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Development and validation of four Leishmania species constitutively expressing GFP protein. A model for drug discovery and disease pathogenesis studies

Published online by Cambridge University Press:  20 November 2013

ASHA PARBHU PATEL ,
ANDREW DEACON  and
GIULIA GETTI
ASHA PARBHU PATEL
Affiliation:
School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK
ANDREW DEACON
Affiliation:
School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK
GIULIA GETTI*
Affiliation:
School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK
*
*Corresponding author: School of Science, University of Greenwich at Medway, Chatham ME4 4TB, UK. E-mail: [email protected]
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Summary

Green fluorescent protein (GFP)-parasite transfectants have been widely used as a tool for studying disease pathogenesis in several protozoan models and their application in drug screening assays has increased rapidly. In the past decade, the expression of GFP has been established in several Leishmania species, mostly for in vitro studies. The current work reports generation of four transgenic parasites constitutively expressing GFP (Leishmania mexicana, Leishmania aethiopica, Leishmania tropica and Leishmania major) and their validation as a representative model of infection. This is the first report where stable expression of GFP has been achieved in L. aethiopica and L. tropica. Integration of GFP was accomplished through homologous recombination of the expression construct, pRib1.2αNEOαGFP downstream of the 18S rRNA promoter in all species. A homogeneous and high level expression of GFP was detected in both the promastigote and the intracellular amastigote stages. All transgenic species showed the same growth pattern, ability to infect mammalian host cells and sensitivity to reference drugs as their wild type counterparts. All four transgenic Leishmania are confirmed as models for in vitro and possibly in vivo infections and represent an ideal tool for medium throughput testing of compound libraries.

Keywords

GFPTHP-1Leishmania aethiopicaLeishmania majorLeishmania mexicanaLeishmania tropicaflow cytometryintracellular amastigote
Type
Research Article
Information
Parasitology , Volume 141 , Issue 4 , April 2014 , pp. 501 - 510
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Copyright
Copyright © Cambridge University Press 2013 

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References

REFERENCES

Alam, A., Goyal, M., Iqbal, M. S., Pal, C., Dey, S., Bindu, S., Maity, P. and Bandyopadhyay, U. (2009). Novel antimalarial drug targets: hope for new antimalarial drugs. Expert Review of Clinical Pharmacology 2, 469–489.CrossRefGoogle ScholarPubMed
Bolhassani, A., Taheri, T., Taslimi, Y., Zamanilui, S., Zahedifard, F., Seyed, N., Torkashvand, F., Vaziri, B. and Rafati, S. (2011). Fluorescent Leishmania species: development of stable GFP expression and its application for in vitro and in vivo studies. Experimental Parasitology 127, 637–645.Google Scholar
Boucher, N., McNicoll, F., Dumas, C. and Papadopoulou, B. (2002). RNA polymerase I-mediated transcription of a reporter gene integrated into different loci of Leishmania . Molecular and Biochemical Parasitology 119, 153–158.Google Scholar
Chan, M. M. Y., Bulinski, J. C., Chang, K. P. and Fong, D. (2003). A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein. Parasitology Research 89, 266–271.Google Scholar
Croft, S. L., Yardley, V. and Kendrick, H. (2002). Drug sensitivity of Leishmania species: some unresolved problems. Transactions of the Royal Society of Tropical Medicine and Hygiene 96(Suppl. 1), S127–S129.Google Scholar
Croft, S. L., Seifert, K. and Yardley, V. (2006). Current scenario of drug development for leishmaniasis. Indian Journal of Medical Research 123, 399–410.Google ScholarPubMed
Dube, A., Gupta, R. and Singh, N. (2009). Reporter genes facilitating discovery of drugs targeting protozoan parasites. Trends in Parasitology 25, 432–439.Google Scholar
Ejazi, S. A. and Ali, N. (2013). Developments in diagnosis and treatment of visceral leishmaniasis during the last decade and future prospects. Expert Review of Anti-Infective Therapy 11, 79–98.CrossRefGoogle ScholarPubMed
Escobar, P., Matu, S., Marques, C. and Croft, S. L. (2002). Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH3 (edelfosine) and amphotericin B. Acta Tropica 81, 151–157.CrossRefGoogle ScholarPubMed
Getti, G., Durgadoss, P., Domínguez-Carmona, D., Martín-Quintal, Z., Peraza-Sánchez, S., Peña-Rodrguez, L. M. and Humber, D. (2009). Leishmanicidal activity of yucatecan medicinal plants on leishmania species responsible for cutaneous leishmaniasis. Journal of Parasitology 95, 456–460.CrossRefGoogle ScholarPubMed
Kain, S. R. (1999). Green fluorescent protein (GFP): applications in cell-based assays for drug discovery. Drug Discovery Today 4, 304–312.Google Scholar
Kamau, S. W., Grimm, F. and Hehl, A. B. (2001). Expression of green fluorescent protein as a marker for effects of antileishmanial compounds in vitro . Antimicrobial Agents and Chemotherapy 45, 3654–3656.Google Scholar
Melby, P. C. (2002). Vaccination against cutaneous leishmaniasis: current status. American Journal of Clinical Dermatology 3, 557–570.Google Scholar
Mißlitz, A., Mottram, J. C., Overath, P. and Aebischer, T. (2000). Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Molecular and Biochemical Parasitology 107, 251–261.CrossRefGoogle ScholarPubMed
Nwaka, S. and Hudson, A. (2006). Innovative lead discovery strategies for tropical diseases. Nature Reviews Drug Discovery 5, 941–955.CrossRefGoogle ScholarPubMed
Okuno, T., Goto, Y., Matsumoto, Y., Otsuka, H. and Matsumoto, Y. (2003). Applications of recombinant Leishmania amazonensis expressing egfp or the β-galactosidase gene for drug screening and histopathological analysis. Experimental Animals 52, 109–118.Google Scholar
Papadopoulou, B. and Dumas, C. (1997). Parameters controlling the rate of gene targeting frequency in the protozoan parasite Leishmania . Nucleic Acids Research 25, 4278–4286.Google Scholar
Pulido, S. A., Muñoz, D. L., Restrepo, A. M., Mesa, C. V., Alzate, J. F., Vélez, I. D. and Robledo, S. M. (2012). Improvement of the green fluorescent protein reporter system in Leishmania spp. for the in vitro and in vivo screening of antileishmanial drugs. Acta Tropica 122, 36–45.Google Scholar
Ready, P. D. and Smith, D. F. (1988). Peanut lectin agglutination and isolation of infective forms of Leishmania major . Transactions of the Royal Society of Tropical Medicine and Hygiene 82, 418.CrossRefGoogle ScholarPubMed
Robinson, K. A. and Beverley, S. M. (2003). Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania . Molecular and Biochemical Parasitology 128, 217–228.CrossRefGoogle ScholarPubMed
Rogers, M. B., Hilley, J. D., Dickens, N. J., Wilkes, J., Bates, P. A., Depledge, D. P., Harris, D., Her, Y., Herzyk, P., Imamura, H., Otto, T. D., Sanders, M., Seeger, K., Dujardin, J. C., Berriman, M., Smith, D. F., Hertz-Fowler, C. and Mottram, J. C. (2011). Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania . Genome Research 21, 2129–2142. doi: 10.1101/gr.122945.111.CrossRefGoogle ScholarPubMed
Sean Ha, D., Schwarz, J. K., Turco, S. J. and Beverley, S. M. (1996). Use of the green fluorescent protein as a marker in transfected Leishmania . Molecular and Biochemical Parasitology 77, 57–64.Google Scholar
Singh, N. and Dube, A. (2004). Short report: Fluorescent Leishmania: application to anti-leishmanial drug testing. American Journal of Tropical Medicine and Hygiene 71, 400–402.Google Scholar
Singh, N., Gupta, R., Jaiswal, A. K., Sundar, S. and Dube, A. (2009). Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening. Journal of Antimicrobial Chemotherapy 64, 370–374.Google Scholar
Tsien, R. Y. (1998). The green fluorescent protein. Annual Review of Biochemistry 67, 509–544.Google Scholar
Utaile, M., Kassahun, A., Abebe, T. and Hailu, A. (2013). Susceptibility of clinical isolates of Leishmania aethiopica to miltefosine, paromomycin, amphotericin B and sodium stibogluconate using amastigote-macrophage in vitro model. Experimental Parasitology 134, 68–75.Google Scholar
Varela, R. E., Lorena Muñoz, D., Robledo, S. M., Kolli, B. K., Dutta, S., Chang, K. P. and Muskus, C. (2009). Leishmania (Viannia) panamensis: an in vitro assay using the expression of GFP for screening of antileishmanial drug. Experimental Parasitology 122, 134–139.Google Scholar
World Health Organization (2010). WHO Technical Report Series. Control of the Leishmaniases. WHO, Geneva, Switzerland.Google Scholar
Zucca, M. and Savoia, D. (2011). Current developments in the therapy of protozoan infections. Open Medicinal Chemistry Journal 5 (Special issue 1), 4–10.CrossRefGoogle ScholarPubMed
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