Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA.
mBio. 2023 Apr 25;14(2):e0326122. doi: 10.1128/mbio.03261-22. Epub 2023 Feb 14.
The apicomplexan parasite is a leading global cause of diarrheal disease, and the infection poses a particularly grave threat to young children and those with weakened immune function. Infection occurs by ingestion of meiotic spores called oocysts, and transmission relies on fecal shedding of new oocysts. The entire life cycle thus occurs in a single host and features asexual as well as sexual forms of replication. Here, we identify and locus tag two Apetala 2-type (AP2) transcription factors and demonstrate that they are exclusively expressed in male and female gametes, respectively. To enable functional studies of essential genes in Cryptosporidium parvum, we develop and validate a small-molecule-inducible gene excision system, which we apply to the female factor AP2-F to achieve conditional gene knockout. Analyzing this mutant, we find the factor to be dispensable for asexual growth and early female fate determination but to be required for oocyst shedding in infected animals . Transcriptional analyses conducted in the presence or absence of AP2-F revealed that the factor controls the transcription of genes encoding crystalloid body proteins, which are exclusively expressed in female gametes. In C. parvum, the organelle is restricted to sporozoites, and its loss in other apicomplexan parasites leads to blocked transmission. Overall, our development of conditional gene ablation in C. parvum provides a robust method for genetic analysis in this parasite that enabled us to identify AP2-F as an essential regulator of transcription required for oocyst shedding and transmission. The parasite infects millions of people worldwide each year, leading to life-threatening diarrheal disease in young children and immunosuppressed individuals. There is no vaccine and only limited treatment. Transmission occurs via the fecal-oral route by an environmentally resilient spore-like oocyst. Infection takes place in the intestinal epithelium, where parasites initially propagate asexually before transitioning to male and female gametes, with sex leading to the formation of new oocysts. The essential role of sexual development for continuous infection and transmission makes it an attractive target for therapy and prevention. To study essential genes and potential drug targets across the life cycle, we established inducible gene excision for C. parvum. We determined that the female-specific transcription factor AP2-F is not required for asexual growth and early female development but is necessary for oocyst shedding . This work enhances the genetic tools available to study gene function.
疟原虫是全球主要的腹泻病病原体之一，对幼儿和免疫功能较弱的人群构成特别严重的威胁。感染是通过摄入称为卵囊的减数分裂孢子引起的，传播依赖于新卵囊的粪便脱落。因此，整个生命周期都发生在单个宿主中，具有无性和有性形式的复制。在这里，我们鉴定并定位了两个 Apetala 2 型（AP2）转录因子，并证明它们分别仅在雄性和雌性配子中表达。为了能够对微小隐孢子虫的必需基因进行功能研究，我们开发并验证了一种小分子诱导的基因切除系统，我们将其应用于雌性因子 AP2-F 以实现条件性基因敲除。分析这种突变体，我们发现该因子对于无性生长和早期雌性命运决定不是必需的，但对于感染动物中的卵囊脱落是必需的。在存在或不存在 AP2-F 的情况下进行的转录分析表明，该因子控制晶体蛋白基因的转录，这些基因仅在雌性配子中表达。在微小隐孢子虫中，该细胞器仅限于孢子虫，而在其他顶复门寄生虫中的缺失会导致传播受阻。总体而言，我们在微小隐孢子虫中成功实现了条件性基因消融，为该寄生虫的遗传分析提供了一种强大的方法，使我们能够鉴定出 AP2-F 作为转录必需因子，对于卵囊脱落和传播是必需的。这种寄生虫每年感染全球数百万人，导致幼儿和免疫抑制个体发生危及生命的腹泻病。目前尚无疫苗，治疗方法也有限。传播通过环境抗逆性孢子样卵囊的粪-口途径发生。感染发生在肠上皮中，寄生虫最初无性繁殖，然后转变为雄性和雌性配子，性别导致新卵囊的形成。性发育对于连续感染和传播的至关重要性使其成为治疗和预防的有吸引力的靶标。为了研究整个生命周期中的必需基因和潜在药物靶点，我们为微小隐孢子虫建立了诱导型基因切除。我们确定雌性特异性转录因子 AP2-F 对于无性生长和早期雌性发育不是必需的，但对于卵囊脱落是必需的。这项工作增强了用于研究基因功能的遗传工具。
