State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, 510642, China.
Parasit Vectors. 2024 Feb 15;17(1):65. doi: 10.1186/s13071-024-06159-y.
Cryptosporidium spp. are common protozoa causing diarrhea in humans and animals. There are currently only one FDA-approved drug and no vaccines for cryptosporidiosis, largely due to the limited knowledge of the molecular mechanisms involved in the invasion of the pathogens. Previous studies have shown that GP60, which is cleaved into GP40 and GP15 after expression, is an immunodominant mucin protein involved in the invasion of Cryptosporidium. The protein is highly O-glycosylated, and recombinant proteins expressed in prokaryotic systems are non-functional. Therefore, few studies have investigated the function of GP40 and GP15.
To obtain recombinant GP40 with correct post-translational modifications, we used CRISPR/Cas9 technology to insert GP40 and GP15 into the UPRT locus of Toxoplasma gondii, allowing heterologous expression of Cryptosporidium proteins. In addition, the Twin-Strep tag was inserted after GP40 for efficient purification of GP40.
Western blotting and immunofluorescent microscopic analyses both indicated that GP40 and GP15 were stably expressed in T. gondii mutants. GP40 localized not only in the cytoplasm of tachyzoites but also in the parasitophorous vacuoles, while GP15 without the GPI anchor was expressed only in the cytoplasm. In addition, a large amount of recTgGP40 was purified using Strep-TactinXT supported by a visible band of ~ 50 kDa in SDS-PAGE.
The establishment of a robust and efficient heterologous expression system of GP40 in T. gondii represents a novel approach and concept for investigating Cryptosporidium mucins, overcoming the limitations of previous studies that relied on unstable transient transfection, which involved complex steps and high costs.
隐孢子虫属是常见的引起人类和动物腹泻的原生动物。目前仅有一种 FDA 批准的药物，没有针对隐孢子虫病的疫苗，这主要是由于对病原体入侵涉及的分子机制了解有限。先前的研究表明，GP60 在表达后被切割成 GP40 和 GP15，是一种参与隐孢子虫入侵的免疫显性粘蛋白蛋白。该蛋白高度 O-糖基化，在原核系统中表达的重组蛋白无功能。因此，很少有研究调查 GP40 和 GP15 的功能。
为了获得具有正确翻译后修饰的重组 GP40，我们使用 CRISPR/Cas9 技术将 GP40 和 GP15 插入弓形虫的 UPRT 基因座，允许异源表达隐孢子虫蛋白。此外，在 GP40 后插入 Twin-Strep 标签，以有效纯化 GP40。
Western blot 和免疫荧光显微镜分析均表明 GP40 和 GP15 在弓形虫突变体中稳定表达。GP40 不仅定位于速殖子的细胞质中，也定位于寄生泡中，而没有 GPI 锚的 GP15 仅在细胞质中表达。此外，使用 Strep-TactinXT 可大量纯化 recTgGP40，SDS-PAGE 上可见约 50 kDa 的可见带。
在弓形虫中建立稳定高效的 GP40 异源表达系统代表了一种研究隐孢子虫粘蛋白的新方法和概念，克服了以前依赖不稳定瞬时转染的研究的局限性，该方法涉及复杂的步骤和高昂的成本。
