Department of Parasitology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.
Department of Infectious Diseases, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Front Cell Infect Microbiol. 2021 Dec 17;11:794152. doi: 10.3389/fcimb.2021.794152. eCollection 2021.
is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress . These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.
是导致人类阿米巴痢疾和肝脓肿的病原体。这种原生动物的寄生生活方式和毒力需要精细的生物学过程，包括囊泡运输和对宿主免疫反应产生的各种活性氧和氮物种的应激管理。尽管已经在分子水平上研究了代表性毒力因子的细胞内运输机制，但仍不清楚细胞内运输是否以及如何参与对活性氧和氮物种的防御。在这里，我们通过对两种同源菌株（一种是经过动物传代的高毒力 HM-1:IMSS Cl6，另一种是维持减毒的非致病株）的比较转录组分析，鉴定了参与囊泡运输调节的 Arf 家族 GTPase 之一 EhArfX2。EhArfX2 被鉴定为高毒力菌株中上调最明显的基因之一。EhArfX2 定位于小囊泡样结构，与跨高尔基网络 SNARE 的标记物 EhYkt6 大量共定位，但与内质网（ER）驻留伴侣 EhBip 或顺式高尔基 SNARE EhSed5 和高尔基腔糖基转移酶 EhGalT 均不共定位。EhArfX2 的显性激活突变体形式的表达导致溶酶体数量增加，而显性负突变体的表达导致溶酶体形成和半胱氨酸蛋白酶向溶酶体的运输缺陷。在毒力菌株中表达显性负突变体导致仓鼠模型中肝脓肿的大小减小。这种肝脓肿形成缺陷至少部分归因于对硝化应激的抗性降低，而不是氧化应激降低。这些结果表明，EhArfX2 介导的运输对于宿主中的硝化应激反应和毒力是必要的。
