Vibrio alginolyticus disease.Finally,the changes of Toll-like receptors and their signaling pathways in macrophages infected by Vibrio alginolyticus were explored.The secretion and transcription of pro-inflammatory cytokines and the expression of key TLR in Vibrio alginolyticus macrophages were detected by ELISA combined with RT-q PCR.A TLR2 inhibitor（C29）block assay was used to determine that TLR2 positively regulated the release of IL-6,IL-12 and TNF-αand pro-IL-1βproduction of NLRP3 inflammasome first signal in Vibrio alginolyticus infected macrophages.The phosphorylation levels of p38,JNK,ERK,AKT and NF-κB proteins were determined by Western blotting.Furthermore,the immunofluorescence assays were used to determine whether NF-κB protein was phosphorylated into the nucleus and whether activation of these pathways was TLR2-dependent and crosstalk between these pathways.The results showed that macrophages stimulation induced phosphorylation of p38,JNK and ERK in TLR2 heterodimer dependent manner.Meanwhile,inhibition of SB203580（p38）,SCH772984（ERK）and SP600125（JNK）significantly reduced the secretion of IL-1β,IL-6,IL-12 and TNF-αinduced by Vibrio alginolyticus.However,blockade with MK-2206 2HCl（AKT）inhibitor negatively regulated the release levels of IL-1β,IL-6,IL-12 and TNF-αinduced by Vibrio alginolyticus.In addition,Vibrio alginolyticus infection with macrophages results in degradation of IκBαand phosphorylation and translocation of NF-κB to the nucleus,which is dependent on TLR2 heterodimer.NF-κB inhibitor BAY 11-7082 was used to block its activation and down-regulate the release of IL-1β,IL-6,IL-12and TNF-αinduced by Vibrio alginolyticus.Therefore,our results suggest that TLR2 heterodimer-mediated activation of downstream NF-κB,MAPK,and AKT signaling pathways are an important mechanism of inflammatory response during Vibrio alginolyticus infection in vitro,and is an important signal axis in regulating pro-IL-1β,the key protein of the first signal of NLRP3inflammasome.In summary,this study combined RPA technology with Exo fluorescent probe and applied it for the first time in the detection of Vibrio alginolyticus.The establishment of the detection method has far-reaching significance for better control of the spread and outbreak of Vibrio alginolyticus.Our study showed that Vibrio alginolyticus can activate NLRP3 inflammasome to regulate the secretion of the host inflammatory cytokine IL-1β,and TLR2 receptor involves in recognition receptor of the first signal of NLRP3 inflammasome and the immune response to Vibrio alginolyticus infection,providing a potential target for treatment and prevention of Vibrio alginolyticus disease.