Deletion Study of DNA Topoisomerase IB from Leishmania donovani: Searching for a Minimal Functional Heterodimer

The substantial differences between trypanosomal and leishmanial DNA topoisomerase IB concerning to their homologues in mammals have provided a new lead in the study of the structural determinants that can be effectively targeted. Leishmania donovani, the causative agent of visceral leishmaniasis, contains an unusual heterodimeric DNA topoisomerase IB. The catalytically active enzyme consists of a large subunit (LdTopIL), which contains the non-conserved N-terminal end and the phylogenetically conserved “core” domain, and of a small subunit (LdTopIS) which harbors the C-terminal region with the characteristic tyrosine residue in the active site. Heterologous co-expression of LdTopIL and LdTopIS genes in a topoisomerase I deficient yeast strain, reconstitutes a fully functional enzyme LdTopIL/S which can be used for structural studies. An approach by combinatorial cloning of deleted genes encoding for truncated versions of both subunits was used in order to find out structural insights involved in enzyme activity or protein-protein interaction. The role played by the non-conserved N-terminal extension of LdTopIL in both relaxation activity and CPT sensitivity has been examined co-expressing the full-length LdTopIS and a fully active LdTopIΔS deletion with several deletions of LdTopIL lacking growing sequences of the N-terminal end. The sequential deletion study shows that the first 26 amino acids placed at the N-terminal end and a variable region comprised between Ala548 to end of the C-terminal extension of LdTopIL were enzymatically dispensable. Altogether this combinatorial approach provides important structural insights of the regions involved in relaxation activity and for understanding the atypical structure of this heterodimeric enzyme

ATP independent type IB topoisomerase of Leishmania donovani is stimulated by ATP: an insight into the functional mechanism

Most type IB topoisomerases do not require ATP and Mg2+ for activity. However, as shown previously for vaccinia topoisomerase I, we demonstrate that ATP stimulates the relaxation activity of the unusual heterodimeric type IB topoisomerase from Leishmania donovani (LdTOP1L/S) in the absence of Mg2+. The stimulation is independent of ATP hydrolysis but requires salt as a co-activator. ATP binds to LdTOP1L/S and increases its rate of strand rotation. Docking studies indicate that the amino acid residues His93, Tyr95, Arg188 and Arg190 of the large subunit may be involved in ATP binding. Site directed mutagenesis of these four residues individually to alanine and subsequent relaxation assays reveal that the R190A mutant topoisomerase is unable to exhibit ATP-mediated stimulation in the absence of Mg2+. However, the ATP-independent relaxation activities of all the four mutant enzymes remain unaffected. Additionally, we provide evidence that ATP binds LdTOP1L/S and modulates the activity of the otherwise ATP-independent enzyme. This study establishes ATP as an activator of LdTOP1L/S in the absence of Mg2+

Clonage et caractérisation du gène TOP1 de Leishmania donovani

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal

‘LeishMan’ topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme

Découvertes de nouveaux mécanismes de résistance au Topotecan, un inhibiteur des topoisomérases, chez Leishmania infantum

Le protozoaire Leishmania est à l’origine d’une maladie tropicale négligée qui peut s’avérer mortelle si elle n’est pas traitée adéquatement. Dans les dernières années, l’efficacité des médicaments utilisés dans le combat de la leishmaniose a grandement diminué en raison de la résistance du parasite à ceux-ci. Bien que le Topotecan (TPT), présentement utilisé comme antitumoral, ait démontré une activité antileishmaniale puissante (EC50 de l’ordre des nanomolaires), son efficacité pourrait être compromise par l’émergence de la résistance du parasite d’autant plus que des résistances tumorales cliniques au TPT ont déjà été rapportées. Dans cette étude, les mécanismes de résistance au TPT par Leishmania infantum ont été caractérisés d’un point de vue moléculaire. Le génome complet des parasites sélectionnés résistants au TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) a été séquencé. Le rôle dans la résistance des différents éléments génétiques identifiés a été confirmé à l’aide d’une complémentation par nucléofection épisomale dans le parasite sauvage et a été étudié avec des simulations computationnelles. Aucune amplification ni délétion n’a été identifiée et seulement une variation mineure du nombre de chromosomes a été observée. Cependant, un polymorphisme d’un seul nucléotide non synonyme a été identifié dans le gène de la grande sous-unité de la topoisomérase IB (TOP IB), la cible du TPT, chez chacun des parasites mutants résistants conférant des niveaux de résistance variables (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > sauvage). De plus, des modélisations in sillico ont permis d’illustrer la proximité de ces substitutions d’acides aminés au site catalytique de TOP IB ainsi qu’au site de liaison du TPT. En conclusion, ces résultats suggèrent qu’une mutation ponctuelle dans la grande sous-unité TOP IB est suffisante pour engendrer des hauts niveaux de résistance (environ 24 x EC50) chez TPT700.1F187Y, TPT700.2G191A et TPT700.3 W232R. TPT pourrait être considéré comme un modèle pharmacologique pour l’étude de la résistance chez Leishmania.Leishmania, a protozoan parasite, causes a neglected tropical disease that is fatal if left untreated. In recent years, the effectiveness of the drugs used to tackle leishmaniasis has decreased dramatically due to the emergence of drug resistant parasites. Even though Topetecan (TPT), currently employed as an anti-tumoral drug, has shown strong anti-leishmanial activity (its EC50 being measured in nanomoles), its efficiency may be compromised by the resistance developped by the parasites, similarly to the resistance already recorded by tumoral cells to the drug. In this study, the mecanisms of resistance to TPT by Leishmania infantum were caracterised at the molecular level. The whole genome of parasites resistant to TPT (16 x EC50) (TPT700.1, TPT700.2, TPT700.3) was sequenced. The role of various genetic elements in the resistance mecanisms was confirmed via a complementation by episomal nucleofection in the wild type and was studied with the help of computational models. Neither amplications nor deletions were identified and only a minor variation in the chromosome number was observed. However, a non-synonymous single nucleotide polymorphism was identified in the gene coding the large subunit of topoisomerase IB, TPT’s target, within each of the resistant mutant parasites confering variable levels of resistance (TPT700.1 F187Y > TPT700.3 W232R > TPT700.2 G191A > wild type). Furthermore, in sillico models highlighted the proximity of these amino acid substitutions to the catalytic site of topoisomerase IB and also to the binding pockets of TPT. In conclusion, these results suggest that a point mutation in the large subunit of TOP IB is sufficient to confer high levels of resistance (about 24 x EC50) to TPT700.1F187Y, TPT700.2G191A and TPT700.3 W232R. Therefore, TPT can be considered a pharmacological tool to study resistance in Leishmania

Studies on a thiol-dependent reductase and ascorbate metabolism of leishmania

The intracellular protozoan parasite Leishmania causes leishmaniasis, a disease which is most prevalent in tropical and sub-tropical countries where it infects some two million people every year and kills around 60,000 of them. For decades pentavalent antimonial compounds have been the standard first-line drugs used to treat the disease and this remains the case despite increasing reports of drug-resistance. The mode of action of these drugs is not entirely understood, although it is generally accepted that in vivo reduction of the compounds from the pentavalent to a trivalent form is required for antileishmanial activity. The site of antimonial conversion and whether the reaction is catalysed by an enzyme remain controversial points. However, it was recently reported that L. donovani amastigotes were capable of reducing pentavalent antimonials to the trivalent form and that drug-resistant parasites were deficient in this activity, suggesting that a parasite enzyme did mediate drug toxicity. The identity of such an enzyme was investigated in this study. Arsenical and antimonial compounds are similar and several classes of proteins that exhibit arsenate reductase activity have been previously identified in other organisms. Whether Leishmania possessed an enzyme akin to one of these was assessed by attempting to purify enzymes from parasite lysates and by searching the L. major genome database for similar sequences to the arsenate reductases. The latter approach was successful and a gene fragment was identified that shared similarity with omega glutathione S-transferases (oGSTs), a class of glutaredoxin-like GSTs which are capable of reducing pentavalent methylated arsenicals in vitro. The sequence of the complete L. major gene was elucidated by 5' RACE, and was found to encode a protein twice the expected size with similar 3' and 5' halves. The protein was named thiol-dependent reductase, or TDRl. Active recombinant protein was successfully produced and its biochemical activities were found to coincide with oGSTs: TDRl was capable of reducing pentavalent arsenical and antimonial compounds to trivalent species, and possessed thioltransferase and dehydroascorbate reductase activities usually associated with glutaredoxins. TDRl, which was shown to probably reside in the parasite cytosol but may also be secreted, was found to be more abundant in amastigote than promastigote forms, which correlates with the antileishmanial stage-specificity of pentavalent antimonials. L. major TDRl knockout mutants were generated, and the protein was also over-expressed in parasites. Both these genetic manipulations resulted in mutants with enhanced infectivity. TDRl knockout parasites were more susceptible than wild type parasites to paraquat, which induces the production of intracellular superoxide. As its glutaredoxin-like in vitro activities suggest, this implies TDRl has a role in protecting the parasites from oxidative stress, although re-expression of TDRl did not reinstate resistance. (Abstract shortened by ProQuest.)

Estudio de las regiones implicadas en la funcionalidad y resistencia a fármacos de la ADN topoisomerasa IB de leishmania

278 p.Las leishmaniosis son un complejo de enfermedades causadas por parásitos del género Leishmania spp. que presentan una elevada prevalencia a nivel mundial. Afectan sobretodo a países en vías de desarrollo, aunque recientemente, debido a diversos factores, se ha producido una propagación hacia regiones aparentemente exentas de riesgo. Por todo ello, la OMS considera la leishmaniosis una enfermedad priorizada dentro de su ¿Special Programme for Research and Training in Tropical Diseases¿. Dado que no existen vacunas eficaces contra estas enfermedades y que los tratamientos médicos actuales están basados en fármacos obsoletos con una elevada toxicidad, es vital abrir nuevas vías de lucha frente a este parásito mediante la identificación de nuevas dianas terapéuticas y el consiguiente diseño de inhibidores específicos. Recientemente, se han identificado las ADN topoisomerasas como prometedoras dianas de intervención terapéutica en tripanosomátidos. Estas enzimas están involucradas en resolver los problemas topológicos generados durante la replicación, trascripción y recombinación del ADN. La ADN topoisomerasa IB de Leishmania spp. es un complejo heterodimérico con escasa homología con respecto a la proteína del hospedador mamífero, por lo que las regiones no conservadas de la enzima de Leishmania constituyen una diana terapéutica altamente selectiva. En el presente trabajo se ha realizado un estudio exhaustivo de las regiones no conservadas de la ADN topoisomerasa IB de Leishmania, identificando las secuencias específicas responsables de las interacciones entre las dos subunidades del heterodímero así como de la conformación del dominio linker de la proteína, probando además la importancia de este dominio tanto en la capacidad de relajación de la enzima como en la sensibilidad a diferentes tipos de inhibidores. En segundo lugar se han identificado y caracterizado las tres secuencias responsables de la translocación de la proteína al interior del núcleo; una en el extremo C-terminal de la subunidad grande y dos en la subunidad pequeña. La primera de ellas correspondió con una secuencia de 10 aminoácidos localizados en el extremo N-terminal, mientras la que la segunda fue delimitada en las inmediaciones del residuo Tyr catalítico del extremo C-terminal. En este estudio no se encontró ninguna señal específica de translocación al kinetoplasto. Finalmente se evaluó la enzima de Leishmania como diana molecular de intervención terapéutica con tres familias de compuestos: derivados de la camptotecina, indenoisoquinolinas y ácidos grasos tanto acetilénicos como alquinoicos. Con ello se demostró que varios derivados camptotecínicos e indenoisoquinolínicos, especialmente el gimatecan y el LMP400, son inhibidores preferenciales de la enzima de Leishmania frente a la humana, encontrándose una buena correlación entre los ensayos in vitro e in vivo realizados. De igual manera se demostró el uso potencial de los ácidos grasos insaturados de cadena larga como inhibidores de la ADN topoisomerasa, por un mecanismo de acción diferente al de la camptotecina, en el que dichos ácidos grasos impiden el corte del ADN mediado por la enzima pero no la unión entre ambos

A Biochemical Approach to Characterize a Divergent Trypanosoma brucei Mitochondrial DNA Polymerase, POLIB

Trypanosoma brucei is a single-celled parasitic protist that causes African sleeping sickness in people and nagana in cattle in sub-Saharan Africa. T. brucei and related trypanosomatid parasites contain an unusual catenated mitochondrial genome known as kinetoplast DNA (kDNA) composed of dozens of 23 kb maxicircles and thousands of 1 kb minicircles. The kDNA structure and replication mechanism are divergent from other eukaryotes and essential for parasite survival. POLIB is one of three Family A DNA polymerases that are independently essential to maintain the kDNA network, and has been implicated in minicircle replication. However, the division of labor among the paralogs, particularly which might be a replicative, proofreading enzyme remains enigmatic. De novo modelling of POLIB suggested a structure that is divergent from all other Family A polymerases in which the thumb subdomain contains a 369 amino acid insertion with homology to DEDDh DnaQ family 3\u27-5\u27 exonucleases. In chapter 2, we explore the polymerase and exonuclease activity of POLIB using purified his-tagged v recombinant variants that have been truncated and codon optimized for expression in E. coli. Using this recombinant protein variants we demonstrated that the 3\u27-5\u27 exonuclease activity of recombinant POLIB prefers DNA vs. RNA substrates and prefers singlestranded vs. double-stranded substrates. POLIB exonuclease activity prevails over polymerase activity on DNA substrates at pH 8.0, while DNA primer extension is favored at pH 6.0. Mutations that ablate POLIB polymerase activity slow the exonuclease rate suggesting crosstalk between the domains. We show that POLIB extends an RNA primer more efficiently than a DNA primer in the presence of dNTPs but does not incorporate rNTPs efficiently using either RNA or DNA primers. Immunoprecipitation of Pol I-like paralogs from T. brucei corroborate the pH selectivity and RNA primer preferences of POLIB and revealed that the other paralogs efficiently extend a DNA primer. We also show that overexpression of the exonuclease-ablated variant of POLIB in T. brucei results in a loss of fitness and impacts kDNA replication. We postulate that this unique enzyme and the machinery associated with it in the process of kDNA replication could be excellent drug targets worthy of further study

Involvement of tyrosine phosphatases in Leishmania differentiation and virulence

Leishmania protozoan parasites, the causative agent of leishmaniasis, a disease endemic in more than 80 countries, undergo two main developmental stages during its life cycle: the extracellular flagellated promastigote residing in the midget of the sandfly vector and the obligate intracellular amastigotes which multiply in the phagolysosome of infected macrophages within the mammalian host. The differentiation process from promastigote to amastigote allows Leishmania parasites to adapt to different environments and is essential for parasite proliferation and survival. However, the molecular events that regulate this process are not well understood.In higher eukaryotes, cellular proliferation, differentiation and function are governed largely by protein phosphorylation, which is controlled by protein kinases and phosphatases. The research described in this thesis has investigated the role of protein tyrosine phosphatase in controlling the differentiation and proliferation of the Leishmania pathogen in different life cycle stages, by analogy to what happens in higher eukaryotes. The focus was on protein phosphatases because in general, there are fewer phosphatases than kinases in the eukaryotic cells and therefore there is less likelihood of redundancy under conditions where it is possible to genetically develop mutants in phosphatase genes.By undertaking a predominantly genetic approach, we show the protein tyrosine phosphatase may play a role in L. donovani differentiation and is clearly required for parasite virulence as defined by survival in the mammalian host.The results from this study suggest that Leishmania PTP1 represents a potential drug target. However it is also revealed that the overall three dimensional structure of the active site of Leishmania PTP1 is very similar to the human PTP1B arguing that it may be difficult to develop parasite specific inhibitors. Taken together this study represents the first genetic analysis of a key regulatory gene in Leishmania, which establishes the foundation for future more biochemical approaches to study protein phosphorylation in Leishmania