Influence Of Salmonella Plasmid Virulence Gene On Infection Process And Autophagy In Zebrafish Model

Objective：Zebrafish larvae are translucent and bacteria dissemination can easily be real-timetracked by fluorescence microscopy. Zebrafish is a good model to study the interactionbetween pathogen and host. In order to study the influence of Salmonella entericaserovar typhimurium (S.typhimurium, STM) on autophagy of zebrafish, the experimentfirst constructed a salmonella-zebrafish infection model. Salmonella plasmid virulencegene (spv) can inhance bacterial virulence. In the second part, to investigate theinfluence of spv gene on infection process and autophagy, wild-type STM strain(STM-WT) harboring spv and spv-deletion STM strain STM-Δspv were used to infectzebrafish model. The results will provide theoretical and experimental basis for furtherstudy on the mechanisms of Salmonella pathogenesis, the prevention of Salmonellainfection and new drug researches.Methods：1. Construction of salmonella-zebrafish infection model and the research onautophagy(1) Determination of the concentration of S.typhimurium to infect zebrafish72hours post-fertilization (hpf) zebrafish larvae were immersed in watercontaining S.typhimurium UF110at viable concentrations. Count zebrafish mortalitywithin7days and draw a survival curve to determine the appropriate concentration.(2) The dissemination and proliferation of S.typhimurium in zebrafish72hpf zebrafish larvae were immersed in water containing GFP-UF110at theappropriate concentration. The dissemination of S.typhimurium in zebrafish wereobserved under a fluorescence microscope. To investigate the proliferation in vivo,whole-larvae colony forming unit (CFU) and intracellular CFU were enumerated at6,8,10,12,14,16and24hours post infection (hpi).(3) The influence of S.typhimurium on autophagy in zebrafish infection model Zebrafish larvae were harvested at6,8,10,12and14hpi, and then the expressionof the autophagy-associated protein microtubule-associated protein1light chain3-Ⅱ(Lc3-Ⅱ) and p62were detected by western-blotting (WB). Ultrastructure of theintestinal epithelial cells was observed by transmission electron microscope (TEM).(4) The influence of Torin1and chloroquine (CQ) on xenophagy in zebrafishTo explore the influence of autophagy agonist Torin1and lysosomal inhibitors CQto zebrafish infection model. Firstly drug effect on bacterial growth curve and survivalrate of zebrafish was detected. Then WB was used to determine the concentration ofTorin1and CQ. After treated by the appropriate concentration of Torin1, CQ or both ofthem, zebrafish were infected by S.typhimurium. The intracellular CFU wereenumerated, and WB tested the expression of Lc3-Ⅱand p62.2. Influence of spv on infection process and autophagy in zebrafish infection model(1) Construction of red fluorescence strainsZebrafish yolk sac has strong green fluorescent background. In order to reduce theinterference of background, this part firstly constructed strains expressing redfluorescent protein (RFP) which have little background interference. Plasmid expressingRFP was transformed into STM-WT and STM-Δspv by the CaCl2method. Bacteria wasidentificated under a fluorescence microscope. The relationship between fluorescenceintensity and the amount of bacteria was determined and the influence of RFP on onbacterial growth curve was detected.(2) The influence of spv on median lethal dose (LD50) in zebrafishAfter infection by different concentrations of STM-WT or STM-Δspv, the deathsof zebrafish within7d were counted. And then LD50in two strains was calculated byReed-Muench method.(3) The influence of spv on proliferation and dissemination of S.typhimurium inzebrafishZebrafish larvae infected by STM-WT or STM-Δspv at different times werecollected, compare the dissemination of two strains under a fluorescence microscope.Compare whole-larvae CFU and intracellular CFU between two strains at4,8,12,16and24hpi through the amikacin exclusion assay.(4) The influence of spv on autophagy in zebrafish infecton modelZebrafish larvae infected by STM-WT or STM-Δspv at different times were collected, then the expression of Lc3-Ⅱ and p62were detected by WB. The larvaetreated with Torin1or CQ for24h were cocultivated with STM-WT or STM-Δspv for8h, and then the expression of Lc3-Ⅱ and p62were detected by WB and the intracellularCFU were also detected by plate counting.Results:1. Construction of salmonella-zebrafish infection model and the research onautophagy(1) Determination of the concentration of S.typhimurium to infect zebrafish：Zebrafish larvae were normal after infection by109cfu/ml within two days. At3dpi, thelarvae’s tail began to curve, and flexibility and coordination ability were destroyed, andsome began to die. According to the research purpose, autophagy occurs at the earlystage after infection,109cfu/ml which had no significant effect on the larvae was theappropriate concentration.(2) The dissemination and proliferation of S.typhimurium in zebrafish: Afterimmersion of zebrafish for4h in a suspension of GFP-expressing S.typhimurium at aconcentration of109cfu/ml, green fluorescence was observed around the mouth andyolk sac under a fluorescence microscope. The fluorescent signal in gastrointestinaltract was detected at8hpi. Prolonging the infection to1dpi resulted in localisation ofS.typhimurium in the eyes and head. Further infecton resulted in green fluorescenceassociated with the whole head at2dpi and the whole fish at3dpi. According to theresults of the bacterial plate counting, whole-larvae CFU is increasing all the time, andthe number at10hpi were significantly higher than that at8hpi (p<0.001). However,intracellular CFU at10hpi were significantly lower than that at8hpi (p<0.001).(3) The influence of S.typhimurium on autophagy in zebrafish infection model：WBdetection showed that the expression of Lc3-Ⅱat8hpi was significantly higher than thecontrol group and other time points (p<0.001). The autophagy substrate p62expressionwas the opposite (p<0.01). The trend is still exist at10hpi (p<0.05). Swollenmitochondria structure, double membrane autophagosome containing bacteria andsingle-layer membrane autolysosome was observed under the TEM.(4) The influence of Torin1and CQ on xenophagy in zebrafish：0.4μmol/L Torin1treated the larvae for24h, the survival rate was100%, and Lc3-Ⅱ expression was the highest (p<0.001);50μmol/L CQ treated the larvae for24h, no larvae died, and p62expression was the highest (p<0.01). The results of the bacterial plate counting showedthat intracellular CFU after interference by Torin1was significantly decreased (p<0.01).However, after interference by CQ (p<0.05) or by the two drugs (p<0.001), intracellularCFU was significantly increased. WB results showed that after interference by Torin1,autophagy marker protein Lc3-Ⅱ expression was significantly higher than the controlgroup (p<0.01), and autophagy substrate p62expression was significantly decreased(p<0.01). But after interference by CQ (p<0.01) or by the two drugs (p<0.001), theexpression of Lc3-Ⅱand p62were both increased.2. Influence of spv on infection process and autophagy in zebrafish infection model(1) Construction of red fluorescence strains： Red rod-shaped bacteria wereobserved under a fluorescence microscope. The fluorescence intensity and the numberof bacteria was linear positive correlation. And RFP had no significant influence onbacterial growth curve with24hours at least.(2) The influence of spv on LD50in zebrafish: LD50of STM-WT was1.17×107cfu/ml，and LD50of STM-Δspv was1.95×108cfu/ml. The results showed that spv cansignificantly decreased LD50of S.typhimurium in zebrafish.(3) The influence of spv on proliferation and dissemination of S.typhimurium inzebrafishAfter immersion of zebrafish for4h or6h in a suspension of RFP-STM-WT orRFP-STM-Δspv at a concentration of109cfu/ml, red fluorescence was around the mouthand yolk sac in both groups. The fluorescent signal in gastrointestinal tract was detectedat8hpi, and the fluorescence intensity of STM-WT was higher than STM-Δspv.Prolonging the infection, the rate of proliferation and dissemination of STM-WT werefaster, which had disseminated to the whole body at2dpi, while STM-Δspvdisseminated to the whole body at3dpi. The results of the bacterial plate countingshowed that whole-larvae CFU of STM-WT infection group were significantly higherthan that of STM-Δspv infection group at the same infection time (p<0.05, p<0.01).Intracellular CFU were significantly decreased, and STM-WT infection group were alsosignificantly higher than that of STM-Δspv infection group (p<0.01, p<0.001).(4) The influence of spv on autophagy of zebrafish infecton modelAt8hpi, WB detection showed that Lc3-Ⅱexpression in STM-WT infection group were significantly lower than that of STM-Δspv infection group (p<0.01), and p62expression in STM-WT infection group were higher (p<0.01). The trend is still exist at10hpi (Lc3-Ⅱ, p<0.01; p62, p<0.05). But no significant difference at6hpi (p>0.05).After treated by Torin1, Lc3-Ⅱexpression in STM-WT infection group weresignificantly increased (p<0.05), but had no significant difference to STM-Δspvinfection group (p>0.05). Pretreated by CQ, Lc3-Ⅱand p62expression in two groupsare both increased (p<0.01). And Lc3-Ⅱexpression in STM-WT infection group wasstill lower in STM-Δspv infection group (p<0.01), but no significant difference werebetween two groups (p>0.05). Bacterial plate counting showed that intracellular CFU inSTM-WT infection group were significantly higher than that in STM-Δspv infectiongroup (p<0.01). After Torin1intervention, intracellular CFU in both groups weredecreased, and the trend was still but smaller (p<0.05). After CQ intervention,intracellular CFU between two groups had no significant difference (p>0.05).Conclusions:1. Construct salmonella-zebrafish infection model which can be used to studyautophagy successfully.2. Autophagic machinery is conserved and zebrafish is also amendable to Torin1and CQ like mammalian cells.3. Salmonella plasmid virulence gene could increase the pathogenicity of the hostbacteria, promote the proliferation and dissemination in vivo, and exacerbate infectionprocess. The spv inhibits autophagy of zebrafish induced by S.typhimurium, and affectsthe early stage of autophagsome formation, prior to degradation at the autolysosome.