Systematic gene silencing identified Cryptosporidium nucleoside diphosphate kinase and other molecules as targets for suppression of parasite proliferation in human intestinal cells

Abstract

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium . In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium . However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15–30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

Proteome-wide prediction and analysis of the Cryptosporidium parvum protein–protein interaction network through integrative methods

“…By testing the influence of C. parvum on host cellular metabolic signatures, Velez et al have reported that glycolysis can be taken as the anti-cryptosporidial target, and also proved glutaminolysis and lactate release as necessities of the parasite replication [72] . Moreover, gene silencing of the nucleoside-diphosphate kinase (NDK) markedly inhibited parasite development [73] . Although our network cannot directly map modules to those pathways, the modules we identified could be a good supplement as they show a high relevance to the parasite development.…”

Revisiting trypanosomatid nucleoside diphosphate kinases

“…(51) Related to human protozoan parasites, in apicomplexa, silencing of NDPK strongly inhibited Cryptosporidium parvum proliferation in human intestinal cells. (52) The importance of NDPKs in the metabolism of nucleotides and their participation in multiple processes make them attractive targets since disrupting a single enzyme will affect several cellular functions simultaneously. As was mentioned, an interesting feature of trypanosomatids is the absence of metabolic pathways for de novo synthesis of purines, (6) which makes nucleotide metabolism a weakness for the parasites and where NDPKs fulfil a central role.…”

“… 51 Related to human protozoan parasites, in apicomplexa, silencing of NDPK strongly inhibited Cryptosporidium parvum proliferation in human intestinal cells. 52 …”

Disseminated Cryptosporidiosis in an Infant with Non-HIV Pediatric Immunodeficiency: First Case Report from Oman

“…3 This parasite is increasingly recognized as an important zoonotic, food-, and waterborne enteric pathogen causing diarrheal illness in children in developing countries. 4 - 7 Oocysts are transmitted fecal-orally, can resist routine chlorination and ozonation of water sources, 8 and are excreted in the stool of an infected host. Although numerous Cryptosporidium species have been identified, humans are most frequently infected with Cryptosporidium hominis and Cryptosporidium parvum .…”

“…Chronic infection is associated with villous atrophy, crypt hyperplasia, and secondary leucocytic infiltration in the lamina propria. 7 In addition to chronic and considerable diarrhea, growth stunting, biliary tract disease, pancreatitis, and respiratory tract disease may occur. 11 Treatment with antiprotozoal agents is usually ineffective in the context of immunodeficiency since they exhibit parasitistatic rather than parasiticidal activity.…”