Castro-Elizalde Kitzia N, Hernández-Contreras Pedro, Ramírez-Flores Carlos J, González-Pozos Sirenia, Gómez de León Carmen T, Mondragón-Castelán Mónica, Mondragón-Flores Ricardo
Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV-IPN), Av. IPN #2508, Delegación Gustavo A. Madero, Col. Zacatenco, C.P. 07360, Ciudad de México (Mexico City), México.
Unidad de Microscopía Electrónica, LaNSE-CINVESTAV, Ciudad de México (Mexico City), México.
Parasitol Res. 2018 Feb;117(2):547-563. doi: 10.1007/s00436-017-5738-x. Epub 2018 Jan 8.
The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well understood. Maintaining cells infected with T. gondii type II and III strains under stress conditions induces the tachyzoite-bradyzoite in vitro differentiation, along with the formation of cyst-like structures. However, due to the long exposure to such conditions required to induce the differentiation, the severe damages in the host cell and the low encystation frequency, it has been difficult to dissect in more detail these processes. Here, we successfully induced the in vitro formation of Toxoplasma cysts-like structures from tachyzoites of the type I RH strain by treating with mycophenolic acid, an inhibitor of the inosine monophosphate dehydrogenase. Mycophenolic acid is a drug widely used for HXGPRT positive selection of Toxoplasma mutant strains along with xanthine incubation in the culture medium; under such conditions, formation of tissue cysts has not been reported. We show that the exposure of extracellular tachyzoites to mycophenolic acid in absence of xanthine, followed by host cell invasion, triggered their differentiation into cyst-like structures. The differential expression of CST1, BAG1, and SAG1 molecules, as well as the structural modifications of infected cells, was characterized during the formation of cyst-like structures in vitro. These findings will allow the characterization of signaling pathways involved in tachyzoite to bradyzoite conversion and formation of tissue cysts.
刚地弓形虫速殖子向缓殖子转化以及组织包囊形成过程中发生的生化和结构变化尚未完全明确。在应激条件下维持感染II型和III型刚地弓形虫菌株的细胞，可诱导速殖子在体外向缓殖子分化，并形成囊样结构。然而，由于诱导分化所需的此类条件暴露时间长、宿主细胞严重受损以及包囊化频率低，很难更详细地剖析这些过程。在此，我们通过用肌苷单磷酸脱氢酶抑制剂霉酚酸处理，成功地诱导了I型RH株速殖子在体外形成刚地弓形虫囊样结构。霉酚酸是一种广泛用于刚地弓形虫突变株HXGPRT阳性选择的药物，同时在培养基中与黄嘌呤一起孵育；在这种条件下，尚未有组织包囊形成的报道。我们表明，在无黄嘌呤的情况下，将细胞外速殖子暴露于霉酚酸，随后进行宿主细胞侵袭，可触发它们分化为囊样结构。在体外囊样结构形成过程中，对CST1、BAG1和SAG1分子的差异表达以及受感染细胞的结构修饰进行了表征。这些发现将有助于表征参与速殖子向缓殖子转化和组织包囊形成的信号通路。
