Department of Biology, University of Washington, Seattle, Washington, United States of America.
Department of Mechanical Engineering, University of Washington, Seattle, Washington, United States of America.
PLoS Pathog. 2022 Mar 25;18(3):e1010433. doi: 10.1371/journal.ppat.1010433. eCollection 2022 Mar.
The deep-branching eukaryote Giardia lamblia is an extracellular parasite that attaches to the host intestine via a microtubule-based structure called the ventral disc. Control of attachment is mediated in part by the movement of two regions of the ventral disc that either permit or exclude the passage of fluid under the disc. Several known disc-associated proteins (DAPs) contribute to disc structure and function, but no force-generating protein has been identified among them. We recently identified several Giardia actin (GlActin) interacting proteins at the ventral disc, which could potentially employ actin polymerization for force generation and disc conformational changes. One of these proteins, Disc and Actin Associated Protein 1 (DAAP1), is highly enriched at the two regions of the disc previously shown to be important for fluid flow during attachment. In this study, we investigate the role of both GlActin and DAAP1 in ventral disc morphology and function. We confirmed interaction between GlActin and DAAP1 through coimmunoprecipitation, and used immunofluorescence to localize both proteins throughout the cell cycle and during trophozoite attachment. Similar to other DAPs, the association of DAAP1 with the disc is stable, except during cell division when the disc disassembles. Depletion of GlActin by translation-blocking antisense morpholinos resulted in both impaired attachment and defects in the ventral disc, indicating that GlActin contributes to disc-mediated attachment. Depletion of DAAP1 through CRISPR interference resulted in intact discs but impaired attachment, gating, and flow under the disc. As attachment is essential for infection, elucidation of these and other molecular mediators is a promising area for development of new therapeutics against a ubiquitous parasite.
蓝氏贾第鞭毛虫是一种深根系真核生物，是一种细胞外寄生虫，通过一种称为腹盘的基于微管的结构附着在宿主肠道上。附着的控制部分是通过腹盘的两个区域的运动来介导的，这两个区域允许或排除盘下的流体通过。几个已知的盘相关蛋白 (DAP) 有助于盘的结构和功能，但在它们中没有发现产生力的蛋白质。我们最近在腹盘上鉴定了几种贾第虫肌动蛋白 (GlActin) 相互作用蛋白，它们可能利用肌动蛋白聚合来产生力和盘构象变化。这些蛋白之一，盘和肌动蛋白相关蛋白 1 (DAAP1)，在先前显示对附着过程中流体流动重要的盘的两个区域高度富集。在这项研究中，我们研究了 GlActin 和 DAAP1 在腹盘形态和功能中的作用。我们通过共免疫沉淀证实了 GlActin 和 DAAP1 之间的相互作用，并使用免疫荧光在整个细胞周期和营养体附着过程中定位这两种蛋白。与其他 DAP 一样，DAAP1 与盘的关联是稳定的，除非在盘分解的细胞分裂期间。通过翻译阻断抗 sense 吗啉寡核苷酸耗尽 GlActin 会导致附着受损和腹盘缺陷，表明 GlActin 有助于盘介导的附着。通过 CRISPR 干扰耗尽 DAAP1 会导致盘完整但附着、门控和盘下流动受损。由于附着对于感染至关重要，因此阐明这些和其他分子介质是开发针对普遍寄生虫的新疗法的有前途的领域。
