Hostname: page-component-78c5997874-mlc7c Total loading time: 0 Render date: 2024-11-03T05:34:09.711Z Has data issue: false hasContentIssue false
  • English
  • Français

Gene expression profiling of Leishmania (Leishmania) donovani: overcoming technical variation and exploiting biological variation

Published online by Cambridge University Press:  12 October 2007

S. DECUYPERE ,
M. VANAERSCHOT ,
S. RIJAL ,
V. YARDLEY ,
L. MAES ,
S. DE DONCKER ,
F. CHAPPUIS  and
J.-C. DUJARDIN
S. DECUYPERE
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of GlasgowG12 8QQ, UK
M. VANAERSCHOT
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium Department of Biomedical Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
S. RIJAL
Affiliation:
B.P. Koirala Institute of Health Sciences, Dharan, Nepal
V. YARDLEY
Affiliation:
Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK
L. MAES
Affiliation:
Laboratory of Microbiology, Parasitology and Hygiene, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium
S. DE DONCKER
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium
F. CHAPPUIS
Affiliation:
Department of Community Medicine, Travel and Migration Medicine Unit, Hôpitaux Universitaires de Genève, Rue Micheli-du-Crest 24, CH-1211, Geneva 14, Switzerland
J.-C. DUJARDIN*
Affiliation:
Department of Parasitology, Unit of Molecular Parasitology, Institute of Tropical Medicine, Nationalestraat 155, Antwerp B-2000, Belgium
*
*Corresponding author: Institute of Tropical Medicine, Unit of Molecular Parasitology, Nationalestraat 155, B-2000 Antwerp, Belgium. Tel: +32 3 2476358. Fax: +32 3 2476359. E-mail: [email protected]
Get access Rights & Permissions [Opens in a new window]

Summary

Gene expression profiling is increasingly used in the field of infectious diseases for characterization of host, pathogen and the nature of their interaction. The purpose of this study was to develop a robust, standardized method for comparative expression profiling and molecular characterization of Leishmania donovani clinical isolates. The limitations and possibilities associated with expression profiling in intracellular amastigotes and promastigotes were assessed through a series of comparative experiments in which technical and biological parameters were scrutinized. On a technical level, our results show that it is essential to use parasite harvesting procedures that involve minimal disturbance of the parasite's environment in order to ‘freeze’ gene expression levels instantly; this is particularly a delicate task for intracellular amastigotes and for specific ‘sensory’ genes. On the biological level, we demonstrate that gene expression levels fluctuate during in vitro development of both intracellular amastigotes and promastigotes. We chose to use expression-curves rather than single, specific, time-point measurements to capture this biological variation. Intracellular amastigote protocols need further refinement, but we describe a first generation tool for high-throughput comparative molecular characterization of patients' isolates, based on the changing expression profiles of promastigotes during in vitro differentiation.

Keywords

Leishmania (Leishmania) donovanistandardizationcomparative expression profiling isolatesvariation gene expression
Type
Research Article
Information
Parasitology , Volume 135 , Issue 2 , February 2008 , pp. 183 - 194
Copyright
Copyright © Cambridge University Press 2007

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Akopyants, N. S., Matlib, R. S., Bukanova, E. N., Smeds, M. R., Brownstein, B. H., Stormo, G. D. and Beverley, S. M. (2004). Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major. Molecular and Biochemical Parasitology 136, 71–86.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. and others (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
Barak, E., Amin-Spector, S., Gerliak, E., Goyard, S., Holland, N. and Zilberstein, D. (2005). Differentiation of Leishmania donovani in host-free system: analysis of signal perception and response. Molecular and Biochemical Parasitology 141, 99–108.Google Scholar
Bates, P. A. and Tetley, L. (1993). Leishmania mexicana: induction of metacyclogenesis by cultivation of promastigotes at acidic pH. Experimental Parasitology 76, 412–423.CrossRefGoogle ScholarPubMed
Beitz, E. (2005). Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy. Biology of the Cell 97, 373–383.Google Scholar
Boucher, N., Wu, Y., Dumas, C., Dube, M., Sereno, D., Breton, M. and Papadopoulou, B. (2002). A common mechanism of stage-regulated gene expression in Leishmania mediated by a conserved 3′-untranslated region element. The Journal of Biological Chemistry 277, 19511–19520.CrossRefGoogle ScholarPubMed
Campbell, D. A., Thomas, S. and Sturm, N. R. (2003). Transcription in kinetoplastid protozoa: why be normal? Microbes and Infection 5, 1231–1240.CrossRefGoogle ScholarPubMed
Chaussabel, D., Semnani, R. T., McDowell, M. A., Sacks, D., Sher, A. and Nutman, T. B. (2003). Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites. Blood 102, 672–681.CrossRefGoogle ScholarPubMed
Clayton, C. E. (2002). Life without transcriptional control? From fly to man and back again. The EMBO Journal 21, 1881–1888.CrossRefGoogle Scholar
Cohen-Freue, G., Holzer, T. R., Forney, J. D. and McMaster, W. R. (2007). Global gene expression in Leishmania. International Journal for Parasitology 37, 1077–1086.CrossRefGoogle ScholarPubMed
Decuypere, S., Rijal, S., Yardley, V., De Doncker, S., Laurent, T., Khanal, B., Chappuis, F. and Dujardin, J. C. (2005). Gene expression analysis of the mechanism of natural Sb(V) resistance in Leishmania donovani isolates from Nepal. Antimicrobial Agents and Chemotherapy 49, 4616–4621.CrossRefGoogle ScholarPubMed
Duncan, R. (2004). DNA microarray analysis of protozoan parasite gene expression: outcomes correlate with mechanisms of regulation. Trends in Parasitology 20, 211–215.CrossRefGoogle ScholarPubMed
Duncan, R., Alvarez, R., Jaffe, C. L., Wiese, M., Klutch, M., Shakarian, A., Dwyer, D. and Nakhasi, H. L. (2001). Early response gene expression during differentiation of cultured Leishmania donovani. Parasitology Research 87, 897–906.Google Scholar
Duncan, R. C., Salotra, P., Goyal, N., Akopyants, N. S., Beverley, S. M. and Nakhasi, H. L. (2004). The application of gene expression microarray technology to kinetoplastid research. Current Molecular Medicine 4, 611–621.CrossRefGoogle ScholarPubMed
Fong, D. and Chang, K. P. (1981). Tubulin biosynthesis in the developmental cycle of a parasitic protozoan, Leishmania mexicana: changes during differentiation of motile and nonmotile stages. Proceedings of the National Academy of Sciences, USA 78, 7624–7628.Google Scholar
Hart, D. T., Vickerman, K. and Coombs, G. H. (1981). A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers. Parasitology 82, 345–355.Google Scholar
Heid, C. A., Stevens, J., Livak, K. J. and Williams, P. M. (1996). Real time quantitative PCR. Genome Research 6, 986–994.Google Scholar
Hellemans, J., Mortier, G., De Paepe, A., Speleman, F. and Vandesompele, J. (2007). qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biology 8, R19.CrossRefGoogle ScholarPubMed
Holzer, T. R., McMaster, W. R. and Forney, J. D. (2006). Expression profiling by whole-genome interspecies microarray hybridization reveals differential gene expression in procyclic promastigotes, lesion-derived amastigotes, and axenic amastigotes in Leishmania mexicana. Molecular and Biochemical Parasitology 146, 198–218.Google Scholar
Hromatka, B. S., Noble, S. M. and Johnson, A. D. (2005). Transcriptional response of Candida albicans to nitric oxide and the role of the YHB1 gene in nitrosative stress and virulence. Molecular Biology of the Cell 16, 4814–4826.CrossRefGoogle ScholarPubMed
Ivens, A. C., Peacock, C. S., Worthey, E. A., Murphy, L., Aggarwal, G., Berriman, M., Sisk, E., Rajandream, M. A., Adlem, E., Aert, R., Anupama, A., Apostolou, Z. and others (2005). The genome of the kinetoplastid parasite, Leishmania major. Science 309, 436–442.Google Scholar
Laurent, T., Rijal, S., Yardley, V., Croft, S., De Doncker, S., Decuypere, S., Khanal, B., Singh, R., Schonian, G., Kuhls, K., Chappuis, F. and Dujardin, J. C. (2007). Epidemiological dynamics of antimonial resistance in Leishmania donovani: Genotyping reveals a polyclonal population structure among naturally-resistant clinical isolates from Nepal. Infection, Genetics and Evolution 7, 206–212.Google Scholar
Martinez-Calvillo, S., Nguyen, D., Stuart, K. and Myler, P. J. (2004). Transcription initiation and termination on Leishmania major chromosome 3. Eukaryotic Cell 3, 506–517.CrossRefGoogle ScholarPubMed
McAleese, F., Wu, S. W., Sieradzki, K., Dunman, P., Murphy, E., Projan, S. and Tomasz, A. (2006). Overexpression of genes of the cell wall stimulon in clinical isolates of Staphylococcus aureus exhibiting vancomycin-intermediate-S. aureus-type resistance to vancomycin. Journal of Bacteriology 188, 1120–1133.CrossRefGoogle ScholarPubMed
McNicoll, F., Drummelsmith, J., Muller, M., Madore, E., Boilard, N., Ouellette, M. and Papadopoulou, B. (2006). A combined proteomic and transcriptomic approach to the study of stage differentiation in Leishmania infantum. Proteomics 6, 3567–3581.CrossRefGoogle Scholar
Monjour, L., Vouldoukis, I., Brandicourt, O., Mazier, D., Alfred, C., Ploton, I. and Gentilini, M. (1984). Rapid, large-scale production and isolation for Leishmania amastigotes. Annals of Tropical Medicine and Parasitology 78, 423–425.Google Scholar
Mottram, J. C., Robertson, C. D., Coombs, G. H. and Barry, J. D. (1992). A developmentally regulated cysteine proteinase gene of Leishmania mexicana. Molecular Microbiology 6, 1925–1932.CrossRefGoogle ScholarPubMed
Peacock, C. S., Seeger, K., Harris, D., Murphy, L., Ruiz, J. C., Quail, M. A., Peters, N., Adlem, E., Tivey, A., Aslett, M., Kerhornou, A., Ivens, A. and others (2007). Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nature Genetics 39, 839–847.CrossRefGoogle ScholarPubMed
Pham, T. V. and Mauel, J. (1987). Studies on intracellular killing of Leishmania major and lysis of host macrophages by immune lymphoid cells in vitro. Parasite Immunology 9, 721–736.Google Scholar
Rijal, S., Yardley, V., Chappuis, F., Decuypere, S., Khanal, B., Singh, R., Boelaert, M., De Doncker, S., Croft, S. and Dujardin, J. C. (2007). Antimonial treatment of visceral leishmaniasis: are current in vitro susceptibility assays adequate for prognosis of in vivo therapy outcome? Microbes and Infection 9, 529–535.Google Scholar
Sacks, D. L. (1989). Metacyclogenesis in Leishmania promastigotes. Experimental Parasitology 69, 100–103.CrossRefGoogle ScholarPubMed
Saxena, A., Lahav, T., Holland, N., Aggarwal, G., Anupama, A., Huang, Y., Volpin, H., Myler, P. J. and Zilberstein, D. (2007). Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation. Molecular and Biochemical Parasitology 152, 53–65.Google Scholar
Saxena, A., Worthey, E. A., Yan, S., Leland, A., Stuart, K. D. and Myler, P. J. (2003). Evaluation of differential gene expression in Leishmania major Friedlin procyclics and metacyclics using DNA microarray analysis. Molecular and Biochemical Parasitology 129, 103–114.Google Scholar
Schena, M., Shalon, D., Davis, R. W. and Brown, P. O. (1995). Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467–470.Google Scholar
Shapira, M., McEwen, J. G. and Jaffe, C. L. (1988). Temperature effects on molecular processes which lead to stage differentiation in Leishmania. The EMBO Journal 7, 2895–2901.Google Scholar
Tintaya, K. W., Ying, X., Dedet, J. P., Rijal, S., De Bolle, X. and Dujardin, J. C. (2004). Antigen genes for molecular epidemiology of leishmaniasis: polymorphism of cysteine proteinase B and surface metalloprotease glycoprotein 63 in the Leishmania donovani complex. The Journal of Infectious Diseases 189, 1035–1043.Google Scholar
Tobie, E. J., Von Brand, T. and Mehlman, B. (1950). Cultural and physiological observations on Trypanosoma rhodesiense and Trypanosoma gambiense. Journal of Parasitology 36, 48–54.Google Scholar
Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A. and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3, research 0034.1–0034.11.Google Scholar
Zakai, H. A., Chance, M. L. and Bates, P. A. (1998). In vitro stimulation of metacyclogenesis in Leishmania braziliensis, L. donovani, L. major and L. mexicana. Parasitology 116, 305–309.CrossRefGoogle ScholarPubMed