缺氧诱导因子1α对不同毒力刚地弓形虫从速殖子到缓殖子转化的影响

Effects of hypoxia—inducible factor 1α on transformation from tachyzoites into bradyzoites of Toxoplasma gondii with differing levels of virulence

目的研究缺氧诱导因子1α(HIF1α)在3种弓形虫虫株由速殖子到缓殖子转化过程中的作用,阐释HIF1α对弓形虫不同毒力虫株转化的影响。方法选取3种不同毒力弓形虫虫株(Ⅰ型RH-GFP,Ⅱ型ME49-GFP和Ⅲ型VEG),分别感染野生型HIF1α细胞(HIF1αWT)和缺陷型HIF1α细胞(HIF1αKO),在碱性条件下进行诱导转化,通过免疫荧光试验(Immunofluorescence assay,IFA)检测不同条件下弓形虫诱导转化情况,通过细胞中RH-GFP和ME49-GFP荧光值的变化分析Ⅰ型和Ⅱ型弓形虫的转化水平。无荧光标记的Ⅲ型VEG虫株选用速殖子特异性表达蛋白SAG1作为弓形虫速殖子阶段的特异性标记。将包裹弓形虫缓殖子囊肿外层的囊壁作为缓殖子的特异性识别标记,用DBA抗体识别,将封片的载玻片置于荧光显微镜下观察并计数。将玻片上总量为300个被弓形虫感染的细胞分为3组:(1)只含有速殖子(GFP);(2)只含有缓殖子(DBA);(3)同时含有速殖子和缓殖子(GFP+DBA)。每个实验条件至少分别计数3张玻片并计算其平均值。结果最佳诱导转化时间为诱导后96h,Ⅰ型RH-GFP弓形虫感染的HIF1αKO细胞中均不包含DBA和GFP+DBA的细胞,而只含有速殖子GFP蛋白标记,RH-GFP感染的HIF1αWT细胞中偶见极少数速殖子到缓殖子的中间转化标识GFP+DBA;Ⅱ型ME49-GFP弓形虫感染的HIF1αKO细胞中无诱导转化,而在HIF1αWT细胞中有20%~25%只含有DBA的细胞,30%左右只含有GFP的细胞,其他大部分细胞均处于转化中期。Ⅲ型VEG弓形虫感染的HIF1αKO细胞中无诱导转化,而大部分HIF1αWT细胞中的VEG发生了速殖子到缓殖子的转化,完全转化为DBA的比例为55%~60%。结论 HIF1α是弓形虫虫株从速殖子转化为缓殖子的必要条件,且虫株转化速率与虫株毒力成反向调控。

Objective To study the role of hypoxia-inducible factor 1α(HIF1α)in the transformation of Toxoplasma gondii from tachyzoites into bradyzoites and to explain the effect of HIF1α on the transformation of strains of T.gondii with different levels of virulence. Methods Toxoplasma strains with different levels of virulence(typeⅠ RH-GFP,type Ⅱ ME49-GFP,and type Ⅲ VEG)were used.Wild-type HIF1α cells(HIF1α WT)and defective HIF1αcells(HIF1αKO)were infected,and the transformation of T.gondii was detected using an immunofluorescence assay(IFA)under alkaline conditions.The extent of transformation of T.gondii typeⅠ and type Ⅱ was directly analyzed based on changes in RH-GFP and ME49-GFP fluorescence in infected cells.The tachyzoite-specific expression protein SAG1 was used as a specific marker for the tachyzoite stage of T.gondii in type Ⅲ VEG strains without fluorescent markers.In addition,the cyst wall of bradyzoite cysts was used as a specific indicator of bradyzoites.DBA antibody was used to detect bradyzoites.A sealed slide was observed under a fluorescence microscope.The total number of cells infected with T.gondii was divided into three groups on the slide:(1)tachyzoites(GFP)only;(2)bradyzoites(DBA)only;and(3)both tachyzoites and bradyzoites(GFP+ DBA).At least three slides were counted and their average values were calculated for each experimental condition.Results The optimum time for inducing transformation was 96 hours after induction.The HIF1αKO cells infected with T.gondii typeⅠ RH-GFP did not contain DBA or GFP+DBA and had tachyzoite GFP protein markers.A few tachyzoites were found in HIF1α WT cells infected with RH-GFP according to the indicators of intermediate transformation GFP+DBA.Transformation was not induced in HIF1αKO cells infected with T.gondii type Ⅱ ME49-GFP.Only 20-25%of HIF1α WT cells contained DBA,and about 30%contained GFP;and other cells were in the middle stages of transformation.Transformation was not induced in HIF1α KO cells infected with T.gondii type Ⅲ.In most HIF1α WT cells,VEG tachyzoites were transformed into bradyzoites,and the proportion of cells containing DBA that were completely transformed was about 55-60%. Conclusion HIF1αis a necessary condition for T.gondii strains to transform from tachyzoites into bradyzoites,and the rate of transformation of T.gondii strains is inversely regulated with the virulence of T.gondii strains.