Key Points
-
Trypanosomatid protozoans are responsible for some of the most devastating and, as yet, poorly controlled diseases of tropical and semi-tropical countries.
-
In the past ten years, a comprehensive molecular genetic 'toolkit' has become available for their study, including efficient methods of transient and stable DNA transfection, expression vectors, homologous gene replacements and transposon mutagenesis.
-
Classical genetics is difficult in African trypanosomes due to the requirement for a tsetse fly colony, and it is impossible in Leishmania, as proved by unsuccessful laboratory crosses.
-
In trypanosomes, RNA interference (RNAi) technology has proved an especially powerful tool as it can be readily applied to inactivate transcripts from any gene. By contrast, RNAi methods have so far been unsuccessful in Leishmania, possibly because it has lost this pathway and therefore normally tolerates significant levels of endogenous double-stranded RNAs.
-
Forward genetics in Leishmania has been established using powerful selections for mutants and functional rescue after cosmid library transfection.
-
RNAi 'knockdown' libraries have recently enabled forward genetics approaches in trypanosomes.
-
Differences in the efficacy of genetic methods in Leishmania and trypanosomes have steered investigators to focus on different aspects of parasite biology in these organisms.
Abstract
Trypanosomatid protozoans cause important diseases of humans and their domestic livestock. Various molecular genetic tools are now allowing rapid progress in understanding many of the unique aspects of the molecular and cell biology of these organisms. Diploidy and the lack or difficulty of sexual crossing has been a challenge for forward genetics, but powerful selections and functional complementation have helped to overcome it in Leishmania. RNA interference has been adapted for forward genetics in trypanosomes, in which it is also a powerful tool for reverse genetics. Interestingly, the efficacy of different genetic tools has steered research into different aspects of the biology of these parasites.
Similar content being viewed by others
References
Borst, P. & Rudenko, G. Antigenic variation in African trypanosomes. Science 264, 1872–1873 (1994).
Clayton, C. E. Genetic manipulation of Kinetoplastida. Parasitol. Today 15, 372–378 (1999). This is a comprehensive review of the methods and approaches used to genetically modify the trypanosomatid protozoan genome.
Swindle, J. & Tait, A. in Molecular Biology of Parasitic Protozoa (eds Smith, D. F. & Parsons, M.) 6–34 (IRL, Oxford, UK, 1996).
Myler, P. J. et al. Genomic organization and gene function in Leishmania. Biochem. Soc. Trans. 28, 527–531 (2000).
Sogin, M. L., Hinkle, G. & Leipe, D. D. Universal tree of life. Nature 362, 795 (1993).
Hannaert, V. et al. Plant-like traits associated with metabolism of trypanosomatid parasites. Proc. Natl Acad. Sci. USA (in the press).
Vickerman, K. in Biology of the Kinetoplastida (eds Lumsden, W. H. R. & Evans, D. A.) 1–76 (Academic, London, 1976).
Fernandes, A. P., Nelson, K. & Beverley, S. M. Evolution of nuclear ribosomal RNAs in kinetoplastid protozoa: perspectives on the age and origins of parasitism. Proc. Natl Acad. Sci. USA 90, 11608–11612 (1993).
Tschudi, C. & Pearce, E. J. (eds) Biology of Parasitism (Kluwer Academic, Boston, Massachusetts, 2000). A recent compendium of essays that describe many areas of molecular parasitology, with several that focus on the biology of the trypanosomatid protozoans.
Tait, A. et al. Genetic analysis of phenotype in Trypanosoma brucei: a classical approach to potentially complex traits. Phil. Trans. R. Soc. Lond. B Biol. Sci. 357, 89–99 (2002). The authors describe the methods that are used to carry out and analyse genetic crosses in trypanosomes, and report their application to phenotypes such as drug resistance.
Beverley, S. M. in Molecular and Medical Parasitology (eds Marr, J. M., Nilsen, T. & Komuniecki, R.) (Academic, New York, in the press). A detailed summary of the methods that are used for the genetic modification of Leishmania , described in table 2 in this review, and of several of the gene systems to which they have been applied.
Tibayrenc, M. & Ayala, F. J. Evolutionary genetics of Trypanosoma and Leishmania. Microbes Infect. 1, 465–472 (1999).
Shi, H. et al. Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA 6, 1069–1076 (2000).
Clayton, C. E. Life without transcriptional control? From fly to man and back again. EMBO J. 21, 1881–1888 (2002). A summary of many of the unusual aspects of transcription and gene expression in the trypanosomatid protozoa, including the apparent lack of RNA polymerase II promoters.
Tyler-Cross, R. E., Short, S. L., Floeter-Winter, L. M. & Buck, G. A. Transient expression mediated by the Trypanosoma cruzi rRNA promoter. Mol. Biochem. Parasitol. 72, 23–31 (1995).
Gay, L. S., Wilson, M. E. & Donelson, J. E. The promoter for the ribosomal RNA genes of Leishmania chagasi. Mol. Biochem. Parasitol. 77, 193–200 (1996).
Patnaik, P. K., Axelrod, N., Van der Ploeg, L. H. T. & Cross, G. A. M. Artificial linear mini-chromosomes for Trypanosoma brucei. Nucleic Acids Res. 24, 668–675 (1996).
Tamar, S. & Papadopoulou, B. A telomere-mediated chromosome fragmentation approach to assess mitotic stability and ploidy alterations of Leishmania chromosomes. J. Biol. Chem. 276, 11662–11673 (2001).
Clayton, C. E. Control of gene expression in trypanosomes. Prog. Nucleic Acids Res. Mol. Biol. 43, 37–66 (1992). The author discusses the unique mechanisms that are used by trypanosomes to control gene expression, such as transcription by RNA polymerase I, elongation, RNA stability and translational control.
Agabian, N. Trans splicing of nuclear pre-mRNAs. Cell 61, 1157–1160 (1990).
Ullu, E., Tschudi, C. & Gunzl, A. in Molecular Biology of Parasitic Protozoa (eds Smith, D. F. & Parsons, M.) 115–133 (IRL, Oxford, UK, 1996).
Zomerdijk, J. C. B. M., Kieft, R. & Borst, P. Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma brucei. Nature 353, 772–775 (1991).
Papadopoulou, B., Roy, G. & Ouellette, M. Autonomous replication of bacterial DNA plasmid oligomers in Leishmania. Mol. Biochem. Parasitol. 65, 39–49 (1994).
LeBowitz, J. H., Smith, H. Q., Rusche, L. & Beverley, S. M. Coupling of poly(A) site selection and trans-splicing in Leishmania. Genes Dev. 7, 996–1007 (1993).
Cruz, A. K. in Gene Targeting (ed. Vega, M. A.) 65–81 (CRC, Boca Raton, Florida, 1995).
Cruz, A. K., Titus, R. & Beverley, S. M. Plasticity in chromosome number and testing of essential genes in Leishmania by targeting. Proc. Natl Acad. Sci. USA 90, 1599–1603 (1993).
Biebinger, S., Wirtz, L. E., Lorenz, P. & Clayton, C. Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei. Mol. Biochem. Parasitol. 85, 99–112 (1997).
Krieger, S. et al. Trypanosomes lacking trypanothione reductase are avirulent and show increased sensitivity to oxidative stress. Mol. Microbiol. 35, 542–552 (2000). This paper describes a now classic application of the tetracycline-inducible system to the study of an important enzyme that is involved in oxidant resistance.
Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc. Natl Acad. Sci. USA 95, 14687–14692 (1998).
Ullu, E., Djikeng, A., Shi, H. & Tschudi, C. RNA interference: advances and questions. Phil. Trans. R. Soc. Lond. B Biol. Sci. 357, 65–70 (2002). A thoughtful essay that summarizes the current knowledge of RNAi in trypanosomes, addressing both its mechanistic features and its application to parasite biology.
Wang, Z., Morris, J. C., Drew, M. E. & Englund, P. T. Inhibition of Trypanosoma brucei gene expression by RNA interference using an integratable vector with opposing T7 promoters. J. Biol. Chem. 275, 40174–40179 (2000). A description of the use of a convenient regulatable RNAi vector that allows the rapid testing of the role of any gene in trypanosomes.
Li, Z. & Wang, C. C. Functional characterization of the 11 non-ATPase subunit proteins in the trypanosome 19S proteasomal regulatory complex. J. Biol. Chem. 277, 42686–42693 (2002).
Bastin, P., Ellis, K., Kohl, L. & Gull, K. Flagellum ontogeny in trypanosomes studied via an inherited and regulated RNA interference system. J. Cell Sci. 113, 3321–3328 (2000).
Lye, L. F., Cunningham, M. L. & Beverley, S. M. Characterization of quinonoid-dihydropteridine reductase (QDPR) from the lower eukaryote Leishmania major. J. Biol. Chem. 277, 38245–38253 (2002).
Chen, D. Q. et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 level in promastigotes and their infection of macrophages in vitro. Infect. Immun. 68, 80–86 (2000).
Somanna, A., Mundodi, V. & Gedamu, L. Functional analysis of cathepsin B-like cysteine proteases from Leishmania donovani complex. Evidence for the activation of latent transforming growth factor-β. J. Biol. Chem. 277, 25305–25312 (2002).
Zhang, W. W. & Matlashewski, G. Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2. Proc. Natl Acad. Sci. USA 94, 8807–8811 (1997).
Zhang, W. W. & Matlashewski, G. Analysis of antisense and double stranded RNA downregulation of A2 protein expression in Leishmania donovani. Mol. Biochem. Parasitol. 107, 315–319 (2000).
Mochizuki, K., Fine, N. A., Fujisawa, T. & Gorovsky, M. A. Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in Tetrahymena. Cell 110, 689–699 (2002).
Ruvkun, G. Molecular biology. Glimpses of a tiny RNA world. Science 294, 797–799 (2001).
Allshire, R. Molecular biology. RNAi and heterochromatin — a hushed-up affair. Science 297, 1818–1819 (2002).
Plasterk, R. H. RNA silencing: the genome's immune system. Science 296, 1263–1265 (2002). A recent commentary on the role and the importance of RNAi in mitigating the harmful effects of transposable elements.
Bringaud, F. et al. Identification of non-autonomous non-LTR retrotransposons in the genome of Trypanosoma cruzi. Mol. Biochem. Parasitol. 124, 73–78 (2002).
Kapler, G. M. & Beverley, S. M. Transcriptional mapping of the amplified region encoding the dihydrofolate reductase–thymidylate synthase of Leishmania major reveals a high density of transcripts, including overlapping and antisense RNAs. Mol. Cell. Biol. 9, 3959–3972 (1989).
Belli, S. et al. Leishmania major: histone H1 gene expression from the sw3 locus. Exp. Parasitol. 91, 151–160 (1999).
Wong, A. K., Curotto de Lafaille, M. A. & Wirth, D. F. Identification of a cis-acting gene regulatory element from the lemdr1 locus of Leishmania enriettii. J. Biol. Chem. 269, 26497–26502 (1994).
Borst, P. & Ouellette, M. New mechanisms of drug resistance in parasitic protozoa. Annu. Rev. Microbiol. 49, 427–460 (1995).
Beverley, S. M. Gene amplification in Leishmania. Annu. Rev. Microbiol. 45, 417–444 (1991).
Stuart, K. D. Circular and linear multicopy DNAs in Leishmania. Parasitol. Today 7, 158–159 (1991).
Tripp, C. A., Wisdom, W. A., Myler, P. J. & Stuart, K. D. A multicopy, extrachromosomal DNA in Leishmania infantum contains two inverted repeats of the 27.5-kilobase LD1 sequence and encodes numerous transcripts. Mol. Biochem. Parasitol. 55, 39–50 (1992).
Patnaik, P. K., Kulkarni, S. K. & Cross, G. A. Autonomously replicating single-copy episomes in Trypanosoma brucei show unusual stability. EMBO J. 12, 2529–2538 (1993).
Aravind, L., Watanabe, H., Lipman, D. J. & Koonin, E. V. Lineage-specific loss and divergence of functionally linked genes in eukaryotes. Proc. Natl Acad. Sci. USA 97, 11319–11324 (2000).
Volpe, T. A. et al. Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science 297, 1833–1837 (2002).
Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. & Plasterk, R. H. Mut-7 of C. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133–141 (1999).
Lindenbach, B. D. & Rice, C. M. RNAi targeting an animal virus: news from the front. Mol. Cell 9, 925–927 (2002).
Armstrong, T. C. & Patterson, J. L. Cultivation of Leishmania braziliensis in an economical serum-free medium containing human urine. J. Parasitol. 80, 1030–1032 (1994).
Gueiros-Filho, F. J. & Beverley, S. M. Selection against the dihydrofolate reductase–thymidylate synthase (DHFR-TS) locus as a probe of genetic alterations in Leishmania major. Mol. Cell. Biol. 16, 5655–5663 (1996).
Hwang, H. Y. & Ullman, B. Genetic analysis of purine metabolism in Leishmania donovani. J. Biol. Chem. 272, 19488–19496 (1997).
Beverley, S. M. & Turco, S. J. Lipophosphoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania. Trends Microbiol. 6, 35–40 (1998). This article reviews the role of LPG in Leishmania biology and the importance of LPG-based methods in the development of forward genetics and functional genetic rescue.
Butcher, B. A. et al. Deficiency in β1,3-galactosyltransferase of a Leishmania major lipophosphoglycan mutant adversely influences the Leishmania–sand fly interaction. J. Biol. Chem. 271, 20573–20579 (1996).
Turco, S. J., Späth, G. F. & Beverley, S. M. Is lipophosphoglycan a virulence factor? A surprising diversity between Leishmania species. Trends Parasitol. 17, 223–226 (2001).
Späth, G. F. et al. Lipophosphoglycan is a virulence factor distinct from related glycoconjugates in the protozoan parasite Leishmania major. Proc. Natl Acad. Sci. USA 97, 9258–9263 (2000).
Ilg, T. Lipophosphoglycan of the protozoan parasite Leishmania: stage- and species-specific importance for colonization of the sandfly vector, transmission and virulence to mammals. Med. Microbiol. Immunol. (Berl.) 190, 13–17 (2001).
Sacks, D. & Kamhawi, S. Molecular aspects of parasite–vector and vector–host interactions in leishmaniasis. Annu. Rev. Microbiol. 55, 453–483 (2001).
Hoyer, C., Mellenthin, K., Schilhabel, M., Platzer, M. & Clos, J. Use of genetic complementation to identify gene(s) which specify species-specific organ tropism of Leishmania. Med. Microbiol. Immunol. (Berl.) 190, 43–46 (2001).
Cotrim, P. C., Garrity, L. K. & Beverley, S. M. Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection. J. Biol. Chem. 274, 37723–37730 (1999).
Nare, B., Hardy, L. W. & Beverley, S. M. The roles of pteridine reductase 1 and dihydrofolate reductase–thymidylate synthase in pteridine metabolism in the protozoan parasite Leishmania major. J. Biol. Chem. 272, 13883–13891 (1997).
Nare, B., Luba, J., Hardy, L. W. & Beverley, S. New approaches to Leishmania chemotherapy: pteridine reductase 1 (PTR1) as a target and modulator of antifolate sensitivity. Parasitology 114, S101–S110 (1997).
Morris, J. C., Wang, Z., Drew, M. E. & Englund, P. T. Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library. EMBO J. 21, 4429–4438 (2002). A seminal paper that describes the application of an RNAi library for forward genetics in trypanosomes, the first such example for any organism.
Gueiros-Filho, F. J. & Beverley, S. M. Trans-kingdom transposition of the Drosophila element mariner within the protozoan Leishmania. Science 276, 1716–1719 (1997). The first example of heterologous expression of a transposase across distantly related organisms in vivo , which enabled the development of similar systems in a wide range of eukaryotic species.
Shi, H., Wormsley, S., Tschudi, C. & Ullu, E. Efficient transposition of preformed synaptic Tn5 complexes in Trypanosoma brucei. Mol. Biochem. Parasitol. 121, 141–144 (2002).
Goyard, S., Tosi, L. R., Gouzova, J., Majors, J. & Beverley, S. M. New Mos1 mariner transposons suitable for the recovery of gene fusions in vivo and in vitro. Gene 280, 97–105 (2001).
Tosi, L. R. & Beverley, S. M. cis and trans factors affecting Mos1 mariner evolution and transposition in vitro, and its potential for functional genomics. Nucleic Acids Res. 28, 784–790 (2000).
Beverley, S. M. et al. Putting the Leishmania genome to work: functional genomics by transposon trapping and expression profiling. Phil. Trans. R. Soc. Lond. B Biol. Sci. 357, 47–53 (2002).
Gull, K. The cell biology of parasitism in Trypanosoma brucei: insights and drug targets from genomic approaches? Curr. Pharm. Des. 8, 241–256 (2002). A thoughtful perspective on the cell biology of trypanosomes that incorporates both genomic and pharmacological information.
Sacks, D. & Noben-Trauth, N. The immunology of susceptibility and resistance to Leishmania major in mice. Nature Rev. Immunol. 2, 845–858 (2002). These authors provide the most up-to-date perspective on the complex interplay between the mammalian immune system and the control (or persistence) of the Leishmania parasite.
Akopyants, N. S. et al. A survey of the Leishmania major Friedlin strain V1 genome by shotgun sequencing: a resource for DNA microarrays and expression profiling. Mol. Biochem. Parasitol. 113, 337–340 (2001).
Matthews, K. R. Developments in the differentiation of Trypanosoma brucei. Parasitol. Today 15, 76–80 (1999).
Acknowledgements
I thank K. Robinson and members of my lab, P. Sharp, J. Donelson, N. Fasel, J. Patterson, R. Tarleton and S. Turco for stimulating discussions and suggestions, and D. Dobson for reading this manuscript. S.M.B. is supported by grants from the National Institutes of Health.
Author information
Authors and Affiliations
Related links
Related links
FURTHER INFORMATION
WHO Special Programme for Research and Training in Tropical Diseases
Glossary
- ANTIGENIC VARIATION
-
The changing of the surface antigens by a pathogen to evade the immune response of the host.
- SPECTRAL DISEASE
-
Infections that can manifest in several forms, often varying greatly in severity and symptoms in different individuals, probably reflecting differences in the immune response of the host.
- PHAGOLYSOSOME
-
A vacuole in a cell in which a phagocytosed particle is digested.
- ENDOSYMBIONT
-
An organism which lives in the cells of a host organism in a mutualistic relationship or while doing no apparent harm.
- POLYCISTRONIC TRANSCRIPTION
-
The transcription of two or more adjacent open reading frames after a single transcription initiation event. In trypanosomatids, this might extend more than 60 kb and encompass dozens of open reading frames.
- BENT DNA
-
Intrinsically bent DNA arises from a series of 8–10 adenine residues on the same strand that is thought to assist in packaging it into a tight network.
- KINETOPLAST
-
An organelle in the mitochondrion that consists of a large concatenated DNA network of both minicircles (each with an intrinsically 'bent' sequence and guide RNAs required for RNA editing) and maxicircles (which encode genes that are typically associated with mitochondrial DNA in other organisms). It is a defining feature of the protozoan order Kinetoplastida, to which the trypanosomes belong.
- GLYCOSOME
-
A subcellular compartment, related to the peroxisome, originally named because it contains glycolytic enzymes, although other enzymes have subsequently been found there.
- ACIDOCALCISOME
-
An organelle that contains high concentrations of calcium and polyphosphates. Its role is poorly understood, but it might function as a reservoir for calcium in intracellular signalling and for energy.
- FLAGELLAR POCKET
-
A 'pocket-like' invagination of the cellular membrane from which the flagellum arises in trypanosomatids.
- EPISOME
-
A replicon that can exist either extrachromosomally or when integrated into the bacterial chromosome.
- ANEUPLOID
-
Having an unbalanced chromosome number. An example is trisomy.
- POLYPLOID
-
Having more than two complete sets of chromosomes (two sets being the prevalent diploid state).
- LOSS OF HETEROZYGOSITY
-
(LOH). A loss of one of the alleles at a given locus as a result of a genomic change, such as mitotic deletion, gene conversion or chromosome mis-segregration.
- PANNING
-
A technique for isolating parasite subpopulations; monoclonal antibodies or lectins are attached to solid supports over which parasite populations are passed and allowed to attach; after washing, bound parasites are eluted and recovered.
- FLUORESCENCE ACTIVATED CELL SORTING
-
(FACS). The separation of cells or chromosomes by their fluorescence and light-scattering properties, which are measured as the particles flow in a liquid stream past laser beams. The stream is then broken into droplets, and selected droplets are electrically charged and deflected into collection vessels as they pass through an electric field.
- MASS SPECTROMETRY
-
A technique that provides accurate information about the molecular mass of complex molecules. It can identify extremely small amounts of proteins by their mass-fragment spectra.
Rights and permissions
About this article
Cite this article
Beverley, S. Protozomics: trypanosomatid parasite genetics comes of age. Nat Rev Genet 4, 11–19 (2003). https://doi.org/10.1038/nrg980
Issue Date:
DOI: https://doi.org/10.1038/nrg980
- Springer Nature Limited
This article is cited by
-
Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains
Acta Parasitologica (2023)
-
Development of a toolbox to dissect host-endosymbiont interactions and protein trafficking in the trypanosomatid Angomonas deanei
BMC Evolutionary Biology (2016)
-
Leishmania-based expression systems
Applied Microbiology and Biotechnology (2016)
-
Knockdown of LdMC1 and Hsp70 by antisense oligonucleotides causes cell-cycle defects and programmed cell death in Leishmania donovani
Molecular and Cellular Biochemistry (2012)
-
Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation
Parasites & Vectors (2011)