Preassembled Single-Stranded RNA–Argonaute Complexes: A Novel Method to Silence Genes in<i>Cryptosporidium</i>

Castellanos-González, Alejandro; Perry, Nicolás; Nava, Samantha; White, A. Clinton

doi:10.1093/infdis/jiv588

Cited by 24 publications

(28 citation statements)

References 31 publications

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“…In addition, we found that preassembling ds siRNA with the Ago2 protein elicited much more potent gene silencing than a single-guide strand-loaded Ago2 when transfecting mammalian cells. The latter format is known to be the minimal RISC, which comprises a 5′-phosphorylated ss RNA and Ago2, and has recently been utilized to transfect RNAi-deficient parasites for fundamental studies on host-parasite interactions via a commercial transfection reagent (17). We reasoned that the preassembly of ds siRNA with Ago2 may help recruit several other intracellular components such as dicer and transactivation response RNA-binding protein, which are known to facilitate siRNA loading (15).…”

Section: Discussionmentioning

confidence: 99%

“…(iii) Biochemists have successfully loaded short, single-stranded small interference RNA (ss-siRNA) or duplex siRNA containing guide and passenger RNAs into Ago2 in vitro for gene-silencing assays (13)(14)(15)(16). (iv) More recently, an ss-siRNA/ Ago2 complex system was used to silence mRNAs in Cryptosporidium, which lacks key components of RNAi, for interrogating parasite-host interactions (17). (v) The availability of recombinant RNA-free human Ago2 has accelerated the efficiency of loading desired siRNA for fundamental research (13).…”

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confidence: 99%

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Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Small interfering RNA (siRNA) represents a promising class of inhibitors in both fundamental research and the clinic. Numerous delivery vehicles have been developed to facilitate siRNA delivery. Nevertheless, achieving highly potent RNA interference (RNAi) toward clinical translation requires efficient formation of RNA-induced gene-silencing complex (RISC) in the cytoplasm. Here we coencapsulate siRNA and the central RNAi effector protein Argonaute 2 (Ago2) via different delivery carriers as a platform to augment RNAi. The physical clustering between siRNA and Ago2 is found to be indispensable for enhanced RNAi. Moreover, by utilizing polyamines bearing the same backbone but distinct cationic side-group arrangements of ethylene diamine repeats as the delivery vehicles, we find that the molecular structure of these polyamines modulates the degree of siRNA/Ago2-mediated improvement of RNAi. We apply this strategy to silence the oncogene STAT3 and significantly prolong survival in mice challenged with melanoma. Our findings suggest a paradigm for RNAi via the synergistic coassembly of RNA with helper proteins.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference

Wang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Cryptosporidium lacks the machinery involved with mammalian gene silencing 6 . In previous studies, we have demonstrated the feasibility to silence Cryptosporidium genes by transfecting oocysts with human Argonaute (with slicer activity) loaded with ssRNA 5 . These initial experiments confirmed reduction at protein levels and pointed out the usefulness of the method to evaluate parasite invasion.…”

Section: Discussionmentioning

confidence: 99%

“…The limitation of tools to genetically manipulate gene expression in this parasite has been identified as a major hurdle for drug and vaccine development 2,4 . We developed a method to silence genes in this parasite by using preassembled complexes of Cryptosporidium single strand RNA and the human enzyme Argonaute 2 (hAgo2) 5 . We hypothesized that this method could be used to study key steps of infection and identify novel targets for drug and vaccine development.…”

Section: Introductionmentioning

confidence: 99%

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

Castellanos-González

Martinez-Traverso

et al. 2019

Preprint

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24 25 26 27 2 28Abstract: 29 Background: Cryptosporidiosis is a major cause of diarrheal disease. However, the only drug 30 approved for cryptosporidiosis does not work well in high risk populations. Therefore, novel 31 drugs are urgently needed. Then, the identification of novel is necessary to develop new 32 therapies against this parasite. Recently, we have developed a rapid method to block gene 33 expression in Cryptosporidium by using pre-assembled complexes of Cryptosporidium 34 antisense RNA and human protein with slicer activity (Argonaute 2). We hypothesized that 35 structural proteins, proteases, enzymes nucleotide synthesis and transcription factors are 36 essential for parasite development, thus in this work we knock down expression of 4 selected 37 genes: Actin, Apicomplexan DNA-binding protein (AP2), Rhomboid protein 1 (Rom 1) and 38 nucleoside diphosphate kinase (NDK) and elucidated its role during invasion, proliferation and 39 egress of Cryptosporidium. 40 41 Methods: We used protein transfection reagents (PTR) to introduce pre-assembled complexes 42 of antisense RNA and human Argonaute 2 into Cryptosporidium parvum oocysts, the complexes 43 blocked expression of Actin (Act), Transcription factor AP2 (AP2), nucleoside diphosphate 44 kinase (DKN), and rhomboid protein 1 (Rom1). After gene silencing, we evaluated parasite 45 reduction using In vitro models of excystation, invasion, proliferation and egress. We evaluated 46 the potency of ellagic acid, a nucleoside diphosphate kinase inhibitor for anti-cryptosporidial 47 activity using a model of in vitro infection with human HCT-8 cells. 48 49 Results: Silencing of Act, AP2, NDK and Rom1 reduce significantly invasion, proliferation and 50 egress of Cryptosporidium. We showed that silencing of NDK markedly inhibited 51 Cryptosporidium proliferation. This was confirmed by demonstration that ellagic acid reduced 52 the number of parasites at micro molar concentrations (EC 50 =15-30 µM) without showing any 53 toxic effect on human cells. 54 55 Conclusions: Overall the results confirmed the usefulness RNA silencing can be used to 56 identify novel targets for drug development against Cryptosporidium. We identified ellagic acid 57 (EA), a nucleoside diphosphate kinase inhibitor also blocks Cryptosporidium proliferation. Since 58 EA is a dietary supplement approved for human use, then this compound should be studied as 59 a potential treatment for cryptosporidiosis. 3 60 Author summary 61 The World Health Organization reports diarrhea kills around 760,000 children under five every 62 year. Cryptosporidium infection is a leading cause of diarrhea morbidity and mortality. Current 63 therapies to treat this infection are suboptimal, therefore novel treatments are urgently needed. 64 We used genetic tools to identify novel targets for drug development, thus in this work we 65 evaluated the role of 4 genes during Cryptosporidium infection. We demonstrated that silencing 66 of nucleoside-diphosphate kinase (NDK) drastically reduced invasion, proliferation and e...

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“…For this study, we first determined the expression patterns of SUB1 and CDPK5 throughout the asexual stage of infection. We then determined the specific roles of SUB1 and CDPK5 by a modified small interfering RNA (siRNA) method (26,27) and evaluated the effects on parasite viability, infection of host intestines, and the egress of merozoites during infection.…”

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confidence: 99%

Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells

2019

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Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P Ͻ 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.

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Preassembled Single-Stranded RNA–Argonaute Complexes: A Novel Method to Silence Genes inCryptosporidium

Cited by 24 publications

References 31 publications

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference

Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference

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

Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells

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