Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis
Drug-Free Approach To Study the Unusual Cell Cycle of Giardia intestinalis
复制标题
DOI：
10.1128/msphere.00384-16
复制
发表时间：
2017-09-01
期刊：
影响因子：
4.8
通讯作者：
Yee, Janet
中科院分区：
文献类型：
--
作者：
Horlock-Roberts, Kathleen;Reaume, Chase;Yee, Janet
贾第鞭毛虫是一种引起贾第虫病的原生动物寄生虫，贾第虫病是一种严重的感染性腹泻。尽管在贾第虫感染期间细胞周期在控制增殖和分化中的重要性，但由于缺乏不会诱导细胞损伤导致伪影的同步程序，因此难以研究该过程。我们利用逆流离心淘洗（CCE），一种基于大小的分离技术，成功地获得了贾第虫培养物中富含G（1）、S和G（2）的组分。与药物诱导的贾第鞭毛虫培养物的同步化不同，CCE不诱导双链DNA损伤或内复制。我们观察到淘析组分中细胞中值体的外观和大小增加，对应于细胞周期从早期G（1）到晚期G（2）的进展。因此，CCE可以用来检查的动力学的中间体和其他结构和细胞器在贾第虫细胞周期。对于细胞周期基因表达研究，通过程序geNorm将肌动蛋白相关基因鉴定为CCE样品逆转录定量PCR（RT-qPCR）分析的最合适标准化物。11个可疑的细胞周期调控基因的CCE组分中的10个在贾第虫中具有类似于高等真核生物的表达谱。然而，这些基因在细胞周期中的RNA水平相差不到4倍到5倍，这可能表明贾第虫不需要基因表达的大变化来调节细胞周期。重要性贾第虫是世界上最常见的肠道原生动物之一，在人类和40多种动物中发现感染。贾第鞭毛虫的生活史在活动滋养体和感染性包囊之间交替。细胞周期的调控控制着贾第鞭毛虫滋养体在活动感染过程中的增殖，并包含着滋养体向包囊分化的限制点。在这里，我们开发了逆流离心淘洗作为一种无药物的方法，以获得贾第虫培养物中富集的细胞从G1，S和G2阶段的细胞周期的馏分。对这些组分的分析表明，细胞没有显示出与用于贾第虫培养物同步化的药物相关的副作用。因此，逆流离心淘洗将推进对贾第虫细胞周期中关键调控事件的研究，并确定潜在的药物靶点，以阻止贾第虫增殖和传播。
Giardia intestinalis is a protozoan parasite that causes giardiasis, a form of severe and infectious diarrhea. Despite the importance of the cell cycle in the control of proliferation and differentiation during a giardia infection, it has been difficult to study this process due to the absence of a synchronization procedure that would not induce cellular damage resulting in artifacts. We utilized counterflow centrifugal elutriation (CCE), a size-based separation technique, to successfully obtain fractions of giardia cultures enriched in G(1), S, and G(2). Unlike drug-induced synchronization of giardia cultures, CCE did not induce double-stranded DNA damage or endoreplication. We observed increases in the appearance and size of the median body in the cells from elutriation fractions corresponding to the progression of the cell cycle from early G(1) to late G(2). Consequently, CCE could be used to examine the dynamics of the median body and other structures and organelles in the giardia cell cycle. For the cell cycle gene expression studies, the actin-related gene was identified by the program geNorm as the most suitable normalizer for reverse transcription-quantitative PCR (RT-qPCR) analysis of the CCE samples. Ten of 11 suspected cell cycle-regulated genes in the CCE fractions have expression profiles in giardia that resemble those of higher eukaryotes. However, the RNA levels of these genes during the cell cycle differ less than 4-fold to 5-fold, which might indicate that large changes in gene expression are not required by giardia to regulate the cell cycle.IMPORTANCE Giardias are among the most commonly reported intestinal protozoa in the world, with infections seen in humans and over 40 species of animals. The life cycle of giardia alternates between the motile trophozoite and the infectious cyst. The regulation of the cell cycle controls the proliferation of giardia trophozoites during an active infection and contains the restriction point for the differentiation of trophozoite to cyst. Here, we developed counterflow centrifugal elutriation as a drug-free method to obtain fractions of giardia cultures enriched in cells from the G1, S, and G2 stages of the cell cycle. Analysis of these fractions showed that the cells do not show side effects associated with the drugs used for synchronization of giardia cultures. Therefore, counterflow centrifugal elutriation would advance studies on key regulatory events during the giardia cell cycle and identify potential drug targets to block giardia proliferation and transmission.