• View in gallery

    A, Epifluorescent microscopic image showing expression of green fluorescent protein (GFP) in an electroporated Leish-mania donovani field isolate. Cells were viewed after washing and fixing in 3.8% formaldehyde in phosphate-buffered saline (magnification ×100). B, Confocal microscopic image of intracellular amasti-gotes expressing GFP from an episomal vector (magnification ×40).

  • View in gallery

    Fluorescence-activated cell sorting analysis of the parasitocidal effect of drugs on a green fluorescent protein (GFP)-transfected Leishmania donovani field isolate. A, pXG-GFP transfected L. donovani promastigotes without any drug. B, pXG-GFP transfected L. donovani with drugs. Purple indicates untransfected L. donovani promastigotes and green indicates transfectants. The histograms represent relative GFP fluorescence plotted against frequency of events per channel (equivalent to cell number).

  • 1

    Berman JD, 1991. Clinical diagnostic and chemical developments in the last 10 years. Clin Infect Dis 24 :684–703.

  • 2

    Desjeux P, 2001. The increase in risk factors for leishmaniasis world-wide. Trans R Soc Trop Med Hyg 95 :239–243.

  • 3

    Buckner FS, Verlinde CL, La Flamme AC, van Voorhis WC, 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob Agents Chemother 40 :2592–2597.

    • Search Google Scholar
    • Export Citation
  • 4

    Collins LA, Torrero MN, Franzblau SG, 1998. Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis. Anti-microb Agents Chemother 42 :344–347.

    • Search Google Scholar
    • Export Citation
  • 5

    McFadden DC, Seeber F, Boothroyd JC, 1997. Use of Toxo-plasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro. Antimicrob Agents Chemother 41 :1849–1853.

    • Search Google Scholar
    • Export Citation
  • 6

    Roy G, Dumas C, Sereno D, Wu Y, Singh AK, Tremblay MJ, Ouellette M, Olivier M, Papadopoulou B, 2000. Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol Biochem Parasitol 110 :195–206.

    • Search Google Scholar
    • Export Citation
  • 7

    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC, 1994. Green fluorescent protein as a marker for gene expression. Science 263 :802–805.

    • Search Google Scholar
    • Export Citation
  • 8

    Singh N, 2002. Is there true Sb (V) resistance in Indian kala azar field isolates? Curr Sci 83 :101–102.

  • 9

    Ha DS, Schwarz JK, Turco SJ, Beverley SM, 1996. Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 77 :57–64.

    • Search Google Scholar
    • Export Citation
  • 10

    Singh N, Singh RT, Sundar S, 2002. Identification of a gene linked to drug resistance in field isolates of Leishmania donovani. Ann Trop Med Parasitol 96 :839–841.

    • Search Google Scholar
    • Export Citation
  • 11

    Ganguly NK, 2002. Oral miltefosine may revolutionize treatment of visceral leishmaniasis. TDR News 68 :2.

  • 12

    Thakur CP, 1993. Diminishing effectiveness of currently used drugs in treatment of kala azar and amphotericin B in antimony and pentamidine resistant kala azar. Bhaduri, AN, Basu MK, Sen AK, Kumar S, eds. Current Trends in Leishmania Research. New Delhi: Publications and Information Directorate, Council of Scientific and Industrial Research, 254–262.

  • 13

    Narain L, Dutta GP, 1978. Cultivation and in vitro chemotherapeutic studies on Leishmania donovani. Indian J Parasitol 2 :83–86.

  • 14

    de Jimenez G, Ercoli N, 1965. Effect of drugs on various leishmania isolates and succinic dehydrogenase inhibition. Exp Parasitol 17 :302–308.

    • Search Google Scholar
    • Export Citation
  • 15

    Lira R, Sundar S, Makharia A, Kenney R, Gam A, Saraiva E, Sacks D, 1999. Evidence that the high incidence of treatment failures in Indian kala azar is due to the emergence of antimony resistant strains of Leishmania donovani. J Infect Dis 180 :564–567.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

SHORT REPORT: FLUORESCENT LEISHMANIA: APPLICATION TO ANTI-LEISHMANIAL DRUG TESTING

View More View Less
  • 1 Division of Biochemistry and Division of Parasitology, Central Drug Research Institute, Lucknow, India

Classic techniques for detecting the susceptibility of Leishmania to different drugs are time-consuming, laborious, and require the use of macrophages. The use of flow cytometry for monitoring Leishmania susceptibility to drugs is beginning to be implemented. Using green fluorescent protein (GFP), we have improved and simplified the screening procedure. We introduced a GFP marker into field strains of Leishmania causing kala-azar (visceral leishmaniasis) and explored the suitability of transgenic L. donovani promastigotes that constitutively express GFP in their cytoplasm as target cells for in vitro screening of anti-leishmanial drugs.

Leishmania are protozoan parasites that affect millions of people worldwide.1 A recent World Health Organization report shows that an estimated 350 million people are at the risk of infection and approximately two million are infected annually (http://www.who.int/emc/diseases/leish/leis.html). Along with Brazil, Sudan, and Bangladesh, India contributes 90% of the global burden of visceral leishmaniasis.2 The current outlook for chemotherapy of leishmaniasis is more promising than it has been for several years with advances in combinatorial chemistry and (high through-put screening methods set to revolutionize lead discovery and lead optimization programmes). In addition, reporter genes will improve assays both in vitro and in vivo for the identification and evaluation of new drugs from these libraries. The use of reporter genes in a number of intracellular microorganisms has facilitated antimicrobial drug discovery and testing.3–5 Luciferase has been transfected into Leishmania promastigotes.6 Green fluorescent protein (GFP) from Aequorea victoria7 is a cytoplasmic protein with low toxicity that can be continuously synthesized and can easily imaged and quantitated. Consequently, we investigated the use of GFP-transfected L. donovani clinical isolates for screening compounds for leishmanicidal activity.

Field isolates of L. donovani were collected and cultured as previously described.8 The Leishmania expression vector pXG-GFP was obtained from Dr. Stephen Beverley (Washington University, St. Louis, MO).9 Electroporation of Leishmania promastigotes was done as previously described.10 Briefly, 20 μg of circular plasmid DNA was added to cells that were immediately transfected by electroporation with a Genepulser II apparatus (Bio-Rad Laboratories, Hercules, CA). After electroporation, cells were placed on ice for two minutes before being transferred to liquid medium. The selection with the drug was initiated 24 hours after electroporation. The growth of cells highly resistant to geneticin disul-fate (G418) was observed after 14–20 days. Cells were analyzed on an FACSscan analytical flow cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with a 15-mV, 488-nm, air-cooled argon ion laser. Thirty thousand cells were acquired for each analysis. Analysis by fluorescence-activated cell sorting (FACS) showed a clear quantitative separation between transfected and control parasites. Since the transfectants were adapted to increasing drug concentrations, their relative fluorescence increased accordingly. The parasites were then routinely grown in 720 μg/mL of G418 because increasing the drug concentration did not result in an increase in arbitrary fluorescence units.

The transfectants were examined for GFP expression by confocal microscopy (Bio-Rad Laboratories). As shown in Figure 1A, GFP was completely localized in the cytosol. Cells were viewed after washing and fixing in 3.8% formaldehyde in phosphate-buffered saline (PBS). These promastigotes were used to infect J774 macrophages at a host:parasite ratio of 1:108. Confocal microscopy showed that the intracellular amastigotes expressing GFP from the episomal vector could be visualized directly without the need for any cumbersome antibody staining step (Figure 1B).

Flow cytometry was used to quantify the parasitocidal effect of commonly used anti-leishmanial drugs. A promastigote suspension (2 × 106 /mL) of transgenic Leishmania (medium M199 supplemented with 20% fetal calf serum) in late log phase was seeded in duplicate into 24-well flat-bottom microtrays. They were co-incubated with different drug concentrations, ranging from 0 to 100 μg. After incubation at 26°C for 24 hours, the parasites were washed and suspended in PBS, followed by flow cytometric analysis. We initiated the study with four antileishmanial standard drugs along with miltefosine, the drug that has been licensed in India and is undergoing phase IV trials.11 Three drugs, pentavalent antimonial, pentamidine, and amphotericin B, are routinely used in the treatment of kala-azar (visceral leishmaniasis). In India, only sodium stibogluconate is used as a first-line drug and only pentamidine is used as a second-line drug. The use of amphotericin B evolved because it is an effective drug for antimony- and pentamidine-resistant cases. Allopurinol has shown a poor efficacy of 5.6%.12 Promastigotes are not susceptible to pentavalent antimonial drugs.13 However, ampho-tericin B and pentamidine are active against the promastigote form of the parasite.14 Our results were consistent with those of these reports. When compared with the control (Figure 2A), which is parasites without drug, allopurinol and sodium stibogluconate (Figure 2B) showed no parasitocidal effect on promastigotes, whereas miltefosine (Figure 2B) and ampho-tericin B showed a greater dose-dependent efficacy than pent-amidine.

Thus, using a GFP-transfected clinical isolate of L. dono-vani, we have demonstrated the parasitocidal effect of drugs as previously reported in literature, but in a quicker, more reliable, simpler, less expensive, and faster manner. This rapid determination of drug activity by FACS required no additional antibody staining. This in vitro technique shows numerous advantages over the traditional drug screening procedures or over the luciferase system because it does not require either substrates or cofactors due to the intrinsically fluorescent nature of the protein. Although animal models are well established for drug testing, they are not suitable for large-scale primary drug screening. Since cells are not killed during this procedure, it is possible to perform high-efficiency screening in microtiter plates. This technique is currently being automated into a 96-well format for high-efficiency screening of compounds from libraries at our Institute. In addition, since it is now established that treatment failures in patients may be due to the unresponsiveness of the parasite to a specific drug,8,15 we have introduced the GFP marker into field strains not responsive to treatment to test whether we can detect resistance in in vitro models and in animals.

Figure 1.
Figure 1.

A, Epifluorescent microscopic image showing expression of green fluorescent protein (GFP) in an electroporated Leish-mania donovani field isolate. Cells were viewed after washing and fixing in 3.8% formaldehyde in phosphate-buffered saline (magnification ×100). B, Confocal microscopic image of intracellular amasti-gotes expressing GFP from an episomal vector (magnification ×40).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 4; 10.4269/ajtmh.2004.71.400

Figure 2.
Figure 2.

Fluorescence-activated cell sorting analysis of the parasitocidal effect of drugs on a green fluorescent protein (GFP)-transfected Leishmania donovani field isolate. A, pXG-GFP transfected L. donovani promastigotes without any drug. B, pXG-GFP transfected L. donovani with drugs. Purple indicates untransfected L. donovani promastigotes and green indicates transfectants. The histograms represent relative GFP fluorescence plotted against frequency of events per channel (equivalent to cell number).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 71, 4; 10.4269/ajtmh.2004.71.400

Authors’ addresses: Corresponding author: Neeloo Singh, Division of Biochemistry, Central Drug Research Institute, Lucknow, India, Telephone: 91-522-212-41118, Fax: 91-522-222-3405, E-mail: neeloo888@yahoo.com. Anuradha Dube, Division of Parasitology, Central Drug Research Institute, Lucknow, India.

Acknowledgment: We are indebted to Stephen Beverley (Washington University, St. Louis, MO) for plasmid pXG-GFP.

REFERENCES

  • 1

    Berman JD, 1991. Clinical diagnostic and chemical developments in the last 10 years. Clin Infect Dis 24 :684–703.

  • 2

    Desjeux P, 2001. The increase in risk factors for leishmaniasis world-wide. Trans R Soc Trop Med Hyg 95 :239–243.

  • 3

    Buckner FS, Verlinde CL, La Flamme AC, van Voorhis WC, 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob Agents Chemother 40 :2592–2597.

    • Search Google Scholar
    • Export Citation
  • 4

    Collins LA, Torrero MN, Franzblau SG, 1998. Green fluorescent protein reporter microplate assay for high-throughput screening of compounds against Mycobacterium tuberculosis. Anti-microb Agents Chemother 42 :344–347.

    • Search Google Scholar
    • Export Citation
  • 5

    McFadden DC, Seeber F, Boothroyd JC, 1997. Use of Toxo-plasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro. Antimicrob Agents Chemother 41 :1849–1853.

    • Search Google Scholar
    • Export Citation
  • 6

    Roy G, Dumas C, Sereno D, Wu Y, Singh AK, Tremblay MJ, Ouellette M, Olivier M, Papadopoulou B, 2000. Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models. Mol Biochem Parasitol 110 :195–206.

    • Search Google Scholar
    • Export Citation
  • 7

    Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC, 1994. Green fluorescent protein as a marker for gene expression. Science 263 :802–805.

    • Search Google Scholar
    • Export Citation
  • 8

    Singh N, 2002. Is there true Sb (V) resistance in Indian kala azar field isolates? Curr Sci 83 :101–102.

  • 9

    Ha DS, Schwarz JK, Turco SJ, Beverley SM, 1996. Use of the green fluorescent protein as a marker in transfected Leishmania. Mol Biochem Parasitol 77 :57–64.

    • Search Google Scholar
    • Export Citation
  • 10

    Singh N, Singh RT, Sundar S, 2002. Identification of a gene linked to drug resistance in field isolates of Leishmania donovani. Ann Trop Med Parasitol 96 :839–841.

    • Search Google Scholar
    • Export Citation
  • 11

    Ganguly NK, 2002. Oral miltefosine may revolutionize treatment of visceral leishmaniasis. TDR News 68 :2.

  • 12

    Thakur CP, 1993. Diminishing effectiveness of currently used drugs in treatment of kala azar and amphotericin B in antimony and pentamidine resistant kala azar. Bhaduri, AN, Basu MK, Sen AK, Kumar S, eds. Current Trends in Leishmania Research. New Delhi: Publications and Information Directorate, Council of Scientific and Industrial Research, 254–262.

  • 13

    Narain L, Dutta GP, 1978. Cultivation and in vitro chemotherapeutic studies on Leishmania donovani. Indian J Parasitol 2 :83–86.

  • 14

    de Jimenez G, Ercoli N, 1965. Effect of drugs on various leishmania isolates and succinic dehydrogenase inhibition. Exp Parasitol 17 :302–308.

    • Search Google Scholar
    • Export Citation
  • 15

    Lira R, Sundar S, Makharia A, Kenney R, Gam A, Saraiva E, Sacks D, 1999. Evidence that the high incidence of treatment failures in Indian kala azar is due to the emergence of antimony resistant strains of Leishmania donovani. J Infect Dis 180 :564–567.

    • Search Google Scholar
    • Export Citation
Save