Warschkau David, Klein Sandra, Schadt Ella, Doellinger Joerg, Schares Gereon, Seeber Frank
FG16: Mycotic and Parasitic Agents and Mycobacteria, Robert Koch Institute, 13353 Berlin, Germany.
Humboldt-Universität zu Berlin, Department of Biology, 10099 Berlin, Germany.
FEMS Microbes. 2024 Dec 23;6:xtae034. doi: 10.1093/femsmc/xtae034. eCollection 2025.
The apicomplexan parasite has a complex life cycle. Access to sexual stages and sporozoite-containing oocysts, essential for studying the parasite's environmental transmission, is limited and requires animal experiments with cats. Thus, alternatives and resource-efficient methods are needed. Several molecular factors and transcriptional switches responsible for differentiation have been identified in recent years. In tachyzoites, drug-induced inhibition of the histone deacetylase HDAC3, or genetic depletion of transcription factors regulating HDAC3, leads to the expression of genes that are specific to sexual stages and oocysts. Here, we applied this concept and showed that the commercially available HDAC3 inhibitor apicidin could be used to identify the hitherto unknown antigen of the sporozoite-specific monoclonal antibody G1/19 in tachyzoites. Using mass spectrometry of immunoprecipitated G1/19 target protein from apicidin-treated cultures, we identified it as SporoSAG. In addition, for the much less abundant sporozoite-specific protein LEA860, apicidin treatment was still sufficient to induce a detectable protein level in immunofluorescence microscopy. We also discuss further applications and the limitations of this approach. This allows to overcome issues with the paucity of material of sexual stages and oocysts from to some extent without the need for cat-derived material.
顶复门寄生虫具有复杂的生命周期。获取对于研究寄生虫环境传播至关重要的有性阶段和含子孢子的卵囊受到限制，并且需要用猫进行动物实验。因此，需要替代方法和资源高效的方法。近年来已经确定了几种负责分化的分子因子和转录开关。在速殖子中，药物诱导的组蛋白脱乙酰酶HDAC3抑制，或调节HDAC3的转录因子的基因缺失，会导致有性阶段和卵囊特异性基因的表达。在这里，我们应用了这一概念，并表明市售的HDAC3抑制剂阿皮西丁可用于鉴定速殖子中迄今未知的子孢子特异性单克隆抗体G1/19的抗原。通过对来自阿皮西丁处理培养物的免疫沉淀G1/19靶蛋白进行质谱分析，我们将其鉴定为SporoSAG。此外，对于丰度低得多的子孢子特异性蛋白LEA860，阿皮西丁处理仍然足以在免疫荧光显微镜下诱导可检测到的蛋白水平。我们还讨论了这种方法的进一步应用和局限性。这在一定程度上允许克服来自有性阶段和卵囊的材料匮乏的问题，而无需猫源材料。
