| Objective:To construct Salmonella typhimurium mutant with plasmid virulence gene spv C knock-out(STM-Δspv C) and spv C complement(STM-c-spv C) strain by λRed recombination system and prokaryotic expression vector p BAD/gⅢ, respectively. Moreover, to investigate the effect of spv C gene on infection process and autophagy using zebrafish and He La cell infection model, and to explore the possible signal pathway so as to provide new ideas and methods for prevention and control of Salmonella infection.Methods:Part One. Construction mutant of Salmonella typhimurium with spv C gene.1. Construction of STM-Δspv C by λRed recombination systemAccording to the downstream sequence of spv C, primers containing spv C homologous sequences at 5 ’end were designed. The linear DNA fragments containing spv C homology arms with kanamycin resistance gene in the middle of it was amplified using the plasmid p KD4 as template. The STM-WT strain transfered with plasmid p KD46 had the ability of recombination after induced by L-arabinose. The linear DNA fragments were transformed into competent STM-WT strain carrying plasmid p KD46 by electroporation, and the positive transformants were selected by LB plate containing kanamycin. The plasmid p CP20 expressing Flp recombinase was used to eliminate the kanamycin resistance gene which was present between the FRT sites. He La cells were infected by both STM-wild type(STM-WT) and STM-Δspv C, Western Blot was used to detect the phosphorylation levels of ERK in He La cells that infected by Salmonella strain.2. Construction of STM-c-spv C with prokaryotic expression vector p BAD/gⅢAccording to spv C sequence in Gene Bank, designed specific spv C-F(Xho I) and spv C-R(Eco RI) primers, amplified the spv C DNA fragments using the STM-WT genome DNA as templates. spv C DNA fragments and prokaryotic expression vector p BAD/gⅢ were digested by DNA restriction enzymes Xho I and Eco RI respectively, products were then ligated by T4 DNA ligase. The recombinant plasmid was transformed into DH5α E. coli, positive clones were selected by PCR and DNA sequencing. The positive recombinant plasmids were transformed to STM-Δspv C, bacteria were collected to extract proteins after induction of L-arabinose and detected the expression Spv C protein by Western Blot.Part Two. The influence of spv C on zebrafish infection and autophagy.STM-WT and STM-Δspv C were used to establish zebrafish infection model. Zebrafish larvae at 5 days post fertilization were immersed in water containing bacteria with a final concentration of 108 CFU/ml. After 6 h post infection(hpi), larvae were washed and transferred to individual 24-well microtiter plate containing fresh autoclaved Holtfreter buffer and incubated in sterile conditions. Zebrafish were collected for experiments at 6, 24, 72, 120 hpi.1. The morphology of zebrafish intestinal tract was observed by light microscopeZebrafish were collected at different time points, washed 3 times with sterile PBS, and the morphology of zebrafish intestinal tract was observed by light microscope.2. The number of Salmonella in zebrafishThe numbers of bacteria in the whole zebrafish larvae and zebrafish cells(amikacin was used to kill extracellular bacteria) were calculated.3. Observation of ultrastructure of zebrafish intestinal epithelia by transmission electron microscopeZebrafish were collected at 72 hpi, ultrastructure of zebrafish intestinal epithelia in different infection groups and the formation of autophagosomes were observed by transmission electron microscope(TEM).4. Evaluation of Beclin 1 and turnover of Lc3 by Western BlotCollected the zebrafish at different infection points, and the zebrafish protein were extracted to detect the expression of Lc3 and Beclin 1 by Western Blot.Part Three. The influence of spv C on autophagy of He La cells and the signal pathway.STM-WT, STM-Δspv C and STM-c-spv C were used to infect He La cells by multiplicity of infection(MOI) 100:1. Besides, established the He La cells infection model with ERK inhibitor(PD0325901) intervention, and infected cells were collected at 0.5, 2 and 6 hpi for experiments.1. Observation of bacteria inside He La cells by Wright-Giemsa stainHe La cells were collected at different time, washed three times with PBS, fixed with methanol for 10 min, dried and stained by Wright-Giemsa solution. Observed the the bacterial invasion and proliferation as well as the morphology of different infected cells.2. Determination of the proper concentration of ERK inhibitor PD0325901He La cells were pretreated for 24 h with a series of concentrations of ERK inhibitor(PD0325901) varied from 0, 5, 10, 20, 50, to 100 n M. The phosphorylation level of ERK and cell viability of He La cells were detected by Western Blot and methyl thiazolyl tetrazolium(MTT), respectively.3. Intracellular colony-forming unit(CFU) countInfected He La cells were collected and the numbers of intracellular CFU were measured by plate counting.4. Detection of the level of LC3 and Beclin 1 by Western BlotHe La cells were harvested and Western blot was used to detect the expression levels of autophagy related proteins LC3 and Beclin 1.5. Bacteria co-localization with p62 were detected by immunofluorescenceHe La cells were harvested at 2 hpi, p62 protein was labeled by antibody with green fluorescent, cell nucleus and bacteria were labeled by hochest, the co-localization of bacteria and p62 in different infection groups were observed by confocal laser scanning microscope(CLSM).6. Observation of LC3 puncta by CLSMThe He La cells which were stability transfected with m RFP-GFP-LC3 plasmid were used to establish infection model. He La cells were collected at 2 hpi, bacteria and cell nucleus were labeled by hochest, the LC3 puncta as well as the co-localization of bacteria and LC3 in different infection groups were observed by CLSM.Results:Part One. Construction mutant of Salmonella typhimurium with spv C gene1. Construction of STM-Δspv C by λRed recombination systemPCR analysis showed spv C gene was knocked out, the level of phosphorylation ERK in He La cells infected with STM-Δspv C was detected by Western Blot, which confirmed the successful construction of spv C knocked out strain.2. Construction of STM-c-spv C with prokaryotic expression vector p BAD/gⅢThe results of PCR and DNA sequencing demonstrated the construction of the recombinant plasmid p BAD-spv C, the expression of Spv C protein was induced by L-arabinose and confirmed by Western Blot analysis after p BAD-spv C transformed into STM-Δspv C strain.Part Two. The influence of spv C on Zebrafish infection and autophagy1. The morphology of zebrafish intestinal tract was observed by light microscopeThe zebrafish infected with STM-WT and STM-Δspv C both had narrower intestinal lumen with the extension of infection time, but it was more serious in the STM-WT infection group, which had intestinal lumen with blurred contents and decreased intestine area.2. The number of Salmonella in zebrafishThe number of bacteria increased significantly in both STM-WT and STM-Δspv C infection groups with the extension of infection time(P < 0.001). The amount of intracellular bacteria in STM-WT infection group was significantly higher than STM-Δspv C infection group at 72 hpi(P < 0.05), and the difference was more pronounced at 120 hpi(P < 0.001).3. Observation of ultrastructure of zebrafish intestine epithelia by TEMAt 72 hpi, the zebrafish infected with STM-WT displayed irregular arrangement of intestinal microvilli and more bacteria within the intestinal lumen; while the zebrafish infected with STM-Δspv C had regular and normal microvilli, and less bacteria within the intestinal lumen. Besides, in the STM-WT infection groups, autophagosome wrapped by double membrane could be observed in intestinal epithelial cells.4. Evaluation of Beclin 1 and turnover of Lc3 by Western BlotAt 24 hpi, the level of Lc3-II/Lc3-I value and Beclin 1 expression were higher in STM-Δspv C infection group than STM-WT infection group(P < 0.05), and this difference was more obvious at 72 hpi(P < 0.01).Part Three. The influence of spv C on autophagy of He La cells and the signal pathway.1. Observation of bacteria inside He La cells by Wright-Giemsa stainThe morphology of He La cells was relative normal with a few intracellular bacteria at 0.5 hpi. The intracellular bacteria significantly increased at 2 hpi, and cytoplasms of some infected cells were loose and destroyed. More cells were disintegrated at 6 hpi, especially those infected with STM-WT and STM-c-spv C.2. Determination of the proper concentration of ERK inhibitor PD0325901It was showed that the phosphorylation ERK of He La cells was obvious with the intervention of 50 n M PD0325901, and the cell survival rate was about 70%. Thus, 50 n M PD0325901 was taken as the proper concentration of ERK inhibitor.3. Intracellular colony-forming unit(CFU) countThe intracellular bacteria CFU weren’t significant different among STM-WT, STM-c-spv C and STM-Δspv C infected cells at 0.5 hpi. However, the number of intracellular bacteria in STM-Δspv C infected cells was significant less than STM-WT and STM-c-spv C infected cells at 2 and 6 hpi(P < 0.05). When He La cells were pretreated with PD0325901, there were no significant difference among the three infected cells.4. Detection of the level of Lc3 and Beclin 1 by Western BlotThe Lc3-II/Lc3-I value and the expression of Beclin 1 were not significantly different among the infected cells at 0.5 and 6 hpi. While LC3-II/LC3-I value and Beclin 1 expression were increased significantly in STM-Δspv C infected cells at 2 hpi(P < 0.05). However, the increase level was suppressed after PD0325901 treatment.5. The co-localization of bacteria and p62It was shown that p62 puncta and p62 positive bacteria increased in STM-Δspv C infected cells(P < 0.05). While there was no significant difference among different infected cells after PD0325901 treatment.6. Observation of LC3 puncta by CLSMMore yellow and red LC3 puncta were observed in STM-Δspv C infected cells than the STM-WT and STM-c-spv C infected groups, and the number of LC3 positive bacteria in STM-Δspv C infected cells were more than the other two groups(P < 0.05). After PD0325901 treatment, yellow and red LC3 puncta in each infected group were reduced, and there was no significant difference among them.Conclusions:1. The mutants of Salmonella typhimurium with spv C gene including STM-Δspv C and STM-c-spv C were constructed by λ Red recombination system and prokaryotic expression vector p BAD/gⅢ, respectively.2. spv C could enhance bacterial virulence, aggravate infection and intestinal pathological changes in zebrafish, and promote Salmonella proliferation and spread by suppressing autophagy.3. spv C could inhibit autophagy through ERK signaling pathway during the early stage of infection, and ultimately promote intracellular bacterial proliferation in He La cells. |
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