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Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites

28 Oct 2008

Marcia Triunfol

Source: International Journal for Parasitology (see original article)

Citation: Franke-Fayard B, Djokovic D, Dooren MW, Ramesar J, Waters AP, Falade MO, Kranendonk M, Martinelli A, Cravo P, Janse CJ (2008) Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites. Int J Parasitol; Jun 12. [Epub ahead of print].

The testing of new anti-malarial drugs requires that at least two steps are undertaken before testing in humans may take place. In the first step, the new drug is tested in vitro and then – if promising results are obtained – it is tested in vivo.

In the first step of the process, assays have been developed that focus on the drug’s ability to affect parasite growth in red cells. In these assays, P. falciparum is the parasite used if the drug is intended for humans. In the second step of the process, animal models, usually rodents, are used to test the drug efficacy. In this step, species-specific parasites such as P. berghei are used.

However, both steps of this process still have limitations, as the procedures currently in use to determine parasite growth (which is the main parameter to evaluate drug efficacy) are time consuming, complicated, and labour intensive, which delays development of a high-throughput method for drug screening.

In an attempt to overcome these obstacles, Franke-Fayard and colleagues developed a fast and simple system to quantify parasite growth, both in vivo and in vitro. They used a system that has been largely used to assess gene expression, but not to quantify plasmodium growth: the luciferase system.

Recent studies have used the GFP system for assessing parasite growth. GFP stands for green fluorescent protein, a protein usually found in corals. The main usefulness of this protein is that it becomes fluorescent when other proteins are attached to it. In other words, if one wants to know if a certain organism is producing a certain protein, a fast and simple way to find out is by using a GFP system in which the protein of interest binds to GFP once it is produced. When the binding takes place GFP produces a fluorescence that can normally be detected by a microplate reader. However, previous studies have used FACS (fluorescence activated cell sorter) to assess parasite growth. The technique is time-consuming and complex. Also, GPF-fluorescence needs to be performed in live parasites, besides the fact that ring-form stages of the parasite containing GFP are difficult to distinguish from unaffected cells, which compromises estimates.

This is where innovation comes in. To be able to quantify parasite growth using a simple microplate reader, the authors used an alternative system known as the luciferase reporter protein system. The luciferase protein is also the result of a light-emitting reaction. To build a DNA construct, the luciferase gene is strategically engineered next to a specific DNA sequence known as a promoter, which is a gene-specific sequence that triggers transcription. The luciferase system is used to investigate whether a promoter of interest is working. If a gene or group of genes that are triggered by that promoter is known, then it is possible to assume whether they are being expressed by analyzing the luciferase activity. When the promoter works, it transcribes the luciferase gene that results in the production of the luciferase protein which then emits light that can be quantified using a micrplater. One can then assume that the other genes that work under the same promoter are also been transcribed in the same rate.

To estimate parasite growth, the authors built cell lines that were infected with DNA constructs carrying the luciferase system. In each of the cell lines a specific promoter was added to the luciferase system, either the ama-1 promoter that is schizont-specific and therefore to be active only in schizont-stage parasites, or the eef1 α α, which is a constitutive promoter that is expected to be working all times.

To test these systems, the effect of well-known anti-malarial drugs on parasite growth was evaluated. The first system (ama-1 promoter) was an in vitro assay used to evaluate the drug effect on schizont growth, whereas the second system (eef1 α α) was an in vivo test evaluated through analysing drug effects on parasite growth during all stages.

The results obtained with these two systems were similar to those obtained with systems used previously, showing that this new strategy of assessing parasite growth luminescence assays is ready to be largely applied in other studies. Hopefully, the development of such assays will foster the testing of new drug candidates to be used against malaria.

Note: This article is published in a journal which is not open access. To see the full article a subscription to the International Journal for Parasitology is therefore required. In some developing countries, readers who are based in institutions may be able to access it through the HINARI programme.

Source

Title:
Simple and sensitive antimalarial drug screening in vitro and in vivo using transgenic luciferase expressing Plasmodium berghei parasites.

Authors:
Franke-Fayard B, Djokovic D, Dooren MW, Ramesar J, Waters AP, Falade MO, Kranendonk M, Martinelli A, Cravo P, Janse CJ

References:
Int. J. Parasitol. Dec 2008: Vol. 38 no. 14, pp. 1651-62

Abstract:
We report two improved assays for in vitro and in vivo screening of chemicals with potential anti-malarial activity against the blood stages of the rodent malaria parasite Plasmodiumberghei. These assays are based on the determination of luciferase activity (luminescence) in small blood samples containing transgenic blood stage parasites that express luciferase under the control of a promoter that is either schizont-specific (ama-1) or constitutive (eef1alphaa). Assay 1, the in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. The second assay, the in vivo drug-luminescence (IVDL) assay, measured parasite growth in vivo in a standard 4-day suppressive drug test, monitored by measuring the constitutive luciferase activity of circulating parasites (eef1alphaa promoter). The IVDL assay generates growth-curves that are identical to those obtained by manual counting of parasites in Giemsa-stained smears. The reading of luminescence assays is rapid, requires a minimal number of handling steps and no experience with parasite morphology or handling fluorescence-activated cell sorters, produces no radioactive waste and test-plates can be stored for prolonged periods before processing. Both tests are suitable for use in larger-scale in vitro and in vivo screening of drugs. The standard methodology of anti-malarial drug screening and validation, which includes testing in rodent models of malaria, can be improved by the incorporation of such assays.

PMID: 18590736
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