Yang Jichao, Zhang Lihong, Diao Huiyan, Xia Ningbo, Zhou Yanqin, Zhao Junlong, Shen Bang
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
Hubei Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.
Front Microbiol. 2017 Nov 13;8:2210. doi: 10.3389/fmicb.2017.02210. eCollection 2017.
is an important zoonotic pathogen infecting one third of the world population and numerous animals. A key factor to its wide distribution is the ability to interconvert between fast replicating tachyzoites and slowly growing bradyzoites, and to establish lifelong chronic infection in intermediate hosts. Although it is well accepted that stage conversion plays key roles in the pathogenesis and transmission of the parasite, little is known about the molecular mechanisms behind it. Using existing gene expression data from TOXODB and published work, we looked for proteins with novel functional domains and whose expression is up-regulated in the bradyzoite stage, hoping to find molecules that have critical roles in regulating stage conversion and bradyzoite formation. In this study we characterized two such proteins ANK1 and DnaK-TPR, both of which are primarily expressed in bradyzoites and contain novel motifs to mediate protein-protein interactions. Through CRISPR/CAS9 directed gene editing technology, both genes were individually knocked out in type 1 strain TgHB2 and type 2 strain ME49. Disruption of neither of these two genes affected the growth or replication of tachyzoites , consistent with their minimal expression at this stage. However, mutants lacking or displayed modest virulence attenuation during mice infection. Surprisingly, inactivation of neither nor seemed to have a significant impact on bradyzoite differentiation or cyst formation . These results suggest that ANK1 and DnaK-TPR probably do not directly contribute to bradyzoite differentiation, but likely affect other aspects of bradyzoite biology.
是一种重要的人畜共患病原体，感染着世界三分之一的人口和众多动物。其广泛传播的一个关键因素是能够在快速复制的速殖子和生长缓慢的缓殖子之间相互转换，并在中间宿主体内建立终身慢性感染。尽管人们普遍认为阶段转换在该寄生虫的发病机制和传播中起关键作用，但对其背后的分子机制却知之甚少。利用来自TOXODB的现有基因表达数据和已发表的研究成果，我们寻找具有新功能域且在缓殖子阶段表达上调的蛋白质，希望找到在调节阶段转换和缓殖子形成中起关键作用的分子。在本研究中，我们对两种这样的蛋白质ANK1和DnaK - TPR进行了表征，它们都主要在缓殖子中表达，并含有介导蛋白质 - 蛋白质相互作用的新基序。通过CRISPR/CAS9定向基因编辑技术，在1型菌株TgHB2和2型菌株ME49中分别敲除了这两个基因。这两个基因的破坏均未影响速殖子的生长或复制，这与它们在此阶段的低表达一致。然而，缺乏ANK1或DnaK - TPR的突变体在小鼠感染期间表现出适度的毒力减弱。令人惊讶的是，ANK1和DnaK - TPR的失活似乎对缓殖子分化或包囊形成均无显著影响。这些结果表明，ANK1和DnaK - TPR可能不直接促进缓殖子分化，但可能影响缓殖子生物学的其他方面。
