The Molecular Mechanism Of Salmonella Plasmid Virulence Gene SpvB Inhibit Autophagy And The Effect On Infected Zebrafish Model

Objective: Salmonella enterica serovar typhimurium(S. typhimurium) is a pathogenic bacterium, which parasitizes the intestinal tract of human and animals and can cause diseases, mainly spreading by contaminated food and water. Salmonella plasmid virulence gene is closely related to the bacterial virulence. The virulence phenotype is mediated by three regions, including regulatory gene spv R, effector genes spv B and pv C. As reported that spv B product has the activity of ADP-ribosylation transferase to nduce the cytoskeleton depolymerization. Our preliminary studies found that spv B nhibited the formation of autophagosomes, but the molecular mechanism is unclear.In this study, infection model was firstly established in human cervical epithelial cells He La cells(human epithelial cell line), through detection of autophagy flux and the interaction of autophagosome formation with cytoskeleton, to study the molecular mechanisms that spv B influenced the formation of autophagosomes. Secondly, the model of infected zebrafish was set up to study the effect of spv B on autophagy and infection process after intestinal epithelial cells infection with Salmonella in vivo, to reveal the function of Salmonella virulence genes and provide new way for treatment and control of intestinal infectious diseases.Methods:Part One. The molecular mechanism of Salmonella plasmid virulence gene spv B inhibits autophagy.Wild-type S. typhimurium strain STM-WT(carrying the spv B genes), spv B mutatedstrain STM-Δspv B(mutated spv B) and STM-c-Δspv B(Δspv B-complemented strain) were co-cultivated with He La cells, construct infection model in vitro. The following experiments were conducted:1. To observe the cellular ultrastructure by transmission electron microscope The cells were harvested after 1 h and 2 h, Ultra-thin sections were made to observe the ultrastructure of cells infected with different bacteria under transmission electron microscopy.2. To observe LC3 puncta by confocal laser scanning microscopy He La cells transfected with m RFP-GFP-LC3 plasmid, were co-incubated with bacteria for 1 h, to observe LC3 puncta by confocal laser microscopy. After pretreatment with rapamycin and Bafilomycin A1, the number of yellow dots LC3 was compared in the cells infected with different bacteria. Co-localization of bacteria with cytoskeleton and autophagy proteins was observed by confocal laser microscopy. The cells were pretreated with rapamycin and Bafilomycin A1. Then the number of yellow dots of LC3 and the change of drug intervention in different infection group were compared.3. To observe the co-localization of bacteria with cytoskeleton and autophagy proteins by confocal laser microscopy Cells were seeded in a 24-well plate with a circular coverslip inside, and cultured to the logarithmic phase, followed by transfected with EGFP-Beclin-1, EGFP-Atg14 and p GFP-ZFYVE 1 plasmid. Transfected cells were co-incubated with bacteria for 1 h, and subjected to staining with Phalloidin-Rhodamine, anti-Salmonella O-Ag serum, and anti-rabbit Ig G-Dy Light 405, followed by observation of the co-localization bacteria with cytoskeleton and autophagy protein under confocal laser microscope.4. Bacteria co-localization with p62 were viewed under fluorescence microscopeThe cells were harvested after infection 1 h. The bacteria with p62 protein localization in infection group after the antibody incubation were observed under fluorescence microscope.5. The level of autophagy related proteins and p70S6 K were determined by Western blotThe cells were harvested after infection 1 h and 2 h. The protein immunoblot(Western blot, WB) method was used to detect the expression level of autophagy related proteins LC3, p62 and Beclin 1 with or without RAPA and Baf drug intervention.The cells were harvested after infection 1 h and 3 h. The expression level of p70S6 K protein which is downstream of m TOR signaling pathways were determined by Western blot. Analysis the changes of phosphorylation expression level between different infection groups.6. The intracellular bacterial survival was detected by plate count methodThe cells were harvested after infection 30 min, 1 h, 3 h and 5 h, calculated cell colony forming unit inside the cell. The number of intracellular bacteria was recorded by colony counting method in different infection groups.Part Two. The effect of Salmonella plasmid virulence gene spv B on autophagy and intestinal lesions in zebrafish infection modelSince the expression covering of STM-c-Δspv B need to be induced by L-Arab sugar, and it was not suitable in vivo, we chose STM-WT and STM-Δspv B as experimental strains in the second part.1. Constructed the intestinal infection model in ZebrafishSelected 5 days post-fertilization zebrafish larvae, diluted bacteria with Holtfreter buffer to 1×109 cfu/ml, 1×108 cfu/ml, 1×107 cfu/ml, 1×106 cfu/ml, 1×105 cfu/ml, 1×104 cfu/ml. Zebrafish larvae were immersed after 6 hours infection, then put them in Holtfreter buffer, counted zebrafish mortality within 7 days, drawed a survival curve and calculated median lethal dose(LD50) to determine the appropriate concentration and the influence of zebrafish to survive at different strains.2. The invasion and spread of the bacteria was detected by plate count method in zebrafishCollected the zebrafish larvae after 6 h, 24 h, 72 h and 120 h infection, calculated the bacteria in the whole zebrafish and performed amikacin exclusion assay. Whole-larvae CFU and intracellular CFU were compared to detect the spread of bacteria and reproduction ability.3. The level of autophagy related proteins in zebrafish were determined byWestern blotZebrafish larvae were collected which were infected by STM-WT or STM-Δspv B at different times, the level of autophagy related proteins LC3、Beclin-1 and P62 were determined by Western blot. Comparison of the autophagy level changes in zebrafish with extension of time after the zebrafish infected with Salmonella typhimurium(STM-WT and STM-Δspv B). The change of zebrafish intestines ultrastructure at 72 h post infection was observed by transmission electron microscopy.4. The pathological changes and level of apoptosis were measured after zebrafish infectionThe changes of zebrafish intestines were observed by optical microscope and HE dyeing to compare the influence of bacteria at different time on zebrafish intestines. The level of apoptosis related protein Bcl-2 was determined by Western blot and its m RNA transcriptional level was detected by real-time fluorescent quantitative PCR.5. The inflammation analysis in zebrafish infection modelZebrafish infected with STM-WT and STM-Δspv B after 6 h, 24 h, 72 h and 120 h were collected to analyze the effect of bacteria on inflammation response. q RT-PCR was used to detect the levels of TNFα,IL-1β,IL-22 and IL-10. Neutral red staining method was used to detect the function of macrophage phagocytosis in zebrafish with the extension of infection time in different infection groups.Results:Part One. The molecular mechanism of Salmonella plasmid virulence gene spv B inhibits autophagy.1. spv B inhibited autophagosome formation: The observation of the cellular ultrastructure by transmission electron microscope showed typically autophagosomes(double-membrane structure) containing undigested bacteria in He La cells infected with STM-Δspv B at 1 h. The autophagosomes subsequently fused with lysosome, and the single-membrane autolysosomes containing STM-Δspv B and partially digested cytoplasmic materials were observed at 2 h post infection. Salmonella-containingvacuoles and intracellular bacteria increased could be observed in STM-WT and STM-c-spv B infection groups.2. spv B inhibited autophagosome formation at the early stages: It was observed that cells had visible yellow LC3 puncta infected by STM-Δspv B, while cells showed less LC3 puncta infected by STM-WT and STM-c-spv B groups. RAPA induced autophagy before infection with STM, the numbers of both yellow(autophagosomes) and red(autolysosomes) puncta in STM-Δspv B infected cells were significantly increased. Baf was used to inhibit the fusion of autophagosome with lysosome, most punctae in STM-Δspv B infected cells are yellow(autophagosome) without a concomitant increase in red puncta.3. spv B influenced the expression of autophagy related proteins: the level of p62 infected with STM-WT and STM-c-spv B was significantly higher than that in which infected by STM-Δspv B. Consistent with the level of p62, both LC3 turnover and Beclin-1 expression increased in STM-Δspv B infected cells, and the treatment of RAPA increased LC3 turnover and Beclin-1 expression in all infected groups, while Baf significantly increased the accumulation of p62 in cells infected with STM-Δspv B. These results indicated that spv B inhibited autophagy at the stage of autophagosome formation in host cells.4. spv B inhibitd p62 positioning with intracellular bacteria: p62 positive bacteria were observed in cells infected by STM-Δspv B, although only a small portion of the intracellular bacteria were targeted. In contrast, few p62 positive Salmonella was observed in other two groups.5. spvB interferes with the initial stage of autophagy-targeting STM by depolymerization of actin cytoskeleton: In cells transfected with GFP-Beclin-1, as well as GFP-ATG14 and GFP-ZFYVE 1, positive structures were frequently co-localized with actin fibers. A few of the intracellular STM-Δspv B bacteria were co-localized with actin fibers. Few F-actin fibers were observed in STM-WT and STM-c-spv B. As a result, more diffused fluorescent protein was observed in these cells. Although more intracellular bacteria were detected in cells infected with STM-WT and STM-c-spv B,the intracellular bacteria were sporadically captured by fluorescent protein.6. spv B suppressed autophagy via m TOR signaling pathway in host cells: Phosphorylation of p70S6 K was downregulated upon STM infection, indicating that the autophagy pathway was activated by STM infection. While the activation of p70S6 K was completely abrogated in He La cells infected with STM-Δspv B after infection 3 h. These results suggested that spv B suppressed autophagy via m TOR signaling pathway in host cell.7. spvB could promote survival of bacteria in host cells: Results of plate counting showed that the number of STM-WT and STM-c-Δspv B multiplied significantly in infected He La cells than STM-Δspv B infected cells.Part Two. The effect of Salmonella plasmid virulence genes spv B on autophagy and intestinal lesions in zebrafish infection model1. The establishment of zebrafish intestinal infection model: A record within 7 d infected with different doses of zebrafish showed that the death of zebrafish decreased with the reduced of infection concentration and increased mortality with longer duration of infection. Using Reed-Muench method to calculate the pathogenicity of zebrafish infected by STM-WT and STM-Δspv B, result showed that the LD50 of STM-WT was 1.33×106cfu/ml and the LD50 of STM-Δspv B was 2.22×106cfu/ml. It indicated that spv B could significantly reduce the LD50 of zebrafish infected Salmonella.2. The observation of the state of spread and reproduction of STM in zebrafish: Results showed that the bacteria in zebrafish increased exponentially with the infection time. Bacteria were all reduced in each group after added Amikacin. The amount of bacteria in zebrafish at 72 hpi was higher in the group of STM-WT infection than the group of STM-Δspv B infection. And this difference was more obvious at 120 hpi. This result indicated that spv B could promote bacteria invasion and enhanced the ability of bacteria to reproduce.3. The detection of the effect of spvB on autophagy of zebrafish infected by Salmonella: Results showed that the expression level of Beclin-1 was obviously higher in the group of STM-Δspv B infected zebrafish than that of STM-WT infection group.And the autophagy substrate protein p62 expression level was significantly lower in STM-Δspv B infected group. Autophagosomes could be observed under transmission electron microscope at 72 hpi in this group. However, Salmonella invaded zebrafish intestinal epithelial mucous membrane layer in STM-WT infection group could be seen, and the intestinal microvilli arranged disorder, even appeared falls off phenomenon and intestinal columnar epithelium cell structure was not clear.4. The observation of morphological changes in zebrafish intestines after Salmonella infection and the measurement of the level of apoptosis. By using ordinary optical microscope, it found that with the extension of infection time, zebrafish intestinal was narrowed, contents blurred and inner lumen area reduced in the wild strains of STM-WT infection group. However, zebrafish intestinal lesions were less in the mutant strains infection group and the arrangement of intestinal epithelium microvilli was ordered with the extension of zebrafish infection time, emerged a large number of inflammatory cells infiltration in 120 hpi. The intestinal lumen lesion was lightened in STM-Δspv B infection group than in STM-WT infection group, while different degrees of lumen stenosis and inflammatory cells infiltration could also be observed. The level of Bcl-2 was significantly lower in STM-WT infection group at 120 hpi.5. Detection of the inflammation responses in zebrafish after infect with Salmonella: Real-time fluorescent quantitative PCR was used to analysis the transcriptional level of TNFα, IL-1β, IL-22 and IL-10 in zebrafish. The results showed that the above m RNA transcriptions were increased with the extension of time in STM-WT infection group. There was no significant difference in STM-WT and STM-Δspv B infection groups at 6 h post infection. The expression of inflammation cytokines was significantly lower in STM-Δspv B infection group than in STM-WT infection group at 120 hpi. Salmonella infection induced innate immune response in zebrafish. Neutral red staining method was used to detect the ability of macrophage phagocytosis after zebrafish infected. The results showed that there was no significant difference in ability of macrophage phagocytosis in zebrafish between STM-WT and STM-Δspv B infection group.Conclusions:1. spv B inhibited the formation of autophagosomes of host cells bdepolymerization of actin cytoskeleton.2. spv B suppressed autophagosomes formation via activating m TOR signaling pathway in host cells.3. spvB enhanced bacterial virulence by affecting autophagy process and inflammation response in zebrafish infection model.