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In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

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Related in: MedlinePlus

Fluorescence evaluation of tdTomato parasites.(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.
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pntd-0000740-g002: Fluorescence evaluation of tdTomato parasites.(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.

Mentions: The tdTomato gene used in these studies was generated by genetically fusing two copies of the gene encoding the Tomato Red Fluorescent protein to create a tandem dimer (td) named tdTomato [25]. This construct possesses many desirable properties relative to previously studied fluorescent proteins, including a faster and more complete maturation and increased brightness [25], [35]. In addition, the tdTomato fluorescence signal can be detected at ≥620 nm (outside the range of of the bulk of animal tissue autofluorescence), which minimizes autofluorescence and greatly increases the penetration of fluorescence signals into the tissues [25], [36]. The expression vector pTrex-Neo [26], carrying the T. cruzi ribosomal promoter, has been shown to provide strong protein expression in T. cruzi and to enable the stable integration of exogenous genes into the ribosomal locus [26], [37]–[38]. Thus the gene encoding tdTomato was cloned into pTrex-Neo and the pTREX-Neo-tdTomato (Figure 1A) construct transfected into epimastigotes of various T. cruzi strains, which were then drug-selected as described in the Materials and Methods. Parasite fluorescence was monitored by microscopy and flow cytometry (Figure 2A–B). Transfectant parasites expressing the tomato protein showed a bright red fluorescence distributed throughout the cell in all life cycle stages (Figure 2A). Moreover, the fluorescence was stable in the absence of antibiotic pressure for >5 months (Figure 2B).


In vitro and in vivo high-throughput assays for the testing of anti-Trypanosoma cruzi compounds.

Canavaci AM, Bustamante JM, Padilla AM, Perez Brandan CM, Simpson LJ, Xu D, Boehlke CL, Tarleton RL - PLoS Negl Trop Dis (2010)

Fluorescence evaluation of tdTomato parasites.(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903469&req=5

pntd-0000740-g002: Fluorescence evaluation of tdTomato parasites.(A) Microscope image showing the tdTomato expressing parasites in all the life stages. (B) The fluorescence intensity in epimastigotes was assessed using a CyAn flow cytometer (DakoCytomation) and analyzed with FlowJo software (Tree Star). No decrease in fluorescence intensity was observed in parasites cultured for >5 months with (red) and without antibiotic (blue). The background fluorescence of WT parasites (green) can also be observed.
Mentions: The tdTomato gene used in these studies was generated by genetically fusing two copies of the gene encoding the Tomato Red Fluorescent protein to create a tandem dimer (td) named tdTomato [25]. This construct possesses many desirable properties relative to previously studied fluorescent proteins, including a faster and more complete maturation and increased brightness [25], [35]. In addition, the tdTomato fluorescence signal can be detected at ≥620 nm (outside the range of of the bulk of animal tissue autofluorescence), which minimizes autofluorescence and greatly increases the penetration of fluorescence signals into the tissues [25], [36]. The expression vector pTrex-Neo [26], carrying the T. cruzi ribosomal promoter, has been shown to provide strong protein expression in T. cruzi and to enable the stable integration of exogenous genes into the ribosomal locus [26], [37]–[38]. Thus the gene encoding tdTomato was cloned into pTrex-Neo and the pTREX-Neo-tdTomato (Figure 1A) construct transfected into epimastigotes of various T. cruzi strains, which were then drug-selected as described in the Materials and Methods. Parasite fluorescence was monitored by microscopy and flow cytometry (Figure 2A–B). Transfectant parasites expressing the tomato protein showed a bright red fluorescence distributed throughout the cell in all life cycle stages (Figure 2A). Moreover, the fluorescence was stable in the absence of antibiotic pressure for >5 months (Figure 2B).

Bottom Line: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use.In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato).This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting.

View Article: PubMed Central - PubMed

Affiliation: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT

Background: The two available drugs for treatment of T. cruzi infection, nifurtimox and benznidazole (BZ), have potential toxic side effects and variable efficacy, contributing to their low rate of use. With scant economic resources available for antiparasitic drug discovery and development, inexpensive, high-throughput and in vivo assays to screen potential new drugs and existing compound libraries are essential.

Methods: In this work, we describe the development and validation of improved methods to test anti-T. cruzi compounds in vitro and in vivo using parasite lines expressing the firefly luciferase (luc) or the tandem tomato fluorescent protein (tdTomato). For in vitro assays, the change in fluorescence intensity of tdTomato-expressing lines was measured as an indicator of parasite replication daily for 4 days and this method was used to identify compounds with IC(50) lower than that of BZ.

Findings: This method was highly reproducible and had the added advantage of requiring relatively low numbers of parasites and no additional indicator reagents, enzymatic post-processes or laborious visual counting. In vivo, mice were infected in the footpads with fluorescent or bioluminescent parasites and the signal intensity was measured as a surrogate of parasite load at the site of infection before and after initiation of drug treatment. Importantly, the efficacy of various drugs as determined in this short-term (<2 weeks) assay mirrored that of a 40 day treatment course.

Conclusion: These methods should make feasible broader and higher-throughput screening programs needed to identify potential new drugs for the treatment of T. cruzi infection and for their rapid validation in vivo.

Show MeSH
Related in: MedlinePlus