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  • 1
    Electronic Resource
    Electronic Resource
    Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus (1998)
    Oxford BSL : Blackwell Science Ltd, UK
    Molecular microbiology 29 (1998), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1998.00968.x
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  • 2
    Electronic Resource
    Electronic Resource
    A developmentally regulated cysteine proteinase gene of Leishmania mexicana (1992)
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 6 (1992), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (Imcpa) has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective meta-cyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPhe-ValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.1992.tb01365.x
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  • 3
    Electronic Resource
    Electronic Resource
    Gene disruptions indicate an essential function for the LmmCRK1 cdc2-related kinase of Leishmania mexicana (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 22 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The generation of homozygous null mutants for the crk1 cdc2-related kinase of Leishmania mexicana was attempted using targeted gene disruption. Promastigote mutants heterozygous for crk1 were readily isolated with a hyg-targeting fragment, but attempts to create null mutants by second-round transfections with a ble-targeting fragment yielded two classes of mutant, neither of which was null. First, the transfected fragment formed an episome; second, the cloned transfectants were found to contain wild-type crk1 alleles as well as hyg and ble integrations. DNA- content analysis revealed that these mutants were triploid or tetraploid. Plasticity in chromosome number following targeting has been proposed as a means by which Leishmania avoids deletion of essential genes. These data support this theory and implicate crk1 as an essential gene, validating CRK1 as a potential drug target. L. mexicana transfected with a Trypanosoma brucei homologue, tbcrk1, was shown to be viable in an lmmcrk1 null background, thus showing complementation of function between these trypanosomatid genes. The expression of crk1 was further manipulated by engineering a six-histidine tag at the C-terminus of the kinase, allowing purification of the active complex by affinity selection on Ni2+–nitriloacetic acid (NTA) agarose.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.00136.x
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  • 4
    Electronic Resource
    Electronic Resource
    A potential role for ICP, a leishmanial inhibitor of cysteine peptidases, in the interaction between host and parasite (2004)
    Oxford, UK : Blackwell Publishing Ltd.
    Molecular microbiology 54 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The biological role of a natural inhibitor of cysteine peptidases (designated ICP) of Leishmania has been investigated by genetic manipulation of the parasite. Null mutants grew normally in vitro, were as infective to macrophages in vitro as wild-type parasites, but had reduced infectivity to mice. Mutants re-expressing ICP from a single gene gave partial restoration of virulence in vivo, whereas mutants overexpressing ICP secreted the inhibitor and showed markedly reduced virulence in mice. Promastigotes of the null mutants had similar cysteine peptidase activities as the wild-type parasites, suggesting that ICP is not required for the expression or processing of the enzymes. The only proteins found to bind to ICP in promastigote cell lysates were fully processed forms of CPA and CPB, showing that ICP does not bind in abundance either to zymogens of the cysteine peptidases or other leishmanial proteins. However, only a small proportion of ICP colocalized with CPA and CPB in the promastigote (in the endoplasmic reticulum and Golgi) and the majority of ICP resided in vesicles that are apparently distinct from endosomes and the multivesicular tubule (MVT)-lysosome. These data suggest that ICP has a role other than modulation of the activity of the parasite’s own cysteine peptidases and their normal trafficking to the MVT-lysosome via the flagellar pocket. The finding that ICP partially colocalized with an endocytosed cysteine peptidase leads us to postulate that ICP has a role in protection of the parasite against the hydrolytic environment of the sandfly gut and/or the parasitophorous vacuole of host macrophages.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04355.x
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  • 5
    Electronic Resource
    Electronic Resource
    Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not for exit from mitosis (2005)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 56 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Two MOB1 genes, MOB1-A and MOB1-B, were identified in Trypanosoma brucei. MOB1-A of T. brucei was shown to form a complex with TbPK50, a functional homologue of the Schizosaccharomyces pombe protein kinase Orb6, and immune precipitated MOB1-A exhibited histone H1 protein kinase activity. MOB1-A and TbPK50 were also shown to bind p12cks1, a cyclin-dependent kinase accessory protein. Immune fluorescence of epitope-tagged MOB1-A and MOB1-B in bloodstream form trypanosomes showed they had a punctate distribution all through the cell cytoplasm and were excluded from the nucleus throughout the cell cycle. Using RNA interference (RNAi), MOB1 was shown to be essential in both bloodstream and procyclic life cycle stages. In the bloodstream form, RNAi of MOB1 resulted, after 8 h, in a significant increase in post-mitotic cells, the majority of which had a visible cleavage furrow. This was followed by the appearance of cells with abnormal complements of nuclei and kinetoplasts, often with the number of nuclei exceeding the number of kinetoplasts. Thus, downregulation of MOB1 in the bloodstream form results in a delay in cytokinesis, and leads to a deregulation of the cell cycle, possibly through an inhibitory effect on kinetoplast replication. In contrast, downregulation of MOB1 in the procyclic form appears to impede the accuracy of cytokinesis, by allowing mispositioning of the cleavage furrow and inappropriate cytokinesis. Unlike its counterpart in budding yeast, T. brucei MOB1 does not appear to be required for mitotic exit.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04542.x
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  • 6
    Electronic Resource
    Electronic Resource
    Generation of Leishmania mutants lacking antibiotic resistance genes using a versatile hit-and-run targeting strategy (2004)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 235 (2004), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The development of a method to create defined mutants of Leishmania parasites lacking foreign genes conferring resistance to antibiotics has both experimental and practical applications. Mutants deficient in specific virulence genes have potential as attenuated live vaccines, but these can only be of clinical relevance if the antibiotic resistance genes used for selection of the mutants are subsequently removed. In addition, the limited number of antibiotic resistance genes that can be used for genetic manipulation of Leishmania means that a system for recycling them for subsequent use would be highly beneficial when multiple genetic modifications are wanted. In the method we report here, a cassette carrying in tandem the hygromycin resistance gene as a positive marker and thymidine kinase gene as a negative marker is first integrated into the locus of interest and then replaced by a null targeting fragment containing no exogenous DNA. The application of this hit-and-run strategy for removal of one allele of the CPB cysteine peptidase gene array of Leishmania infantum is described.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2004.tb09571.x
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  • 7
    Electronic Resource
    Electronic Resource
    Characterisation of the QM gene of Trypanosoma brucei (2002)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 211 (2002), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The QM protein has been reported to have roles in both tumour suppression and transcription factor regulation in vertebrate cells, and in ribosome stability in both yeast and mammals. The present study isolated the QM gene of Trypanosoma brucei and determined its sequence. Alignment with QM sequences from Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Homo sapiens revealed greater than 60% identity. Southern blot analysis revealed multiple copies of QM within the trypanosome genome. An epitope tag was inserted into the C-terminus of the T. brucei QM and the protein expressed under inducible control in procyclic form trypanosomes. Immune fluorescence microscopy revealed co-localisation with the GPI:protein transamidase component, GPI8, a distribution indicative of ribosome association with the rough endoplasmic reticulum.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2002.tb11213.x
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  • 8
    Electronic Resource
    Electronic Resource
    Stable transformation of trypanosomatids through targeted chromosomal integration of the selectable marker gene encoding blasticidin S deaminase (2000)
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 186 (2000), S. 0 
    Details
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: The susceptibilities of the protozoan parasites Leishmania mexicana and Trypanosoma brucei to the nucleoside antibiotic blasticidin S were assessed. A concentration of 10 μg ml−1 was sufficient to cause cell death within 72 h of L. mexicana promastigotes and bloodstream forms of T. brucei in vitro. The gene encoding blasticidin S deaminase (BSD) was therefore incorporated into cassettes for targeting to the cysteine proteinase C locus of L. mexicana (CPC::BSD) and the tubulin locus of T. brucei (tub::RAD51-BSR). Following transfection of mutant parasites that contained other well-established selectable marker genes (HYG, NEO, BLE, PAC and SAT), clones resistant to 10 μg ml−1 blasticidin S were shown by PCR and Southern blotting to have integrated the cassettes by homologous recombination. The results confirm that BSD can be used as a selectable marker gene for targeted chromosomal integration during genetic manipulations of trypanosomatids.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1574-6968.2000.tb09119.x
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  • 9
    Electronic Resource
    Electronic Resource
    Cysteine or serine proteinase? (1989)
    [s.l.] : Nature Publishing Group
    Nature 342 (1989), S. 132-132 
    Details
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] SIRá€"Higgins et al. 'showed that the 11 IK antigen of Plasmodium falciparum has significant similarity to cysteine proteinases. We wish to point out that the cysteine labelled as the putative active-site residue is in fact involved in a disulphide bridge in papain, actinidin ...
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1038/342132c0
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  • 10
    Electronic Resource
    Electronic Resource
    Parasite cysteine proteinases (1996)
    Springer
    Perspectives in drug discovery and design 6 (1996), S. 99-118 
    Details
    ISSN: 1573-9023
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Cysteine proteinases of protozoan and helminth parasites are considered to have a high potential as targets for novel antiparasite agents. This has stimulated research on the enzymes and a large body of data has now been accumulated. This Perspective provides an overview of the current situation, with recent advances being highlighted. Emphasis is given to the Type I cysteine proteinases of trypanosomatid protozoa, which are atypical in having an extra C-terminal domain, and the asparaginyl endopeptidase ofSchistosoma, for which the only homologues known are those in plants. The locations of parasite cysteine proteinases are described, with emphasis on the extralysosomal sites, and the putative roles of the enzymes in host-parasite interactions, including parasite nutrition and host invasion, are discussed. The major advances being made in elucidating cysteine proteinase function in protozoan parasites through genetic manipulation, including targeted gene deletion, are presented, with the Type I cysteine proteinases ofLeishmania being used as an example. The importance of this approach in the validation of the enzymes as drug targets is discussed. The current status of attempts to exploit parasite cysteine proteinases with drugs is presented, and future prospects are outlined.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02174048
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