RESUMO
Sexual reproduction of Toxoplasma gondii, confined to the felid gut, remains largely uncharted owing to ethical concerns regarding the use of cats as model organisms. Chromatin modifiers dictate the developmental fate of the parasite during its multistage life cycle, but their targeting to stage-specific cistromes is poorly described1,2. Here we found that the transcription factors AP2XII-1 and AP2XI-2 operate during the tachyzoite stage, a hallmark of acute toxoplasmosis, to silence genes necessary for merozoites, a developmental stage critical for subsequent sexual commitment and transmission to the next host, including humans. Their conditional and simultaneous depletion leads to a marked change in the transcriptional program, promoting a full transition from tachyzoites to merozoites. These in vitro-cultured pre-gametes have unique protein markers and undergo typical asexual endopolygenic division cycles. In tachyzoites, AP2XII-1 and AP2XI-2 bind DNA as heterodimers at merozoite promoters and recruit MORC and HDAC3 (ref. 1), thereby limiting chromatin accessibility and transcription. Consequently, the commitment to merogony stems from a profound epigenetic rewiring orchestrated by AP2XII-1 and AP2XI-2. Successful production of merozoites in vitro paves the way for future studies on Toxoplasma sexual development without the need for cat infections and holds promise for the development of therapies to prevent parasite transmission.
Assuntos
Gatos , Técnicas In Vitro , Estágios do Ciclo de Vida , Toxoplasma , Animais , Gatos/parasitologia , Humanos , Cromatina/genética , Cromatina/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Técnicas In Vitro/métodos , Estágios do Ciclo de Vida/genética , Merozoítos/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/fisiologia , Toxoplasmose/genética , Toxoplasmose/parasitologia , Toxoplasmose/transmissão , Transcrição GênicaRESUMO
The reported number of cases of Acanthamoeba amebic keratitis (AK) is continually increasing. Molecular diagnosis has become the first choice of ophthalmologists for identifying and confirming this clinically problematic diagnosis. However, in-house molecular diagnostic procedures are time-consuming and may not be compatible with the urgency of the situation. In this study, a previous in-house AK-PCR technique was adapted for use on BD MAX (Becton Dickinson, Heidelberg, Germany), a fully integrated, automated platform for molecular biology, for the rapid routine diagnosis of AK. Different protocols were compared to optimize DNA extraction from Acanthamoeba cysts. The analytical parameters of the AK-BD MAX PCR were evaluated. Thirty-two samples were simultaneously tested with AK-BD MAX PCR and the original AK-PCR from which it was developed. A thermal-shock pretreatment protocol was validated. The analytical parameters obtained with BD MAX were similar to those obtained with the previous in-house AK-PCR method. The performance of AK-BD MAX PCR was then assessed for routine testing on 40 clinical samples, mostly corneal scrapings. Frozen, ready-to-use, in-house PCR premixes were stable over 8 months. Overall, 34 of the 40 clinical samples (85%) were considered to be true negatives; 4 (10%), probable AK; and 2 (5%), possible AK. This newly developed AK-BD MAX PCR is reliable, rapid, and efficient, and should facilitate Acanthamoeba keratitis diagnosis.
