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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: 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).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

Crovirin effects on T. cruzi trypomastigotes (A) and intracellular amastigotes (B), L. amazonensis promastigotes (C) and intracellular amastigotes (D), and T. brucei rhodesiense PCF (procyclic form, in E) and BSF (bloodstream forms, in F).T. cruzi trypomastigotes were treated with crovirin in RPMI medium with 10% FCS for 24 h only, since they do not survive in growth medium for longer and do not divide. T. brucei BSF and PCF and L. amazonensis promastigotes were treated with crovirin in complete growth media, for up to 72 h. Amastigotes of T. cruzi and Leishmania were treated with crovirin as dividing intracellular forms (infecting peritoneal mouse macrophages). Error bars represent standard deviation the mean of 3 independent experiments.
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pntd-0003252-g004: Crovirin effects on T. cruzi trypomastigotes (A) and intracellular amastigotes (B), L. amazonensis promastigotes (C) and intracellular amastigotes (D), and T. brucei rhodesiense PCF (procyclic form, in E) and BSF (bloodstream forms, in F).T. cruzi trypomastigotes were treated with crovirin in RPMI medium with 10% FCS for 24 h only, since they do not survive in growth medium for longer and do not divide. T. brucei BSF and PCF and L. amazonensis promastigotes were treated with crovirin in complete growth media, for up to 72 h. Amastigotes of T. cruzi and Leishmania were treated with crovirin as dividing intracellular forms (infecting peritoneal mouse macrophages). Error bars represent standard deviation the mean of 3 independent experiments.

Mentions: We tested crovirin activity against the two infective T. cruzi forms, trypomastigotes and amastigotes. Trypomastigote forms do not multiply and do not remain viable after several days in culture media at 37°C. Therefore, the effect of crovirin towards T. cruzi trypomastigotes was evaluated as the ability of the protein to lyse cells after 24 h of treatment (Fig. 4A). The calculated LD50 of crovirin for trypomastigotes was 1.10±0.13 µg/ml (Table 1). This concentration displayed the second higher selectivity index (18.2) (Table 1) among all crovirin treatments.


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)

Crovirin effects on T. cruzi trypomastigotes (A) and intracellular amastigotes (B), L. amazonensis promastigotes (C) and intracellular amastigotes (D), and T. brucei rhodesiense PCF (procyclic form, in E) and BSF (bloodstream forms, in F).T. cruzi trypomastigotes were treated with crovirin in RPMI medium with 10% FCS for 24 h only, since they do not survive in growth medium for longer and do not divide. T. brucei BSF and PCF and L. amazonensis promastigotes were treated with crovirin in complete growth media, for up to 72 h. Amastigotes of T. cruzi and Leishmania were treated with crovirin as dividing intracellular forms (infecting peritoneal mouse macrophages). Error bars represent standard deviation the mean of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003252-g004: Crovirin effects on T. cruzi trypomastigotes (A) and intracellular amastigotes (B), L. amazonensis promastigotes (C) and intracellular amastigotes (D), and T. brucei rhodesiense PCF (procyclic form, in E) and BSF (bloodstream forms, in F).T. cruzi trypomastigotes were treated with crovirin in RPMI medium with 10% FCS for 24 h only, since they do not survive in growth medium for longer and do not divide. T. brucei BSF and PCF and L. amazonensis promastigotes were treated with crovirin in complete growth media, for up to 72 h. Amastigotes of T. cruzi and Leishmania were treated with crovirin as dividing intracellular forms (infecting peritoneal mouse macrophages). Error bars represent standard deviation the mean of 3 independent experiments.
Mentions: We tested crovirin activity against the two infective T. cruzi forms, trypomastigotes and amastigotes. Trypomastigote forms do not multiply and do not remain viable after several days in culture media at 37°C. Therefore, the effect of crovirin towards T. cruzi trypomastigotes was evaluated as the ability of the protein to lyse cells after 24 h of treatment (Fig. 4A). The calculated LD50 of crovirin for trypomastigotes was 1.10±0.13 µg/ml (Table 1). This concentration displayed the second higher selectivity index (18.2) (Table 1) among all crovirin treatments.

Bottom Line: 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).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