Studies into the suitability of the cell-penetrating peptide octaarginine as a transmembrane vehicle for DNA transfection of Cryptosporidium parvum and to improve the antiprotozoan efficacy of Nitazoxanide

Introduction: Cryptosporidium parvum is one of the most common causes of diarrhea worldwide in neonatal calves. This pathogen is also life-threatening in malnourished children and immunodeficient patients. There is no vaccine and a single drug nitazoxanide (NTZ), of only the moderate efficacy has been approved by FDA for cryptosporidiosis treatment in human. Octaarginine is known to facilitate the transport of other molecules across cell membranes and has been use to transfect protozoan organisms. It is also proposed to increase the efficacy of drugs against intracellular pathogens. Aims of the study: The capacity of octaarginine to support transfection of C. parvum as an alternative to electroporation was evaluated. Furthermore, it was studied whether octaarginine covalently bound to NTZ (NTZ-R8) improves efficacy against the parasite. Animals, materials and methods: FAM-octaarginine was added to either intact oocysts, short-time excystation exposed (STE) oocysts, excysted sporozoites, intracellular stages of C. parvum to assess the permeability of the Cryptosporidium membrane to the peptide. The optimal conditions for condensation of plasmid for transfection experiments were evaluated by testing different N/P ratios applying by gel retardation assay. The transfection complex octaarginine/polyethyleneimine (PEI)/DNA was also incubated with intact oocysts, STE oocysts, and excysted sporozoites. Transfected parasites were transferred to HCT-8 cell cultures and further incubated for 24 h. Immunoflourescence assay (IFA) was performed to detect successfully transfected parasites. To evaluate the suitability of octaarginine as a vehicle supporting transport of NTZ across membranes, octaarginine was coupled to NTZ to produce NTZ-R8. Cryptosporidium oocysts were inoculated into HCT-8 cell monolayers in the presence of NTZ and NTZ-R8 at concentrations of 1, 5, 10, 50, 100 or 1000 ng/ml. Parasite growth was monitored by RT- qPCR after RNA extraction from C. parvum exposed HTC-8 cell cultures. RT-qPCR was performed on the target gene 18S rRNA of Cryptosporidium and normalized to the expression of the housekeeping gene 18S rRNA of host cells. To evaluate the efficacy of NTZ-R8 in vivo, IFN-γ knockout mice were orally inoculated with 1000 oocysts each, except for the non-infected controls. Infected mice were treated with NTZ (10 mg/kg BW) or NTZ-R8 (2 mg/kg BW) in 7 days. The efficacy of treatment was evaluated by oocyst excretion, survival rate, clinical symptoms, and histopathological changes in the ileum. Results: Octaarginine easily penetrated into Cryptosporidium sporozoites and STE oocysts, and intracellular stages while the membrane of intact oocysts remained impermeable. The optimal N/P ratio for the full DNA plasmid condensation starts from 10 when octaarginine was also added to the complex. Successful transfection of excysted sporozoites and STE oocysts was observed with only 1µg plasmid in the transfection complex. Transfection was not achieve when intact oocysts were used. The half-maximal inhibition concentration (IC50) of NTZ and NTZ-R8 was 60.54 ng/ml (197 nM) and 4.499 ng/ml (2.9 nM), respectively. Therefore, octaarginine significantly improved inhibition C. parvum growth by NTZ around 68 times (P 0.05). Conclusion: DNA condensation by PEI and DNA delivery by octaarginine allows simple and rapid transfection that requires a small amount of plasmid DNA only and does not depend on sophisticated equipment. Best results were obtained using STE oocysts. Octaarginine also successfully transported the anticryptosporidial compound NTZ into extracellular and intracellular stages of C. parvum and is therefore a suitable vehicle for drug delivery, thus being a promising tool for improvement of treatment efficacy