부산대학교 도서관

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. Author summary: Cryptosporidium species are intracellular intestinal parasites that cause prolonged diarrhea in immunocompromised people and children. Recent in vitro studies of the Cryptosporidium life cycle indicate the parasite undergoes three rounds of asexual replication followed by an obligatory sexual cycle. This life cycle suggests the novel possibilities of therapy or prevention by targeting sexual differentiation and reproduction. To identify drug-like inhibitors and tool compounds, we developed a screening assay for female gametocytogenesis and screened the ReFRAME library. Our screen identified selective sexual differentiation inhibitors. We then studied the impacts of inhibitor treatment on RNA expression. Comparison of our results to publicly available female-specific gene expression data confirmed that the inhibitors selectively modulated differentiation. We then found that ribosomal protein expression falls during Cryptosporidium female gametocytogenesis, and we experimentally demonstrated corresponding translational repression. Further analysis of promoter regions of coordinately regulated genes implicated a conserved promoter motif and an Apetala 2 transcription factor in female gametocytogenesis. This study identifies a set of tool compounds that selectively modulate Cryptosporidium female gametocytogenesis, provides new insights into female differentiation, and lays the groundwork for future investigations to determine if obligatory sexual reproduction is a vulnerability that can be exploited for therapy or prevention of cryptosporidiosis. [ABSTRACT FROM AUTHOR]