Dolgikh V V, Senderskiĭ I V, Pavlova O A, Beznusenko G V
Tsitologiia. 2010;52(1):5-11.
Long adaptation of microsporidia, a large group fungi-related protozoa, to intracellular lifestyle has resulted in a drastic minimization of parasite cell. Ultrastructural analysis has shown that the Golgi complex of the microsporidia Paranosema (Antonospora) grylli and P. locustae appears as branching or varicose networks of thin tubules. These tubular networks are connected to endoplasmic reticulum, plasma membrane and forming polar tube but have no vesicles. Vesicles were not found even if ultra-fast cryofixation and membrane fusion/uncoating inhibition were used. However, a limited number of genes involved in vesicular transport were found in microsporidia genomes. In this study we used RT-PCR to analyze the content of mRNA transcripts encoding beta and beta' subunits COPI coatomer complex, Sec13 and Sec31 subunits COPII, SNARE-proteins synaptobrevin and syntaxin-like member of SFT family in P. locustae intracellular stages. The level of expression of studied genes was comparable with that of gene encoding alternative oxidase, enzyme envolved in microsporidia core metabolism. Moreover, polyclonal antibodies raised against recombinant Sec13 subunit COPII, expressed in B Escherichia coli, has shown accumulation of the protein is spores and stages of intracellular development as well as its association with membranes. The presence of components of vesicular transport machinery in avesicular microsporidia cells requires their functional analysis.
微孢子虫是一大类与真菌相关的原生动物，长期适应细胞内生活方式导致其寄生细胞大幅简化。超微结构分析表明，蝗虫微孢子虫（Paranosema (Antonospora) grylli）和蝗虫微粒子虫（P. locustae）的高尔基体呈现为细管分支或静脉曲张样网络。这些管状网络与内质网、质膜相连并形成极管，但没有囊泡。即使采用超快速冷冻固定和膜融合/脱壳抑制方法也未发现囊泡。然而，在微孢子虫基因组中发现了少量参与囊泡运输的基因。在本研究中，我们使用逆转录聚合酶链反应（RT-PCR）分析了蝗虫微粒子虫细胞内阶段编码COP I包被蛋白复合物β和β'亚基、COP II的Sec13和Sec31亚基、SNARE蛋白突触小泡蛋白以及SFT家族类Syntaxin成员的mRNA转录本含量。所研究基因的表达水平与编码交替氧化酶（参与微孢子虫核心代谢的酶）的基因相当。此外，针对在大肠杆菌中表达的重组COP II的Sec13亚基产生的多克隆抗体显示，该蛋白在孢子和细胞内发育阶段积累，并且与膜相关。无囊泡微孢子虫细胞中存在囊泡运输机制成分，需要对其进行功能分析。
