Purpose

Cryptosporidium parvum is an Apicomplexa parasite that causes watery diarrhea (cryptosporidiosis), especially in children and immunocompromised adults (the latter in a very severe form). No effective treatment exists against infection by this parasite. Phosphatases participate in the regulation of various cellular functions and are thus considered potential therapeutic targets in many diseases. The aim of the present study was to indirectly identify and in silico characterize a protein phosphatase 2C of C. parvum.

Methods

Western blot and indirect immunofluorescence microscopy were performed with a polyclonal antibody against Leishmania major PP2C. Possible cross-reactivity with LmPP2C was assessed by in silico sequence homology to analyze phylogenetic relationships between distinct C. parvum PP2Cs. In addition, another bioinformatics approach was used to predict the possible relationship and function of C. parvum PP2C in the regulation of several cellular processes.

Results

Western blotting showed a protein of approximately 72 kDa. With immunofluorescence, PP2C was localized in the nucleus of oocysts (with some additional labeling in the cytoplasm) and at the apical region of sporozoites. By aligning C. parvum PP2C with known ortholog sequences and carrying out PPI analysis, a determination could be made of the degree of conservation of these enzymes, their possible relationship, and their function in the regulation of several cellular processes associated with a likely nuclear location.

Conclusion

Microscopic localization by immunofluorescence identified CpPP2C at the nucleus in oocysts and at the apical end of the sporozoite body. Hence, this enzyme could be associated with proteins that have an important role in the regulation of transcription and other processes orchestrated by MAPK kinases, according to in silico analysis.

中文翻译：

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小球隐孢子虫蛋白磷酸酶2C（PP2C）的细胞鉴定和计算机表征。

目的

隐孢子虫是一种寄生虫，会引起水样腹泻（隐孢子虫病），特别是在儿童和免疫功能低下的成年人（后者的病情很严重）中。没有有效的方法可以抵抗这种寄生虫的感染。磷酸酶参与各种细胞功能的调节，因此被认为是许多疾病的潜在治疗靶标。本研究的目的是间接识别和在计算机芯片上表征的蛋白磷酸酶2C微小隐孢子虫。

方法

用针对主要利什曼原虫PP2C的多克隆抗体进行蛋白质印迹和间接免疫荧光显微镜检查。通过计算机序列同源性评估与Lm PP2C可能的交叉反应，以分析不同的小隐隐线虫PP2C之间的系统发生关系。此外，另一种生物信息学方法被用来预测小球藻PP2C在调节几个细胞过程中的可能关系和功能。

结果

Western印迹显示约72kDa的蛋白质。通过免疫荧光，PP2C定位在卵囊核（在细胞质中有一些附加标记）和子孢子的顶端区域。通过将小球藻PP2C与已知的直系同源序列进行比对并进行PPI分析，可以确定这些酶的保守程度，它们的可能关系以及它们在调控与可能的核位置相关的几个细胞过程中的功能。

结论

通过免疫荧光的显微定位在卵囊核和子孢子体的顶端鉴定了Cp PP2C。因此，根据计算机分析，该酶可能与在转录调控和其他由MAPK激酶策划的其他过程中起重要作用的蛋白质相关。