O fenômeno de amplificação gênica é um mecanismo de auto-preservação celular observado com freqüência no protozoário parasita Leishmania quando submetido a estímulos negativos como, por exemplo, a presença de drogas. A região H do genoma de Leishmania (Leishmania) major é um dos loci mais estudados que leva à amplificação em resposta a drogas não relacionadas, como a terbinafina. Foram isoladas linhagens de L. (L.) major e Leishmania (Viannia) braziliensis resistentes à terbinafina. Análises por corridas curtas de eletroforese em campo pulsado (PFGE) e Southern blotting revelaram que a resistência observada nestas linhagens não foi gerada pela amplificação do locus H. Sendo assim, a resistência ao inibidor da esqualeno epoxidase está sendo gerada por um outro mecanismo uma vez que outros loci podem estar envolvidos na resistência à terbinafina e através da análise diferencial do perfil de proteínas, os mutantes resistentes apresentaram diferenças na expressão de proteínas. O alvo inicial para a elucidação da resistência à terbinafina nas linhagens selecionadas foi a via de biossíntese de ergosterol. Foram escolhidos os genes da 3-cetoacil-CoA tiolase (ERG10), esqualeno sintase (ERG9 ou SQS1), esqualeno epoxidase (ERG1), oxidoesqualeno-lanosterol ciclase (ERG7) e lanosterol 14-demetilase (ERG11), de L. major e L. braziliensis, além do gene YIP1 de L. braziliensis. Para isso foram sintetizados oligonucleotídeos a partir das seqüências geradas pelo projeto genoma destas espécies depositadas em bancos de dados. Os genes ERG10, SQS1, ERG1, ERG7 e ERG11 de L. major e de L. braziliensis além do YIP1 de L. braziliensis amplificados por PCR foram clonados no vetor pGEM-T Easy, que possibilitou a construção de reagentes para ruptura de todos genes pela inserção da marca HYG e, com exceção do gene ERG7 de L. braziliensis, os genes foram subclonados no vetor pXG1. Fragmentos de restrição dos genes clonados no vetor pGEM-T Easy foram utilizados para analisar o nível de seus transcritos por northern blot. Também verificamos através de corridas curtas de PFGE e análises de Southern, que as linhagens em estudo não apresentam amplificação dos loci em estudo. A participação de genes da via de biossíntese de ergosterol de L. major e L. braziliensis e do gene YIP1 de L. braziliensis na resistência ou susceptibilidade à terbinafina e/ou anfotericina B foi verificada através de experimentos funcionais. Com esse objetivo, os genes YIP1, ERG10 e ERG1 de L. braziliensis foram transfectados na linhagem LB2904 de L. braziliensis, e os genes ERG10, SQS1, ERG1, ERG7 e ERG11 e a ruptura do gene ERG1 pelo cassete HYG de L. major foram transfectados na linhagem LT252 de L. major. Foram realizados experimentos para analisar a susceptibilidade à anfotericina B associada ou não à terbinafina, das linhagens selvagens de L. major e L. braziliensis, e a partir destes experimentos iniciais, foram selecionadas linhagens destas duas espécies resistentes à anfotericina B. Para analisar a possível função regulatória dos elementos RIME 5/2/6 de L. braziliensis, foi feita a amplificação por PCR da repetição invertida LbRIME 5/2/6b que permitiu a clonagem deste fragmento no vetor pGEM-T Easy e a sua ruptura pela marca SAT. A clonagem no vetor pGEM possibilitou a análise da interação de proteínas nucleares à repetição LbRIME 5/2/6b através do gel shift. / Gene amplification is a common phenomenon observed in Leishmania cell lines subjected to drug pressure. The H locus of Leishmania (Leishmania) major is normally found amplified in cell lines selected in unrelated drugs, as terbinafine. We selected cell lines of L. (L.) major and Leishmania (Viannia) braziliensis resistant to terbinafine. Short-run Pulsed Field Gel Electrophoresis (PFGE) and Southern blotting analysis showed that this resistance was not generated by H locus amplification. Resistance to squalene epoxidase inhibiter is being generated by other mechanism once others loci can be involved in the terbinafine resistance and through protein partner differential analysis, mutants resistant showed differences in protein expression. The initial target to terbinafine resistance elucidation in the cell lines selected was ergosterol biosynthesis pathway. We choose genes: 3-ketoacyl-CoA thiolase (ERG10), squalene synthase (ERG9 or SQS1), squalene epoxidase (ERG1), oxidosqualene-lanosterol cyclase (ERG7), and lanosterol 14-demethylase (ERG11), of L. major and L. braziliensis, besides YIP1 gene of L. braziliensis. For this, primers were synthesized using the sequences generated by genome project of these species inserted in gene bank. The genes ERG10, SQS1, ERG1, ERG7 and ERG11 of L. major and of L. braziliensis besides YIP1 of L. braziliensis were amplified by PCR and cloned into pGEM-T Easy vector that enabled all genes disruption by insertion of HYG cassette and, with exception of the ERG7 gene of L. braziliensis, genes were subcloned into shuttle-vector pXG1. Restrictions fragments of these genes were used in Northern analysis to verify the transcripts level. We verified through short-run PFGE and Southern analysis that the resistant cell lines do not show amplification of studied loci. The participation of ergosterol biosynthesis pathway genes of L. major and L. braziliensis and YIP1 gene of L. braziliensis in the resistance to terbinafine was verified in functional experiments. With this objective, the genes YIP1, ERG10 and ERG1 of L. braziliensis were transfected into LB2904 cell line of L. braziliensis, and the genes ERG10, SQS1, ERG1, ERG7 and ERG11 and the ERG1 gene disruption by HYG mark of L. major were transfected into LT252 cell line of L. major. Experiments that analyze the susceptibility to amphotericin B associated or not to terbinafine were performed using the wild type cell lines of L. major and L. braziliensis, and with this initials experiments, we selected cell lines of these two species resistant to amphotericin B. In order to analyze the possible regulatory function of the RIME 5/2/6 elements of L. braziliensis, the repeat LbRIME 5/2/6b was amplified by PCR and cloned into pGEM-T Easy vector and with this clone, the element was disrupted with a SAT cassette. The cloning into vector pGEM enabled to analyze the interaction of the nuclear protein with the repeat LbRIME 5/2/6b through gel shift assay.

The opportunistic fungus Candida albicans is a commensal member of the human microflora and is the most common causative agent of fungal-related disease with particular significance in immunocompromised individuals. Emerging drug resistance is a major problem in Candida, contributed by enzymes involved in the detoxification of xenobiotics and pharmacological agents. One such enzyme, cytochrome b5 reductase (cb5r), has a high pharmacological significance owing to its role in fatty acid elongation, ergosterol (or cholesterol in mammals) biosynthesis, and cytochrome P450-mediated detoxification of xenobiotics. We have compared the kinetic, biochemical, and pharmacological characteristics of C. albicans cb5r isoforms, Cbr1 and Mcr1, as compared to the mammalian control, rat cb5r. We have observed two key structural differences between the fungal and mammalian proteins that may account for decreased thermal stability and inhibitor specificity of C. albicans Cbr1. Substrate binding affinity and catalytic efficiencies, as well as investigation in the flavin-binding environment, were comparable between the fungal and rat enzymes. In S. cerevisiae, CBR1 and MCR1 knockout strains have been challenged with environmental stressors and subsequently shown to have a role in azole and amphotericin B resistance. Our results of potential protein interactions of C. albicans Cbr1 describe proteins involved in the weak acid stress response, implying a novel role of the protein in pathogenicity. Conclusively, this report describes potential inhibitors of the fungal protein, as well as elaborating upon its important role in ergosterol biosynthesis and possible mechanisms of CYP450-mediated drug detoxification.

O fenômeno de amplificação gênica é um mecanismo de auto-preservação celular observado com freqüência no protozoário parasita Leishmania quando submetido a estímulos negativos como, por exemplo, a presença de drogas. A região H do genoma de Leishmania (Leishmania) major é um dos loci mais estudados que leva à amplificação em resposta a drogas não relacionadas, como a terbinafina. Foram isoladas linhagens de L. (L.) major e Leishmania (Viannia) braziliensis resistentes à terbinafina. Análises por corridas curtas de eletroforese em campo pulsado (PFGE) e Southern blotting revelaram que a resistência observada nestas linhagens não foi gerada pela amplificação do locus H. Sendo assim, a resistência ao inibidor da esqualeno epoxidase está sendo gerada por um outro mecanismo uma vez que outros loci podem estar envolvidos na resistência à terbinafina e através da análise diferencial do perfil de proteínas, os mutantes resistentes apresentaram diferenças na expressão de proteínas. O alvo inicial para a elucidação da resistência à terbinafina nas linhagens selecionadas foi a via de biossíntese de ergosterol. Foram escolhidos os genes da 3-cetoacil-CoA tiolase (ERG10), esqualeno sintase (ERG9 ou SQS1), esqualeno epoxidase (ERG1), oxidoesqualeno-lanosterol ciclase (ERG7) e lanosterol 14-demetilase (ERG11), de L. major e L. braziliensis, além do gene YIP1 de L. braziliensis. Para isso foram sintetizados oligonucleotídeos a partir das seqüências geradas pelo projeto genoma destas espécies depositadas em bancos de dados. Os genes ERG10, SQS1, ERG1, ERG7 e ERG11 de L. major e de L. braziliensis além do YIP1 de L. braziliensis amplificados por PCR foram clonados no vetor pGEM-T Easy, que possibilitou a construção de reagentes para ruptura de todos genes pela inserção da marca HYG e, com exceção do gene ERG7 de L. braziliensis, os genes foram subclonados no vetor pXG1. Fragmentos de restrição dos genes clonados no vetor pGEM-T Easy foram utilizados para analisar o nível de seus transcritos por northern blot. Também verificamos através de corridas curtas de PFGE e análises de Southern, que as linhagens em estudo não apresentam amplificação dos loci em estudo. A participação de genes da via de biossíntese de ergosterol de L. major e L. braziliensis e do gene YIP1 de L. braziliensis na resistência ou susceptibilidade à terbinafina e/ou anfotericina B foi verificada através de experimentos funcionais. Com esse objetivo, os genes YIP1, ERG10 e ERG1 de L. braziliensis foram transfectados na linhagem LB2904 de L. braziliensis, e os genes ERG10, SQS1, ERG1, ERG7 e ERG11 e a ruptura do gene ERG1 pelo cassete HYG de L. major foram transfectados na linhagem LT252 de L. major. Foram realizados experimentos para analisar a susceptibilidade à anfotericina B associada ou não à terbinafina, das linhagens selvagens de L. major e L. braziliensis, e a partir destes experimentos iniciais, foram selecionadas linhagens destas duas espécies resistentes à anfotericina B. Para analisar a possível função regulatória dos elementos RIME 5/2/6 de L. braziliensis, foi feita a amplificação por PCR da repetição invertida LbRIME 5/2/6b que permitiu a clonagem deste fragmento no vetor pGEM-T Easy e a sua ruptura pela marca SAT. A clonagem no vetor pGEM possibilitou a análise da interação de proteínas nucleares à repetição LbRIME 5/2/6b através do gel shift. / Gene amplification is a common phenomenon observed in Leishmania cell lines subjected to drug pressure. The H locus of Leishmania (Leishmania) major is normally found amplified in cell lines selected in unrelated drugs, as terbinafine. We selected cell lines of L. (L.) major and Leishmania (Viannia) braziliensis resistant to terbinafine. Short-run Pulsed Field Gel Electrophoresis (PFGE) and Southern blotting analysis showed that this resistance was not generated by H locus amplification. Resistance to squalene epoxidase inhibiter is being generated by other mechanism once others loci can be involved in the terbinafine resistance and through protein partner differential analysis, mutants resistant showed differences in protein expression. The initial target to terbinafine resistance elucidation in the cell lines selected was ergosterol biosynthesis pathway. We choose genes: 3-ketoacyl-CoA thiolase (ERG10), squalene synthase (ERG9 or SQS1), squalene epoxidase (ERG1), oxidosqualene-lanosterol cyclase (ERG7), and lanosterol 14-demethylase (ERG11), of L. major and L. braziliensis, besides YIP1 gene of L. braziliensis. For this, primers were synthesized using the sequences generated by genome project of these species inserted in gene bank. The genes ERG10, SQS1, ERG1, ERG7 and ERG11 of L. major and of L. braziliensis besides YIP1 of L. braziliensis were amplified by PCR and cloned into pGEM-T Easy vector that enabled all genes disruption by insertion of HYG cassette and, with exception of the ERG7 gene of L. braziliensis, genes were subcloned into shuttle-vector pXG1. Restrictions fragments of these genes were used in Northern analysis to verify the transcripts level. We verified through short-run PFGE and Southern analysis that the resistant cell lines do not show amplification of studied loci. The participation of ergosterol biosynthesis pathway genes of L. major and L. braziliensis and YIP1 gene of L. braziliensis in the resistance to terbinafine was verified in functional experiments. With this objective, the genes YIP1, ERG10 and ERG1 of L. braziliensis were transfected into LB2904 cell line of L. braziliensis, and the genes ERG10, SQS1, ERG1, ERG7 and ERG11 and the ERG1 gene disruption by HYG mark of L. major were transfected into LT252 cell line of L. major. Experiments that analyze the susceptibility to amphotericin B associated or not to terbinafine were performed using the wild type cell lines of L. major and L. braziliensis, and with this initials experiments, we selected cell lines of these two species resistant to amphotericin B. In order to analyze the possible regulatory function of the RIME 5/2/6 elements of L. braziliensis, the repeat LbRIME 5/2/6b was amplified by PCR and cloned into pGEM-T Easy vector and with this clone, the element was disrupted with a SAT cassette. The cloning into vector pGEM enabled to analyze the interaction of the nuclear protein with the repeat LbRIME 5/2/6b through gel shift assay.

O ergosterol é um componente responsável por manter a integridade e a fluidez das membranas de Leishmania spp. A partir de uma metodologia que consiste em seleção por superexpressão gênica, foram isolados nove diferentes loci de L. (L.) major relacionados com a resistência a dois inibidores da via de biossíntese do ergosterol: Terbinafina (TBF) e Itraconazol (ITZ). Análises funcionais individuais desses nove loci na presença de TBF e ITZ (ou do análogo Cetoconazol - CTZ) apresentaram níveis significantes de resistência após transfecção em células selvagens de L. (L.) major. Nesse trabalho apresentamos a metodologia de isolamento de um desses loci (cItz2), bem como a análise in silico das regiões cromossômicas correspondentes aos insertos dos nove cosmídios no genoma de L. (L.) major / Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated nine different loci of L. (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, Terbinafine (TBF) and Itraconazole (ITZ). Individual functional analysis of these nine loci in the presence of TBF and/or ITZ (or the ITZ analog Ketoconazole, CTZ), have showed significant levels of resistance after transfection into L. (L.) major wild-type cells, followed by over expression induction. In this work, we show the insert mapping and chromosomal identification of one of these loci (cItz2), as well as discuss the in silico chromosomal analysis of the nine correspondent inserts in the L. (L.) major genome

O ergosterol é um componente responsável por manter a integridade e a fluidez das membranas de Leishmania spp. A partir de uma metodologia que consiste em seleção por superexpressão gênica, foram isolados nove diferentes loci de L. (L.) major relacionados com a resistência a dois inibidores da via de biossíntese do ergosterol: Terbinafina (TBF) e Itraconazol (ITZ). Análises funcionais individuais desses nove loci na presença de TBF e ITZ (ou do análogo Cetoconazol - CTZ) apresentaram níveis significantes de resistência após transfecção em células selvagens de L. (L.) major. Nesse trabalho apresentamos a metodologia de isolamento de um desses loci (cItz2), bem como a análise in silico das regiões cromossômicas correspondentes aos insertos dos nove cosmídios no genoma de L. (L.) major / Ergosterol is an important compound responsible to maintain integrity and fluidity of Leishmania spp. membranes. Starting from an overexpression/selection method, our group has isolated nine different loci of L. (L.) major related to resistance against two inhibitors of the ergosterol biosynthesis pathway, Terbinafine (TBF) and Itraconazole (ITZ). Individual functional analysis of these nine loci in the presence of TBF and/or ITZ (or the ITZ analog Ketoconazole, CTZ), have showed significant levels of resistance after transfection into L. (L.) major wild-type cells, followed by over expression induction. In this work, we show the insert mapping and chromosomal identification of one of these loci (cItz2), as well as discuss the in silico chromosomal analysis of the nine correspondent inserts in the L. (L.) major genome

La lutte chimique est la principale méthode utilisée pour contrôler les maladies causées par les champignons phytopathogènes. Dans certains cas, desphénomènes de résistance envers les fongicides se développent au sein despopulations, altérant parfois l’efficacité des molécules. La compréhension du moded’action des fongicides et des mécanismes de résistance sous-jacents participe à élaboreret à adapter des stratégies de management anti résistance ; et ainsi permettre depérenniser la durée de vie des molécules. Le fenhexamid est un fongicide récent (BayerCropScience, 2000), avec un mode d’action unique. Il est le seul fongicide commercialisébloquant l’étape de C4-déméthylation de la biosynthèse de l’ergostérol. Plusieurs typesde résistance (naturelle et acquises) ont été détectées dans les vignobles européens chez lecomplexe d’espèces Botrytis spp. responsable de la pourriture grise de la vigne. Lestravaux développés durant la thèse s’inscrivent dans l’objectif de la caractérisation dumode d’action et de l’élucidation des mécanismes de résistance. Le premier axe s’estattaché à la caractérisation fonctionnelle de deux gènes impliqués dans la C-4déméthylation de la biosynthèse de l’ergostérol : le gène erg27 codant la 3-céto réductase,cible du fenhexamid, et le gène erg28 codant une protéine qui interagirait en partie avecla 3-céto réductase. Concernant la résistance au fenhexamid, il a été démontré que, pargénétique inverse, les mutations détectées dans le gène erg27 de différents types d'isolatsrésistants issus du vignoble (phénotypes de résistance HydR3- et HydR3+) conféraient larésistance. Par ailleurs, une analyse de fitness du phénotype le plus préoccupant(phénotype HydR3+) a été réalisée en conditions contrôlées sur des souches isogéniquesartificielles afin d’apporter une réponse sur la persistance possible de ces souches auvignoble. Une méthode fine de quantification moléculaire de ces mêmes isolats aégalement été mise au point pour faciliter le suivi de leur évolution et de la persistancedes populations naturelles à l’échelle des vignobles. Cette nouvelle méthode, nomméeASPPAA PCR, exploite le polymorphisme nucléotidique du gène erg27, à l’origine de larésistance. Enfin, la résistance naturelle au fenhexamid de l’espèce apparentée à Botrytiscinerea, appelée Botrytis pseudocinerea a été élucidée. La résistance au fongicide de cetteespèce a été expliquée par la combinaison de modifications de cible (mécanismeminoritaire) et d’une dégradation du fongicide par un cytochrome P450 nomméCyp68.4 (mécanisme majeur). Il s’agit de la première identification et caractérisationgénétique d’un mécanisme de résistance à un fongicide conférée par un processus dedétoxification chez un champignon phytopathogène. / Chemical control is the main method used to control diseases caused byphytopathogenic fungi. In some cases, the resistance phenomena towardfungicides occur within fungal populations, which might alter practicalefficiency of molecules. Understanding modes of action of fungicides andunderlying resistance mechanisms participate to the development and adaptationof management strategies against resistance, and thus help to sustain the life ofmolecules. Fenhexamid is a recent fungicide (Bayer CropScience, 2000), with aparticular mode of action. It is the only fungicide marketed blocking the C4-demethylation step of ergosterol biosynthesis. Several types of resistance (naturaland acquired) were detected in European vineyards in the Botrytis spp speciescomplex, causing grey mold disease. This work focused on the characterization ofthe mode of action and the elucidation of resistance mechanisms. The first aspectinvestigated the functional characterization of two genes involved in the C4-demethylation of ergosterol biosynthesis. The erg27 gene potentially encoding the3-keto reductase which is the fenhexamid target and the erg28 gene encoding aprotein that interact in part with the 3-ketoreductase. Concerning fenhexamidresistance, we shown by reverse genetics that mutations detected in the erg27 genefrom different resistant isolates from the vineyards (phenotypes HydR3- andHydR3+) confer resistance. Furthermore, a fitness analysis under controlledconditions on the most worrying resistant phenotype (HydR3+) was performed onisogenic artificial strains in order to predict the possible persistence of these strainsin vineyards. A fine molecular method to quantify these isolates was developed tofacilitate the follow-up of evolution and persistence of resistant populations in thevineyard. This new method, named ASPPAA PCR is based on the nucleotidepolymorphism of the erg27 gene, responsible for fenhexamid resistance. Finally,the natural resistance to fenhexamid of the related species to Botrytis cinerea, B.pseudocinerea, was elucidated. Fungicide resistance of this species is explained bythe combination of target site modifications (minor mechanism) and fungicidedegradation mediated by a cytochrome P450 named Cyp68.4 (major mechanism).This is the first characterization of a genetic resistance mechanism to a fungicideconferred by detoxification in a phytopathogenic fungus.