The neglected human diseases caused by trypanosomatids are currently treated with toxic therapy with limited efficacy. In search for novel anti-trypanosomatid agents, we showed previously that the Crotalus viridis viridis (Cvv) snake venom was active against infective forms of Trypanosoma cruzi. Here, we describe the purification of crovirin, a cysteine-rich secretory protein (CRISP) from Cvv venom with promising activity against trypanosomes and Leishmania.
Crude venom extract was loaded onto a reverse phase analytical (C8) column using a high performance liquid chromatographer. A linear gradient of water/acetonitrile with 0.1% trifluoroacetic acid was used. The peak containing the isolated protein (confirmed by SDS-PAGE and mass spectrometry) was collected and its protein content was measured. T. cruzi trypomastigotes and amastigotes, L. amazonensis promastigotes and amastigotes and T. brucei rhodesiense procyclic and bloodstream trypomastigotes were challenged with crovirin, whose toxicity was tested against LLC-MK2 cells, peritoneal macrophages and isolated murine extensor digitorum longus muscle. We purified a single protein from Cvv venom corresponding, according to Nano-LC MS/MS sequencing, to a CRISP of 24,893.64 Da, henceforth referred to as crovirin. Human infective trypanosomatid forms, including intracellular amastigotes, were sensitive to crovirin, with low IC50 or LD50 values (1.10–2.38 µg/ml). A considerably higher concentration (20 µg/ml) of crovirin was required to elicit only limited toxicity on mammalian cells.
This is the first report of CRISP anti-protozoal activity, and suggests that other members of this family might have potential as drugs or drug leads for the development of novel agents against trypanosomatid-borne neglected diseases.
The pathogenic trypanosomatid parasites of the genera Leishmania and Trypanosoma infect over 20 million people worldwide, with an annual incidence of ∼3 million new infections. An additional 400 million people are at risk of infection by exposure to parasite-infected insects which act as disease vectors. Trypanosomatid-borne diseases predominant in poorer nation and are considered neglected, having failed to attract the attention of the pharmaceutical industry. However, novel therapy is sorely needed for Trypanosoma and Leishmania infections, currently treated with ‘dated’ drugs that are often difficult to administer in resource-limiting conditions, have high toxicity and are by no means always successful, partly due to the emergence of drug resistance. The last few decades have witnessed a growing interest in examining the potential of bioactive toxins and poisons as drugs or drug leads, as well as for diagnostic applications. In this context, we isolated and purified crovirin, a protein from the Crotalus viridis viridis (Cvv) snake venom capable to inhibiting and/or lysing infective forms of trypanosomatid parasites, at concentrations that are not toxic to host cells. This feature makes crovirin a promising candidate protein for the development of novel therapy against neglected diseases caused by trypanosomatid pathogens.