Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

Treating cryptosporidiosis: A review on drug discovery strategies.

Despite several decades of research on therapeutics, cryptosporidiosis remains a major concern for human and animal health. Even though this field of research to assess antiparasitic drug activity is highly active and competitive, only one molecule is authorized to be used in humans. However, this molecule was not efficacious in immunocompromised people and the lack of animal therapeutics remains a cause of concern. Indeed, the therapeutic arsenal needs to be developed for both humans and animals. Our work aims to clarify research strategies that historically were diffuse and poorly directed. This paper reviews in vitro and in vivo methodologies to assess the activity of future therapeutic compounds by screening drug libraries or through drug repurposing. It focuses on High Throughput Screening methodologies (HTS) and discusses the lack of knowledge of target mechanisms. In addition, an overview of several specific metabolic pathways and enzymatic activities used as targets against Cryptosporidium is provided. These metabolic processes include glycolytic pathways, fatty acid production, kinase activities, tRNA elaboration, nucleotide synthesis, gene expression and mRNA maturation. As a conclusion, we highlight emerging future strategies for screening natural compounds and assessing drug resistance issues.

Avaliaçäo da influência do formol e do hipoclorito de sódio na pesquisa de oocistos de Cryptosporidium nas fezes, através do método de heine / The evaluation of the effect of formol and sodium hypochlorite on the demonstration of Cryptosporidium oocysts in feces by Heine's method

Cryptosporidium oocysts were searched by Heine's method in stools of nine calves with cryptosporidiosis after stool treatment with two disinfectants, 10 per cent paraformaldehyde solution and 14.5 per cent sodium hypochlorite solution. After 30 minutes exposition to sodium hypochlorite solution oocysts became non refractile and acquired a reddish tinge, making their identification difficult. No morphological alterations occurred in oocysts after paraformaldehyde treatment. We recommend paraformaldehyde at 10 per cent concentration as means of human immunodeficiency virus (HIV) inactivation for routine use in stool examinations and therefore making safer those type of procedures for laboratory personnel, when using Heine's method

Efecto de los corticosteroides sobre una infección experimental con Cryptosporidium / Corticosteroids effect on Crystosporidium experimental infection

Ratones blancos de la cepa NGP (20-30 g de peso) fueron inoculados vía oral con 10 elevado a 1, 10 elevado a 3 y 10 elevado a 5, ooquistes de Cryptosporidium sp. aislado de un caso humano. Algunos de ellos fueron tratados semanalmente con 5 ó 10 mg de acetato de cortisona, durante 1 mes. Infecciones muy altas con el parásito fueron obtenidas en los animales tratados con el inmunosupresor, los cuales también mostraron una patología clara, no sólo a través de la apariencia general sino por los parámetros de supervivencia y curva de peso corporal. Se establece un modelo de estudio de la criptosporidiosis usando ratones NGP adultos, acetato de cortisona como inmunosupresor y un inóculo tan bajo como 10 elevado a 3, ooquistes lo que facilita cualquier investigación. Se discuten estos hallazgos, a la luz del uso de los corticosteroides en el tratamiento de las enfermedades infecciosas del hombre