Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Apr 15;213(8):1307-14.
doi: 10.1093/infdis/jiv588. Epub 2015 Dec 11.

Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium

Alejandro Castellanos-Gonzalez  1 , Nicolas Perry  1 , Samantha Nava  1 , A Clinton White Jr  1
Affiliations

Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium

Alejandro Castellanos-Gonzalez et al. J Infect Dis. .

Abstract

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.

Keywords: Argonaute; Cryptosporidium; Cryptosporidium parvum; cryptosporidiosis; siRNA; silencing.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Complex assembling and human Argonaute 2 (hAgo2) activity. A, In human cells, dicer enzyme processes double-stranded RNA (dsRNA; left). The resulting small interfering RNA (siRNA) is loaded by dicer and transactivation response RNA-binding protein onto hAgo2 (RISC complex) to producing target degradation. hAgo2 and single-stranded RNA (ssRNA) are preassembled in vitro, and the complex is introduced into organisms without RISC enzymes, such as Cryptosporidium, to produce silencing (right). B, Complex assembly was confirmed by the electrophoretic mobility-shift assay assay, using ssRNA labeled with FAM. Lanes 1–3, Samples incubated with hAgo2 (0, 50, 250 ng). Lane 4, Molecular markers. C, ssRNA-hAgo2 complex activity is demonstrated by denaturing gel electrophoresis, using an RNA target (50 nucleotides) labeled with carboxyfluorescein. 1, Cp15 target; 2, Cp15-ssRNA with no target; 3, Cp15-ssRNA with hAgo2 alone; 4, Cp15 target incubated with ssRNA alone; 5, Cp15 target incubated with hAgo2 alone; 6, Cp15 target loaded with 0.01 µM of Cp15-ssRNA; 7, Cp15 target loaded with 0.1 µM of Cp15-ssRNA; 8, Cp15 target loaded with 1 µM of Cp15-ssRNA; 9, unrelated target (60 nucleotides) with 1 µM of Cp15-ssRNA. Abbreviation: mRNA, messenger RNA.
Figure 2.
Figure 2.
Cryptosporidium transfection and viability assays. A, Fluorescent immunoglobulin G (IgG) labeled with phycoerythrin (red) was encapsulated within protein transfection reagent (PTR) and then used to transfect Cryptosporidium sporozoites within oocysts. Oocysts were incubated with anti-Cryptosporidium oocyst antibody-FITC (Oocyst Ab-FITC; green). IgG was only introduced into samples treated with PTR (top). No signal was detected in the parasites with samples incubated only with IgG (bottom). B, PTR was loaded with labeled human Argonaute 2 (hAgo2)–NHS-rhodamine (red) and then used to transfect oocysts. Cyst wall was stained with oocyst antibody (green). hAgo2 was detected in oocysts (white triangle; top) or in released sporozoites after excystation (white triangle; bottom). C, We stained oocysts with the vital dye carboxyfluorescein succinimidyl ester (CFSE; green). Stained and motile sporozoites were observed (white triangles). White bar, 5 µm. D, Viability was quantified by flow cytometry for untransfected parasites (wild type), heat-killed parasites, parasites treated with empty PTR, or parasites transfected with complexes of hAgo2 preloaded with scramble single-stranded RNA (ssRNA), calcium-dependent kinase 1 (CDPK)–ssRNA, inosine-5-monophosphate dehydrogenase (Ino-5mon)–ssRNA, and glycoprotein (Gp900)–ssRNA.
Figure 3.
Figure 3.
Specific RNA slicing in Cryptosporidium was demonstrated by rapid amplification of 5′ complementary DNA ends (RACE). A, Capped RNA (red circle) does not link the primer adaptor (red rectangle), the adaptor is linked only if the RNA target is sliced by human Argonaute 2 (hAgo2)—single-stranded RNA (ssRNA) complex (green), and a RACE–polymerase chain reaction (PCR) product is obtained only from sliced products by using an adaptor and gene-specific primer (GSP; green rectangle) but not with capped RNA. B, Electrophoresis of PCR-RACE products of parasites transfected only with protein transfection reagent (uncut) or with specific hAgo2-ssRNA (sliced). C, The PCR-RACE product was cloned and sequenced. The sequence of the sliced target (underlined in red) is located immediately after the adapter (underlined black), where the cleavage site is indicated (black triangle). Abbreviation: MW, molecular weight. This figure is available in black and white in print and in color online.
Figure 4.
Figure 4.
Silenced genes in Cryptosporidium. A, Quantitative polymerase chain reaction (qPCR; top) shows a fold reduction in messenger RNA target from transfected parasites with human Argonaute 2 (hAgo2) loaded with single-stranded RNA (ssRNA) specific for glycoprotein (Gp900; black bar). Control parasites were treated only with transfection reagent (wild type [WT]). Western blot analysis of Gp900 (bottom) in parasites transfected (ssRNA Gp900) or not transfected (WT control). B, qPCR analysis was used to calculate the percentage reduction in expression, relative to basal expression, for the Cryptosporidium targets Cp15, glycoprotein (Gp900), calcium-dependent kinase 1 (CDPK), inosine-5-monophosphate dehydrogenase (Ino-5mon), and Cp17 (gray bars). C, Transfected parasites were used to evaluate the role of silenced genes during infection. Percentages are relative to control (parasites were transfected only with protein transfection reagent [PTR]). In panels B and C, data are mean ± SD of qPCR triplicates and are representative of at least 3 individual experiments with similar results.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kotloff KL, Nataro JP, Blackwelder WC et al. . Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet 2013; 382:209–22. - PubMed
    1. Checkley W, White AC Jr, Jaganath D et al. . A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect Dis 2015; 15:85–94. - PMC - PubMed
    1. Xu P, Widmer G, Wang Y et al. . The genome of Cryptosporidium hominis. Nature 2004; 431:1107–12. - PubMed
    1. Baum J, Papenfuss AT, Mair GR et al. . Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites. Nucleic Acids Res 2009; 37:3788–98. - PMC - PubMed
    1. Lye LF, Owens K, Shi H et al. . Retention and loss of RNA interference pathways in trypanosomatid protozoans. PLoS Pathog 2010; 6:e1001161. - PMC - PubMed

Publication types

MeSH terms