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Differential gene expression analysis in antimony-unresponsive Indian kala azar (visceral leishmaniasis) clinical isolates by DNA microarray

Published online by Cambridge University Press:  19 February 2007

N. SINGH ,
R. ALMEIDA ,
H. KOTHARI ,
P. KUMAR ,
G. MANDAL ,
M. CHATTERJEE ,
S. VENKATACHALAM ,
M. K. GOVIND ,
S. K. MANDAL  and
S. SUNDAR
N. SINGH*
Affiliation:
Central Drug Research Institute, Lucknow, India
R. ALMEIDA
Affiliation:
Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge
H. KOTHARI
Affiliation:
Central Drug Research Institute, Lucknow, India
P. KUMAR
Affiliation:
Central Drug Research Institute, Lucknow, India
G. MANDAL
Affiliation:
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India
M. CHATTERJEE
Affiliation:
Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India
S. VENKATACHALAM
Affiliation:
Connexios Life Sciences, Bangalore, India
M. K. GOVIND
Affiliation:
Connexios Life Sciences, Bangalore, India
S. K. MANDAL
Affiliation:
Central Drug Research Institute, Lucknow, India
S. SUNDAR
Affiliation:
Banaras Hindu University, Varanasi, India
*
*Corresponding author: Central Drug Research Institute, Lucknow, India. E-mail: [email protected]
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Summary

In this study, cDNA microarray analysis of a closely related species, Leishmania major, was used as a screening tool to compare antimonial-resistant and susceptible clinical isolates of Leishmania donovani in order to to identify candidate genes on the basis of antimony resistance. Clinically confirmed resistant isolate 39 and sensitive isolate 2001 were used in this study. Many differentially regulated genes were identified whose expression levels differ in sodium antimony gluconate (SAG)-treated patients. Interestingly, genes on the array, showing changes in expression of over 2-fold revealed the identity of ABC transporters, which are known determinants of drug resistance in laboratory mutants. The functionality of the transporters was validated by flow cytometry which, being biologically informative, provides direct clues to gene function. The results suggest that isolate 39 could have developed resistance by an increased multidrug resistance protein (MRP)-like pump. This study provides preliminary clues to the role of a thiol-dependent efflux system in antimonial resistant clinical isolates of Leishmania donovani.

Keywords

Leishmaniamicroarraydrug resistanceclinical isolatesflow cytometry
Type
Research Article
Information
Parasitology , Volume 134 , Issue 6 , June 2007 , pp. 777 - 787
Copyright
Copyright © Cambridge University Press 2007

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References

REFERENCES

Akopyants, N. S., Matlib, R. S., Bukanova, E. N., Smeds, M. R., Brownstein, B. H., Stormo, G. D. and Beverly, S. M. (2004). Expression profiling using random genomic DNA microarrays identifies deferentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major. Molecular and Biochemical Parasitology 136, 71–86.CrossRefGoogle Scholar
Alexander, J. and Russell, D. G. (1992). The interaction of Leishmania species with macrophages. Advances in Parasitology 31, 175–254.CrossRefGoogle ScholarPubMed
Almeida, R., Gilmartin, B. J., McCann, S. H., Norrish, A., Ivens, A. C., Lawson, D., Levick, M. P., Smith, D. F., Dyall, S. D., Vetrie, D., Freeman, T. C., Coulson, R. M., Sampaio, I., Schneider, H. and Blackwell, J. M. (2004). Expression profiling of the Leishmania life cycle: cDNA arrays identify developmentally regulated genes present but not annotated in the genome. Molecular and Biochemical Parasitology 136, 87–100.CrossRefGoogle Scholar
Beverley, S. M., Akopyants, N. S., Goyard, S., Matlib, R. S., Gordon, J. L., Brownstein, B. H., Stormo, G. D., Bukanova, E. N., Hott, C. T., Li, F., Macmillan, S., Muo, J. N., Schwertman, L. A., Smeds, M. R. and Wang, Y. (2002). Putting the Leishmania genome to work: functional genomics by transposon trapping and expression profiling. Philosophical Transactions of The Royal Society of London, B 357, 47–53.CrossRefGoogle ScholarPubMed
Callahan, H. L. and Beverley, S. M. (1991). Heavy metal resistance: a new role for P-glycoproteins in Leishmania. Journal of Biological Chemistry 266, 18427–18430.CrossRefGoogle ScholarPubMed
Cheok, M. H., Yang, W., Pui, C. H., Downing, J. R., Cheng, C., Naeve, C. W., Relling, M. V. and Evens, W. E. (2003). Treatment specific changes in gene expression discriminate in vivo drug response in human leukemia cells. Nature Genetics 34, 85–90.CrossRefGoogle ScholarPubMed
Chiquero, M. J., Perez-Victoria, J. M., O'valle, F., Gonzalez-Ros, J. M., del Moral, R. G., Ferragut, J. A., Castanys, S. and Gamarro, F. (1998). Altered drug membrane permeability in a multidrug-resistant Leishmania tropica line. Biochemical Pharmacology 55, 131–139.CrossRefGoogle Scholar
Chomezynski, P. and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162, 156–159.Google Scholar
Chulay, J. D. and Bryceson, A. D. M. (1983). Quantitation of amastigotes of Leishmania donovani in smears of splenic aspirates from patients with visceral leishmaniasis. Americal Journal of Tropical Medicine and Hygyiene 32, 475–479.CrossRefGoogle ScholarPubMed
Cleveland, W. S. (1979). Robust locally weighted regression and smoothing scatterplots. Journal of the American Statistical Association 74, 829–836.CrossRefGoogle Scholar
Croft, S. L. and Coombs, G. H. (2003). Leishmaniasis- current chemotherapy and recent advances in the search for novel drugs. Trends in Parasitology 19, 502–508.CrossRefGoogle ScholarPubMed
Dey, S., Papadopoulou, B., Haimeur, A., Roy, G., Grondin, K., Dou, D., Rosen, B. P. and Ouellette, M. (1994). High level of arsenite resistance in Leishmania tarentolae is mediated by an active extrusion system. Molecular and Biochemical Parasitology 67, 49–57.CrossRefGoogle ScholarPubMed
Diehl, S., Diehl, F., El-Sayed, N. M., Clayton, C. and Hoheisel, J. D. (2002). Analysis of stage specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA- microarray. Molecular and Biochemical Parasitology 123, 115–123.CrossRefGoogle ScholarPubMed
Dube, A., Singh, N., Sundar, S. and Singh, N. (2005). Refractoriness to the treatment of sodium stibogluconate in Indian kala-azar field isolates persists in in vitro and in vivo experimental models. Parasitology Research 96, 216–223.CrossRefGoogle Scholar
El Fadili, K., Messier, N., Leprohon, P., Roy, G., Guimond, C., Trudel, N., Sarvia, N. G., Papadopoulou, B., Legare, D. and Ouellette, M. (2005). Role of the ABC transporter MRPA (PGPA) in antimony resistance in Leishmania infantum axenic and intracellular amastigotes. Antimicrobial Agents and Chemotherapy 49, 1988–1993.CrossRefGoogle ScholarPubMed
Ephros, M., Bitnun, A., Shaked, P., Waldman, E. and Zilberstein, D. (1999). Stage-specific activity of pentavalent antimony against Leishmania donovani axenic amastigotes. Antimicrobial Agents and Chemotherapy 43, 278–282.CrossRefGoogle ScholarPubMed
Essodaigui, M., Broxterman, H. J. and Garnier-Suillerot, A. (1998). Kinetic analysis of calcein and calcein-acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein. Biochemistry 37, 2243–2250.CrossRefGoogle ScholarPubMed
Gourbal, B., Sonuc, N., Bhattacharjee, H., Legare, D., Sundar, S., Ouellette, M., Rosen, B. P. and Mukhopadhyay, R. (2004). Drug uptake and modulation of drug resistance in Leishmania by an aquaglyceroporin. Journal of Biological Chemistry 279, 31010–31017.CrossRefGoogle ScholarPubMed
Grondin, K., Haimeur, A., Mukhopadhayay, R., Rosen, B. P. and Ouellette, M. (1997). Co-amplification of the gamma – glutamylcysteine synthetase gene gsh1 and of the ABC transporter gene pgpA in arsenite – resistant Leishmania tarentole. EMBO Journal 16, 3057–3065.CrossRefGoogle Scholar
Guimond, C., Trudel, N., Brochu, C., Marquis, N., El Fadili, A., Peytavi, R., Briand, G., Richard, D., Messier, N., Papadopoulou, B., Corbeil, J., Bergeron, M. G., Legare, D. and Ouellette, M. (2003). Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays. Nucleic Acids Research 31, 5886–5896.CrossRefGoogle ScholarPubMed
Haimeur, A., Brochu, C., Genest, P., Papadopoulou, B. and Ouellette, M. (2000). Amplification of the ABC transporter gene PGPA and increased trypanothione levels in potassium antimonyl tartrate Sb(III) resistant Leishmania tarentole. Molecular and Biochemical Parasitology 108, 131–135.CrossRefGoogle Scholar
Hochberg, Y. (2002). A sharper Bonferroni procedure for multiple test of significance. Biometrika 75, 800–802.CrossRefGoogle Scholar
Homolya, L., Hollo, Z., Germann, U. A., Pastan, I., Gottesman, M. M. and Sarkadi, B. (1993). Fluorescent cellular indicators are extruded by the multidrug resistance protein. Journal of Biological Chemistry 268, 21493–21496.CrossRefGoogle ScholarPubMed
Ilgoutz, S. C. and McConville, M. J. (2001). Function and assembly of the Leishmania surface coat. International Journal for Parasitology 31, 899–908.CrossRefGoogle ScholarPubMed
Kerr, M. K. and Churchill, G. A. (2001). Bootstrapping cluster analysis: assessing the reliability of conclusions from microarray experiments. Proceedings of the National Academy of Sciences, USA 98, 8961–8965.CrossRefGoogle ScholarPubMed
Kothari, H., Kumar, P., Sundar, S. and Singh, N. (2006). Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani. Parasitology International (in the Press).Google ScholarPubMed
Newton, M. A., Kendziroski, C. M., Richmond, C. S., Blattner, F. R. and Tsui, K. W. (2001). On differential variability of expression ratios: improving statistical inference about gene expression changes from microarray data. Journal of Computational Biology 8, 37–52.CrossRefGoogle ScholarPubMed
Pavlidis, P. and Noble, W. S. (2001). Analysis of strain and regional variation in gene expression in mouse brain. Genome Biology 2, r0042.1–r0042.15.CrossRefGoogle ScholarPubMed
Perez-Victoria, F. J., Castanys, S. and Gamarro, F. (2003). Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug. Antimicrobial Agents and Chemotherapy 47, 2397–2403.CrossRefGoogle ScholarPubMed
Perez-Victoria, J. M., Parodi-Talice, A., Torres, C., Gamarro, F. and Castanys, S. (2001). ABC transporters in the protozoan parasite Leishmania. International Microbiology 4, 159–166.CrossRefGoogle ScholarPubMed
Perez-Victoria, J. M., Perez-Victoria, F. J., Parodi-Talice, A., Jimenez, I. A., Ravelo, A. G., Castanys, S. and Gamarro, F. (2001). Alkyl-lysophospholipid resistance in multidrug resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein like transporter modulator. Antimicrobial Agents and Chemotherapy 45, 2468–2474.CrossRefGoogle ScholarPubMed
Quackenbush, J. (2002). Microarray data normalization and transformation Nature Genetics 32 (Suppl.), 496–501.CrossRefGoogle ScholarPubMed
Rathod, P. K., Ganesan, K., Hayward, R. E., Bozdech, Z. and Derisi, J. L. (2002). DNA microarrays for malaria. Trends in Parasitology 18, 39–45.CrossRefGoogle ScholarPubMed
Rutherford, K., Parkhill, J., Crook, J., Horsnell, T., Rice, P., Rajandream, M. A. and Barrel, B. (2000). Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945.CrossRefGoogle ScholarPubMed
Singh, N. (2002). Is there true Sb (V) resistance in Indian kala-azar field isolates? Current Science 83, 101–102.Google Scholar
Singh, N. (2006). Drug resistance mechanisms in clinical isolates of Leishmania donovani. Indian Journal of Medical Research 123, 411–422.Google ScholarPubMed
Singh, N., Singh, R. T. and Sundar, S. (2003). Novel mechanisms of drug resistance in kala azar field isolates. Journal of Infectious Diseases 188, 600–607.CrossRefGoogle ScholarPubMed
Singh, R., Kumar, D., Ramesh, V., Negi, N. S., Singh, S. and Salotra, P. (2006). Visceral leishmaniasis, or kala azar (KA): high incidence of refractoriness to antimony is contributed by anthroponotic transmission via post-KA dermal leishmaniasis. Journal of Infectious Diseases 194, 302–306.CrossRefGoogle ScholarPubMed
Sundar, S. (2001). Drug resistances in Indian visceral leishmaniasis. Tropical Medicine and International Health 6, 849–854.CrossRefGoogle ScholarPubMed
Sundar, S. and Murray, H. W. (2005). Availability of miltefosine for the treatment of kala-azar in India. Bulletin of the World Health Organization 83, no. 5, pp. 394–395.Google ScholarPubMed
Tan, P. K., Downey, T. J., Spitznagel, E. L. Jr., Eu, P., Fu, D., Dimitroy, D. S., Lempicki, R. A., Raaka, B. M. and Cam, M. C. (2003). Evaluation of gene expression measurements from commercial microarray platforms. Nucleic Acids Research 31, 5676–5684.CrossRefGoogle ScholarPubMed
Versantvoor, C. H. M., Broxterman, H. J., Pinedo, H. M., De Vries, E. G., Feller, N., Kuiper, C. M. and Lankelma, J. (1992). Energy-dependent processes involved in reduced drug accumulation in multidrug-resistant human lung cancer cell lines without P-glycoprotein expression. Cancer Research 52, 17–23.Google Scholar
Wilson, M., Derisi, J., Kristensen, H. H., Imboden, P., Rane, S., Brown, P. O. and Schoolnik, G. K. (1999). Exploring drug induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization. Proceedings of the National Academy of Sciences, USA 96, 12833–12838.CrossRefGoogle ScholarPubMed
Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J. and Speed, T. P. (2002). Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Research 30, e15.CrossRefGoogle Scholar