Gene silencing in Cryptosporidium: A rapid approach to identify novel targets for drug development

Abstract Background Cryptosporidiosis is a major cause of diarrheal disease. However, the only drug approved for cryptosporidiosis does not work well in high risk populations. Therefore, novel drugs are urgently needed. Then, the identification of novel is necessary to develop new therapies against this parasite. Recently, we have developed a rapid method to block gene expression in Cryptosporidium by using pre-assembled complexes of Cryptosporidium antisense RNA and human protein with slicer activity (Argonaute 2). We hypothesized that structural proteins, proteases, enzymes nucleotide synthesis and transcription factors are essential for parasite development, thus in this work we knock down expression of 4 selected genes: Actin, Apicomplexan DNA-binding protein (AP2), Rhomboid protein 1 (Rom 1) and nucleoside diphosphate kinase (NDK) and elucidated its role during invasion, proliferation and egress of Cryptosporidium. Methods We used protein transfection reagents (PTR) to introduce pre-assembled complexes of antisense RNA and human Argonaute 2 into Cryptosporidium parvum oocysts, the complexes blocked expression of Actin (Act), Transcription factor AP2 (AP2), nucleoside diphosphate kinase (DKN), and rhomboid protein 1 (Rom1). After gene silencing, we evaluated parasite reduction using In vitro models of excystation, invasion, proliferation and egress. We evaluated the potency of ellagic acid, a nucleoside diphosphate kinase inhibitor for anti-cryptosporidial activity using a model of in vitro infection with human HCT-8 cells. Results Silencing of Act, AP2, NDK and Rom1 reduce significantly invasion, proliferation and egress of Cryptosporidium. We showed that silencing of NDK markedly inhibited Cryptosporidium proliferation. This was confirmed by demonstration that ellagic acid reduced the number of parasites at micro molar concentrations (EC 50 =15-30 µM) without showing any toxic effect on human cells. Conclusions Overall the results confirmed the usefulness RNA silencing can be used to identify novel targets for drug development against Cryptosporidium. We identified ellagic acid (EA), a nucleoside diphosphate kinase inhibitor also blocks Cryptosporidium proliferation. Since EA is a dietary supplement approved for human use, then this compound should be studied as a potential treatment for cryptosporidiosis. Author summary The World Health Organization reports diarrhea kills around 760,000 children under five every year. Cryptosporidium infection is a leading cause of diarrhea morbidity and mortality. Current therapies to treat this infection are suboptimal, therefore novel treatments are urgently needed. We used genetic tools to identify novel targets for drug development, thus in this work we evaluated the role of 4 genes during Cryptosporidium infection. We demonstrated that silencing of nucleoside-diphosphate kinase (NDK) drastically reduced invasion, proliferation and egress of this parasite. To validate these finding we used the Ellagic acid (EA) an inhibitor of NDK to treat infected intestinal cells. Our results confirmed that the EA blocks parasite proliferation on infected cells. Interestingly we observed that the ellagic acid also has anti cryptosporidial activity by inducing apoptosis. Since EA is a dietary supplement already approved for human use, then this compound has potential to be used as a rapid alternative to treat Cryptosporidiosis.