Dolgikh V V, Senderskiĭ I V, Pavlova O A, Timofeev S A, Naumov A M
Parazitologiia. 2012 Nov-Dec;46(6):479-92.
Microsporidia is a large group of fungi-related unicellular parasites with obligate intracellular lifestyle. Unlike other protozoan intracellular parasites (Kinetoplastida and Apicomplexa), most microsporidian species develop in direct contact with the host cell cytoplasm. This fact, acquisition of unique transporters to exploit host metabolic system (alongside the strong minimization of own machinery) and predicted repertoire of microsporidia secretome altogether suggest an active role of parasite proteins in the control of infected cell. Lack of information about secretome of microsporidia intracellular stages is largely due to the methodological difficulties of working with the obligate intracellular parasites. An important problem of such study is the contamination of preparations of host cell cytoplasm by inner (nonsecreted) parasite proteins. Even the homogenization of infected tissue in mild conditions and removal of parasite cells by low-speed centrifugation may result in their partial disruption. We expressed the fragments of three Hsp70 family chaperones from the microsporidium Paranosema (Antonospora) locustae in bacteria Escherichia coli. Immunoblotting with proteins of microsporidia intracellular stages and infected host tissue (locust fat bodies) demonstrated that antibodies against recombinant polypeptides may be used to monitor the integrity of parasite cells during homogenization of infected host tissue and subsequent removal of parasites by centrifugation.
微孢子虫是一大类与真菌相关的单细胞寄生虫，具有专性细胞内生活方式。与其他原生动物细胞内寄生虫（动质体目和顶复门）不同，大多数微孢子虫物种在与宿主细胞质直接接触的情况下发育。这一事实、获取独特转运蛋白以利用宿主代谢系统（同时自身机制大幅简化）以及微孢子虫分泌组的预测组成，都表明寄生虫蛋白在控制受感染细胞方面发挥着积极作用。关于微孢子虫细胞内阶段分泌组的信息匮乏，很大程度上是由于研究专性细胞内寄生虫存在方法上的困难。此类研究的一个重要问题是宿主细胞质制剂会被寄生虫内部（非分泌）蛋白污染。即使在温和条件下对受感染组织进行匀浆，并通过低速离心去除寄生虫细胞，也可能导致它们部分破裂。我们在大肠杆菌中表达了来自微孢子虫蝗虫副 Nosema（Antonospora） locustae 的三个 Hsp70 家族伴侣蛋白的片段。用微孢子虫细胞内阶段的蛋白和受感染宿主组织（蝗虫脂肪体）进行免疫印迹分析表明，针对重组多肽的抗体可用于监测在对受感染宿主组织进行匀浆以及随后通过离心去除寄生虫过程中寄生虫细胞的完整性。
