Molecular Mechanism Of MCR-1 Regulating Caspase-1 Activation In Macrophages

Objective:MCR-1 is a plasmid-mediated colistin resistance gene first reported in the world.Plasmids carrying this gene can spread rapidly and widely among bacteria through conjugation,causing huge economic losses to the aquaculture industry.At present,most of the studies on colistin resistance caused by MCR-1gene focus on epidemiological studies,and there are few studies in natural immunity.Previous research by the research group found that MCR-1 affects IL-1 βsecretion by macrophages.In this study,we explored the molecular mechanism of caspase-1 activation in macrophages by MCR-1,and provided new ideas for the prevention and treatment of colistin-resistant strains.Methods and results :(1)In this study,E.coli carrying MCR-1 and its control bacteria,E.coli 25922 p BAD-MCR-1 and E.coli 25922 p BAD,were constructed by electrotransformation for subsequent experiments.(2)In order to explore whether MCR-1 affects the growth of E.coli,the growth curves of E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1 were drawn and the survival of the two strains in infected macrophages was detected.The results showed that MCR-1inhibited the growth of E.coli in vitro.(3)In order to investigate whether MCR-1has a regulatory effect on the secretion of IL-1β by macrophages,different macrophages were infected with E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1,and the secretion of IL-1β and the expression of pro-IL-1β was detected at different time points.The results showed that MCR-1 up-regulated the protein expression of pro-IL-1β and the secretion of IL-1β in the early stage and down-regulated in the later stage.(4)To investigate whether the effect of MCR-1 on the secretion of IL-1β by macrophages was dependent on the activation of caspase-1,different macrophages were infected with E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1,and the activation of caspase-1 was detected at different time points.The results showed that MCR-1 promoted the activation of caspase-1 at the early stage of infection.MCR-1 inhibited the activation of caspase-1 in the late stage of infection.Subsequently,BMDM of wild type and caspase-1 knockout mice were infected with E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1,respectively.The secretion of IL-1β was detected at different time points.The protein expression of pro-IL-1β and the activation of caspase-1 were detected.The results showed that caspase-1 was knocked out.MCR-1 had no effect on the protein expression of pro-IL-1β and the secretion of IL-1β,indicating that MCR-1 regulated the secretion of IL-1β by macrophages depending on the activation of caspase-1.(5)In order to investigate whether MCR-1 has an effect on caspase-1-activated ’ platform ’-NLRP3 inflammasome,different macrophages were infected with E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1,and the protein expression levels of NLRP3,ASC and pro-caspase-1 were detected at different time points.The transcription levels of NLRP3,ASC and pro-caspase-1 were detected.The results showed that MCR-1 had no effect on the transcription and protein levels of NLRP3,ASC and pro-caspase-1.MCR-1 had no effect on the protein expression of NLRP3,ASC and pro-caspase-1 after infection with BMDM of wild-type and caspase-1 knockout mice.(6)In order to explore the effect of MCR-1 on the activation of NF-κB signaling pathway and the m RNA expression level of related cytokines,different macrophages were infected with E.coli 25922 p BAD and E.coli 25922 p BAD-MCR-1,and the expression level of p65,p-p65 protein and the m RNA expression levels of IL-1β,IL-18,IL-6 and TNF-α were detected bolt at different time points.The results showed that MCR-1 inhibits the activation of the NF-κB signaling pathway,up-regulated the m RNA expression of IL-1β,IL-6 and TNF-α in the early stage of infection,and down-regulated its m RNA expression in the late stage of infection.Conclusions: MCR-1 inhibits the in vitro growth of E.coli and affects the immune regulation after infection of macrophages-MCR-1 inhibits the activation of NF-κB signaling pathway,and regulates the transcription of related cytokines(IL-1β,TL-6 and TNF-α)with early promotion and later inhibition,which then affects the activation of caspase-1 and finally regulates the secretion of IL-1β.