Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum.
Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum.
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隐孢子虫是一种顶复形寄生虫，是导致疟疾的全球主要原因，这种感染对幼儿和免疫功能低下的人构成特别严重的威胁。感染是通过摄入被称为卵囊的减数分裂孢子发生的，而传播依赖于新卵囊的粪便脱落。因此，整个生命周期发生在一个单一的主机和功能无性以及有性形式的复制。在这里，我们确定和位点标记两个Apetala 2型（AP 2）转录因子，并证明它们只在男性和女性配子中表达，分别。为了研究微小隐孢子虫中必需基因的功能，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AP 2-F，以实现条件性基因敲除。分析这个突变体，我们发现该因子在体外是无性生长和早期雌性命运决定的关键，但在体内是感染动物卵囊脱落所需的。在存在或不存在AP 2-F的情况下进行的转录分析显示，该因子控制编码晶体蛋白的基因的转录，晶体蛋白仅在雌性配子中表达。In C.在寄生虫中，该细胞器仅限于子孢子，并且其在其他顶复门寄生虫中的损失导致传播受阻。总之，我们在C. parvum为这种寄生虫的遗传分析提供了一种强大的方法，使我们能够将AP 2-F鉴定为卵囊脱落和传播所需的重要转录调节因子。
The apicomplexan parasite Cryptosporidium 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 in vitro but to be required for oocyst shedding in infected animals in vivo. 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.
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