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Experimental Parasitology 119 (2008) 301–303 www.elsevier.com/locate/yexpr
Research brief
Pyrrhocoricin as a potential drug delivery vehicle for Cryptosporidium parvum Annika Boxell a, Stephanie Hui Chin Lee a, Ryan Jefferies b, Paul Watt b,c, Richard Hopkins b,c, Simon Reid a, Anthony Armson d, Una Ryan a,* b
a Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch University, Murdoch, WA 6150, Australia Drug Discovery Technology Group, Telethon Institute for Child Health Research, Subiaco, School of Paediatrics and Child Health, University of Western Australia, Australia c Phylogica Ltd., Perth, WA, Australia d Division of Health Sciences, School of Nursing, Murdoch University, Murdoch, WA 6150, Australia
Received 31 August 2007; received in revised form 8 February 2008; accepted 12 February 2008 Available online 26 February 2008
Abstract This study analysed the intracellular delivery capacity of insect derived pyrrhocoricin with a peptide cargo in Cryptosporidium parvum in vitro using fluorescence microscopy. Results revealed that pyrrhocoricin was capable of acting as a delivery vehicle in transducing peptides across the parasite cell membrane for multiple life-cycle stages. The successful transduction may aid in target validation and the delivery of future peptide-based drugs against this important human pathogen. Ó 2008 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Cryptosporidium; Pyrrhocoricin; Delivery vehicle; Peptides
1. Introduction Cryptosporidium is an enteric parasite, which has a global impact on the health, survival and economic development of millions of people and animals world-wide (Fayer et al., 2000; Fayer, 2004). Currently there is no effective chemotherapy for Cryptosporidium infection (Carey et al., 2004; Fayer et al., 2000; Fayer, 2004). The oocysts produced by Cryptosporidium are extremely hardy, easily spread via water, and difficult to inactivate or remove from water intended for consumption without the use of filtration (Fayer et al., 2000). For these reasons, Cryptosporidium is categorised as an NIH category B biodefense agent (Puiu et al., 2004; Striepen and Kissinger, 2004). In recent years, peptides have been developed as potential therapeutic agents in drug discovery (Watt, 2006). However, trans*
Corresponding author. Fax: +61 8 9310 4144. E-mail address:
[email protected] (U. Ryan).
0014-4894/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2008.02.005
porting such peptides into enteric parasites is a major obstacle to be overcome, which has limited the pharmaceutical research in this area. Recently, the short, proline-rich insect-derived antimicrobial peptide (AMP), pyrrochoricin (isolated from the European sap-sucking bug, Pyrrhocoris apterus) and its synthetic analogues have been identified as having antibacterial activity and postulated as a drug delivery vehicle (Cociancich et al., 1994; Kragol et al., 2002; Cudic et al., 2003). The present study was undertaken to analyse the intracellular delivery capacity of pyrrhocoricin with a peptide cargo in Cryptosporidium parvum in vitro using fluorescence microscopy. 2. Material and methods Fluorescein-labelled pyrrhocoricin (Fl-Pyr) C-terminal half, residues 11–20 (TPPRPIYNRN) (Kragol et al., 2002), a fluorescein-labelled random peptide (GDPNCF ADNMPQ) from the Shigella flexneri genome (Fl- Pep)
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Fig. 1. Microscopic analysis of 5 lg/ml Fl-Pyr-Pep delivery into C. parvum after a 60 min incubation period. (a and b) nomarski (left) and fluorescent images (right), (c) nomarski (top) and fluorescent image (bottom); (a) oocyst clearly showing pyrrhocoricin uptake into the four internal sporozoites, (b) clump of five trophozoites undergoing division into meronts with nuclear localisation of fluorescence, (c) microgamete. Scale bar = 5 lm.
and a C-terminal half pyrrhocoricin-random peptide fusion (Fl-Pyr-Pep) were commercially synthesised by Mimotopes, Australia. The fluorescein-labelled random peptide (Fl-Pep) was included for use as a negative control. Escherichia coli was chosen as a positive control cellular target as it has been previously reported that the pyrrhocoricin peptides could act as a delivery vehicle into E. coli (Kragol et al., 2002). Peptide transduction assays were performed using a modification of the method described by Kragol et al. (2002). Briefly, E. coli cells and host-cell free cultured C. parvum (Hijjawi et al., 2004) were washed twice with sterile 1 PBS, pH 7.0, and resuspended with each of the synthesised peptides at final concentrations of 5 lg/ml and 100 ng/ml, respectively, in 100 ll. Cryptosporidium and E. coli were incubated at 37 °C for 20 min and 60 min. Thereafter, cells were washed twice again with 1 PBS, pH 7.0, resuspended in 50 ll and examined directly by fluorescence microscopy using an Olympus BX 51 photomicroscope with an Olympus DP 70 camera (100 objectives magnification). The fluorescein-labelled peptides were visualised with epi-fluorescence using a blue excitation filter at a wavelength of 460–490 nm and an emission filter at a wavelength of 520 nm. Fluorescence images were processed using the Olympus DP70-BSW software. 3. Results and discussion In all treated cells (C. parvum and E. coli), no fluorescence was detected using untreated cells and the negative control (100 ng/ml and 5 lg/ml Fl-Pep) after both 20 and 60 min incubation periods, which indicated that random peptides by themselves, are not transducible (data not shown). In E. coli, faint fluorescence was detected in cells treated with 100 ng/ml Fl-Pyr and Fl-Pyr-Pep for 60 min, but maximal fluorescence was detected with 5 lg/ml Fl-Pyr and Fl-Pyr-Pep (data not shown). In C. parvum treated cells, maximal fluorescence was also detected in cells treated for 60 min with 5 lg/ml Fl-Pyr and Fl-Pyr-Pep (Fig. 1). Nucleus and cytoplasmic localisation were observed with cells treated with Fl-Pyr and Fl-Pyr-Pep in various asexual and sexual life-cycle stages indicating that the pyrrhocoricin were transducible into C. parvum. The present study has demonstrated for the first
time that fluorescein-labelled pyrrhocoricin can be successfully introduced into C. parvum along. It is assumed that the peptide cargo is also introduced but in the absence of an assay which specifically detects the peptide (e.g. a C-terminal flurophore or peptide-specific label, etc.) or any biological read-out which is dependent upon internalisation of the peptide, its impossible to confirm the random peptide moiety is still attached. Future studies should label fusion peptides with a C-terminal fluorophore in order to confirm the integrity of the Pyr-Pep fusion. Fluorescein-labelled pyrrhocoricin was also successfully introduced into E. coli supporting a previous study done by Kragol and his colleagues (Kragol et al., 2002), which showed that pyrrhocoricin was capable of transduction across the bacterial (i.e. E. coli) cell membrane and accumulated in the cells (Kragol et al., 2002). Pyrrhocoricin is also capable of transduction across mammalian cells with limited cytotoxic effects (Kragol et al., 2002) and therefore the potential of this peptide to target C. parvum within intestinal epithelial cells also warrants further investigation. To date very little peptide-mediated delivery carrier research has been conducted on protozoan parasites. The findings of the present study warrant further research into using pyrrhocoricin as a delivery vehicle for antiparasitic peptides, for use as knock-out or knock-down agents in target validation. Acknowledgments The authors thank Daniel Shaw for technical assistance. This study was financially supported by an Australian Research Council Linkage Grant No. LP0349255 and by Phylogica Ltd., Perth, WA. References Carey, C.M., Lee, H., Trevors, J.T., 2004. Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst. Water Research 38, 818–862. Cociancich, S., Dupont, A., Hegy, G., Lanot, R., Holder, F., Hetru, C., Hoffmann, J.A., Bulet, P., 1994. Novel inducible antibacterial peptides from a hemipteran insect, the sap-sucking bug Pyrrhocoris apterus. Biochemical Journal 300, 567–575.
A. Boxell et al. / Experimental Parasitology 119 (2008) 301–303 Cudic, M., Lockatell, C.V., Johnson, D.E., Otvos Jr., L., 2003. In vitro and in vivo activity of an antibacterial peptide analog against uropathogens. Peptides 24, 807–820. Fayer, R., 2004. Cryptosporidium: a water-borne zoonotic parasite. Veterinary Parasitology 126, 37–56. Fayer, R., Morgan, U., Upton, S.J., 2000. Epidemiology of Cryptosporidium: transmission, detection and identification. International Journal for Parasitology 30, 1305–1322. Hijjawi, N.S., Meloni, B.P., Ng’anzo, M., Ryan, U.M., Olson, M.E., Cox, P.T., Monis, P.T., Thompson, R.C., 2004. Complete development of Cryptosporidium parvum in host cell-free culture. International Journal for Parasitology 34, 769–777.
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Kragol, G., Hoffmann, R., Chattergoon, M.A., Lovas, S., Cudic, M., Bulet, P., Condie, B.A., Rosengren, K.J., Montaner, L.J., Otvos Jr., L., 2002. Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin. European Journal for Biochemistry 269, 4226–4237. Puiu, D., Enomoto, S., Buck, G.A., Abrahamsen, M.S., Kissinger, J.C., 2004. CryptoDB: the Cryptosporidium genome resource. Nucleic Acids Research 32 (Database issue), D329–D331. Striepen, B., Kissinger, J.C., 2004. Genomics meets transgenics in search of the elusive Cryptosporidium drug target. Trends in Parasitology 20, 355–358. Watt, P.M., 2006. Screening for peptide drugs from the natural repertoire of biodiverse protein folds. Nature Biotechnology 24, 177–183.
