类肌动蛋白MreB在河蟹螺原体形态变化、运动及致病中的功能解析

Spiroplasma is a tiny bacterium without cell wall and with typical spiral morphological character. Its cytockeleton has been most attractive in tiny model organisms. Howerver, the studies on the cytockeleton of spiroplasma are most restricted to morphological observation and speculations deduced by mathematical and physical methods. Biological functions study should be recommended. According to our previous study on the Chinese mitten crab Eriocheir sinensis pathogen Spiroplasma eriocheiris, we found that MreB, which is a primary cytockeleton protein of spiroplasma, has a close relationship with its typical spiral morphologic feature and motility. The present proposal will start with this protein and study on the changes of ultrastructural features of cytoskeleton and the role of MreB in different phenotypes of spherical and spiral of S. eriocheiris by applying mass spectrometry, light and electron microscopy, western blot and molecular methods so on. Then, probe into the relationship between the cytockeleton and motility of S. eriocheiris using the inhibitor A22 and accelerant phalloidine respectively. With the help of CRISPR/Cas9 and homologous recombination, 5 MreB genes will be knockout one by one or multiple combination knockout and then will be compared with the rescued strain and the wild strain respectively for studying the MreB functions in morphologic change and motility. At the end, these three different strains will be tested their pathogenicity in vitro on primary cultured crab hemolymph cells as well as in vivo by inoculating into healthy crabs. From above all, we can reveal the roles of cytoskeleton protein MreB (including 5 homologs) in morphologic change, motility and pathogenicity of S. eriocheiris.

微小、无细胞壁、具螺旋结构的螺原体作为最具吸引力的小型模式生物，其细胞骨架是近年研究热点，但相关研究只局限在形态学研究及数理方法推导，缺乏生物学功能研究。前期研究发现河蟹螺原体Spiroplasma eriocheiris骨架蛋白MreB与其螺旋结构和运动密切相关，本项目从该蛋白入手，通过质谱分析、光镜、免疫电镜及分子生物学等技术，研究S. eriocheiris在螺旋形和球形两种表型下的骨架超微结构特征及MreB变化规律；用MreB特异性抑制剂A22和促进剂鬼笔环肽探讨MreB与形态和运动的关系；分别用CRISPR/Cas9和同源重组技术进行5个MreB基因的单个和多个组合敲除，并与野生菌株和回补菌株比较，验证MreB在细胞形态变化和运动中的功能及5个MreB同系物是如何组合并行使功能；同时在体外和体内水平上比较确证以上菌株的致病性，最终揭示MreB在螺原体细胞形态、运动及致病中的作用。

本项目聚焦微小、无细胞壁、具螺旋结构的螺原体细胞骨架作为研究对象，首先研究了渗透压、鬼笔环肽和A22影响河蟹螺原体形态和MreB蛋白表达的影响，螺原体在PBS中呈现典型的螺旋形，但在水中呈现球形，与螺旋形相比球形时螺原体MreB表达量显著降低。螺旋形螺原体的骨架与其形态一致也是长的螺旋形状，而球形螺原体的骨架表现为一个圆环状。MreB在螺原体的细胞膜外表面有分布。鬼笔环肽处理后螺原体长度明显增长，而A22处理的螺原体长度明显缩短。A22处理后MreB4表达量显著降低。然后研究了鬼笔环肽和A22对螺原体致病性的影响，发现A22能够显著延缓螺原体的生长速度，螺原体出现破碎和死亡现象，使中华绒螯蟹的死亡率降低。鬼笔环肽或A22处理螺原体后影响血淋巴细胞的形态、细胞活力、细胞PO活性、细胞坏死和凋亡。以上研究表明，MreB促进剂和抑制剂的作用下，螺原体的致病力发生了变化，鬼笔环肽处理后螺原体的致病力显著增强，而A22处理后螺原体致病力显著降低。本项目原计划与日本大阪市立大学生物学系Makoto Miyata (宫田真人)教授实验室进行合作对螺原体Mreb蛋白在运动中的功能进行研究，但是由于疫情的影响，该部分的研究延期执行；本项目还计划利用CRISPR/Cas9、同源重组等技术构建MreB的敲除株和回补株，但在项目实施过程中经过很多次尝试，这些技术在螺原体上的应用都以失败告终，在将来的研究中本实验室会继续进行尝试，以促进螺原体的基础研究。本项目新增一些研究内容，如低氧对螺原体致病性的影响，结果表明，低氧胁迫下中华绒螯蟹死亡率升高、组织被破坏、易被螺原体感染，同时血淋巴细胞的细胞凋亡增加，预示着低氧加速螺原体对河蟹的感染。

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