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Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Adade CM, Carvalho AL, Tomaz MA, Costa TF, Godinho JL, Melo PA, Lima AP, Rodrigues JC, Zingali RB, Souto-Padrón T - PLoS Negl Trop Dis (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus

Cytotoxicity analyses of crovirin on LLC-MK2 cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.
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pntd-0003252-g003: Cytotoxicity analyses of crovirin on LLC-MK2 cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.

Mentions: LLC-MK2 cells were treated with crovirin for 72 h and examined for viability using a trypan blue exclusion assay (Fig. 3A). None of the tested crovirin concentrations (4.8, 10 or 20 µg/ml) were capable of inducing significant loss of cell viability, even after 72 h of treatment. In addition, we tested the activity of crovirin against murine peritoneal macrophages to investigate its cytotoxicity towards primary host cells. Treated cells were examined using an MTS assay, and no significant toxicity (p≤0.05) was observed in any treatment conditions (Fig. 3B). Creatine kinase (CK) activity was measured before and two hours after extensor digitorum longus (EDL) muscle exposure to 10 µg/ml crovirin. We did not observed significant CK release from treated EDL muscles compared to control (saline) after 2 hours of incubation with crovirin, indicating that this protein did not generated appreciable myotoxicity at the concentration tested.


Crovirin, a snake venom cysteine-rich secretory protein (CRISP) with promising activity against Trypanosomes and Leishmania.

Adade CM, Carvalho AL, Tomaz MA, Costa TF, Godinho JL, Melo PA, Lima AP, Rodrigues JC, Zingali RB, Souto-Padrón T - PLoS Negl Trop Dis (2014)

Cytotoxicity analyses of crovirin on LLC-MK2 cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4199522&req=5

pntd-0003252-g003: Cytotoxicity analyses of crovirin on LLC-MK2 cells (A) and peritoneal macrophages (B) by trypan blue cell viability and MTS assay, respectively.No tested concentrations induced significant lost of cell viability in either cell type. Error bars represent standard deviation of the mean of 3 independent experiments.
Mentions: LLC-MK2 cells were treated with crovirin for 72 h and examined for viability using a trypan blue exclusion assay (Fig. 3A). None of the tested crovirin concentrations (4.8, 10 or 20 µg/ml) were capable of inducing significant loss of cell viability, even after 72 h of treatment. In addition, we tested the activity of crovirin against murine peritoneal macrophages to investigate its cytotoxicity towards primary host cells. Treated cells were examined using an MTS assay, and no significant toxicity (p≤0.05) was observed in any treatment conditions (Fig. 3B). Creatine kinase (CK) activity was measured before and two hours after extensor digitorum longus (EDL) muscle exposure to 10 µg/ml crovirin. We did not observed significant CK release from treated EDL muscles compared to control (saline) after 2 hours of incubation with crovirin, indicating that this protein did not generated appreciable myotoxicity at the concentration tested.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem, Rio de Janeiro, Brazil.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions: 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.

No MeSH data available.


Related in: MedlinePlus