In Vitro Screening Test Using Leishmania Promastigotes Stably Expressing mCherry Protein

Abstract

Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instruments.P rotozoan parasites of the genus Leishmania are the causative agents of a wide spectrum of human and animal diseases. The clinical manifestations of Leishmania infections range from lesions of the skin and mucous membranes to lethality, the latter caused by visceral species, including Leishmania donovani (1). Leishmania species cause morbidity and mortality throughout large areas of the Old and New World. Leishmaniasis is an emerging disease with an annual incidence of 2 million cases, and more than 12 million people are infected in over 80 countries where the disease is endemic (2), causing 80,000 deaths per year, and 350 million people are at risk of infection and disease. The increased prevalence of Leishmania-human immunodeficiency virus (HIV) coinfection is the major reason for the recent emergence of leishmaniasis in the Western world (3).Since their discovery in the 1940s, the highly toxic pentavalent antimonials [Sb(V)] have been the primary first-line treatment for all types of leishmaniasis in most parts of the world. However, the increasing frequency of relapse in leishmaniasis patients is forcing the use of other chemotherapeutic agents, including amphotericin B, isethionate pentamidine, paromomycin, and miltefosine (4, 5). Unfortunately, the majority of these antiparasitic drugs present severe side effects, there is no guarantee of cure, and some of them are frequently accompanied by emergence of drug resistance (6-8).Alternative treatments are needed; however, advances in drug discovery approaches have not been satisfactory. Although the use of axenic promastigote cultures of Leishmania as a primary screening method has being validated (7-11), the traditional approaches employed to determine the drug effect in culture present several shortcomings. Microscopic counting is prone to error, time-consuming, and requires trained personnel; the use of tetrazolium salts such as MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] to measure cell viability is labor-intensive, and results are not always reliable (12). The more sensitive and less labor-intensive colorimetric resazurin-based method may present variable metabolic behavior under different cell culture conditions. Furthermore, it is not appropriate to perform kinetic experiments due to the significant cytotoxicity that results from prolonged exposure to the reagent (13-15). Other disadvantages of these metabolic assays are the requirement to incubate the substrate with viable cells at 37°C for a sufficient time to generate a measurable signal. This may lead to undesirable artifacts resulting from interactions between the reagent, the tested drug, and the biochemistry ...

Genomic Appraisal of the Multifactorial Basis forIn VitroAcquisition of Miltefosine Resistance in Leishmania donovani

“…S1 in the supplemental material). In order to fully determine drug susceptibility, an EC 50 assay was performed using the resazurin-based CellTiter-Blue method (Promega) (18). As expected, cultures grown in the presence of higher HePC concentrations displayed reduced susceptibilities (2.4-to 7.7-fold more resistant) to the drug, in contrast to cultures grown under lower HePC concentrations (Fig.…”

“…The leishmanicidal effects of HePC and the reference compounds amphotericin B (AmpB) (Sigma), pentamidine isethionate (PI) (Sigma), paromomycin (Pm) (Sigma), and potassium antimony(III) tartrate (SbIII) (Sigma) were evaluated in the resistant strains and compared to those in WT cultures. Culture viability was measured by using the resazurin-based method (CellTiter-Blue [Promega]) (18). Briefly, 1 ϫ 10 6 parasites/ml were seeded in a 96-well plate and incubated in the presence of increasing drug concentrations for 48 h at 26°C, along with appropriate solvent controls.…”

A mitochondrial HSP70 (HSPA9B) is linked to miltefosine resistance and stress response in Leishmania donovani

“…The leishmanicidal effect of HePC (Sigma-Aldrich, St. Louis, USA), pentamidine isethionate (Sigma-Aldrich, St. Louis, USA), amphotericin B (Sigma-Aldrich, St. Louis, USA), potassium antimony (III) tartrate hydrate (Sigma-Aldrich, St. Louis, USA) and paromomycin sulfate salt (Sigma-Aldrich, St. Louis, USA) was also evaluated. For this purpose, culture viability was measured by using the resazurin-based method CellTiter-Blue (Promega, Fitchburg, USA) as described previously [26]. Briefly, 200 μl 1 × 10 6 parasites/ml were seeded in each well of a 96-well plate and incubated in the presence of increasing drug concentrations for 48 h at 26 °C along with appropriate solvent controls.…”

Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major

“…Toxicity against L. major promastigotes and intracellular amastigotes was assessed using the fluorometric resazurin-based method CellTiter-Blue (Promega) as previously described (35). For promastigotes, 10 6 cells/ml were seeded in a 96-well plate and incubated in the presence of increasing concentrations of ZnDPA complex for 72 h at 27°C, along with the appropriate solvent controls.…”

“…Leishmania major axenic promastigotes (MHOM/JL/80/Friedlin), a fluorescent transgenic L. major strain constitutively expressing mCherry (mCherry-L. major), and a fluorescent transgenic strain of mCherry-L. donovani axenic amastigotes were maintained at 27°C in M199 medium supplemented with 10% fetal bovine serum (FBS; Atlanta Biolabs) (35).…”