Identification of novel arsenic resistance genes in yeast

Arsenic is a toxic metalloid that affects human health by causing numerous diseases and by being used in the treatment of acute promyelocytic leukemia. Saccharomyces cerevisiae (budding yeast) has been extensively utilized to elucidate the molecular mechanisms underlying arsenic toxicity and resistance in eukaryotes. In this study, we applied a genomic DNA overexpression strategy to identify yeast genes that provide arsenic resistance in wild-type and arsenic-sensitive S. cerevisiae cells. In addition to known arsenic-related genes, our genetic screen revealed novel genes, including PHO86, VBA3, UGP1, and TUL1, whose overexpression conferred resistance. To gain insights into possible resistance mechanisms, we addressed the contribution of these genes to cell growth, intracellular arsenic, and protein aggregation during arsenate exposure. Overexpression of PHO86 resulted in higher cellular arsenic levels but no additional effect on protein aggregation, indicating that these cells efficiently protect their intracellular environment. VBA3 overexpression caused resistance despite higher intracellular arsenic and protein aggregation levels. Overexpression of UGP1 led to lower intracellular arsenic and protein aggregation levels while TUL1 overexpression had no impact on intracellular arsenic or protein aggregation levels. Thus, the identified genes appear to confer arsenic resistance through distinct mechanisms but the molecular details remain to be elucidated

Arsenic and Antimony Transporters in Eukaryotes

Arsenic and antimony are toxic metalloids, naturally present in the environment and all organisms have developed pathways for their detoxification. The most effective metalloid tolerance systems in eukaryotes include downregulation of metalloid uptake, efflux out of the cell, and complexation with phytochelatin or glutathione followed by sequestration into the vacuole. Understanding of arsenic and antimony transport system is of high importance due to the increasing usage of arsenic-based drugs in the treatment of certain types of cancer and diseases caused by protozoan parasites as well as for the development of bio- and phytoremediation strategies for metalloid polluted areas. However, in contrast to prokaryotes, the knowledge about specific transporters of arsenic and antimony and the mechanisms of metalloid transport in eukaryotes has been very limited for a long time. Here, we review the recent advances in understanding of arsenic and antimony transport pathways in eukaryotes, including a dual role of aquaglyceroporins in uptake and efflux of metalloids, elucidation of arsenic transport mechanism by the yeast Acr3 transporter and its role in arsenic hyperaccumulation in ferns, identification of vacuolar transporters of arsenic-phytochelatin complexes in plants and forms of arsenic substrates recognized by mammalian ABC transporters

Molecular mechanisms involved in arsenic perception and tolerance in "Arabidopsis thaliana"

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-11-2014Arsenate is a toxic metalloid to living organisms. The incorporation of this toxic element to soil and water by anthropogenic and volcanic activities pose continuous threat to sessile organisms. Because of close chemical similarity between arsenate [As(V)] and phosphate (Pi), As(V) uses the Pi transporter to enter plant cell, which makes plants vulnerable for this metalloid. Suppression of Pi transporters is a strategy widely use by plants in nature to prevent arsenate uptake. However, closing Pi-transporters compromises phosphate availability and thus plant survival. Currently it is completely unknown, how plants manage this delicate situation preventing arsenic uptake without impairing phosphate acquisition. Here we found that in response to arsenate, the Pi/As(V) transporter PHT1;1 rapidly repressed its expression during initial 2 to 8 hrs of As(V) exposure. The early repression of the Pi-transporter is regulated by WRKY6, a repressor that is upregulated by As(V). The repression is coordinated by the degradation of PHR1, a transcriptional activator of PHT1;1. The repression is also accompanied by delocalization of the transporter from the plasma membrane. All these responses leads to an instant suppression of As(V) uptake. PHR1 degradation is mediated by ASK18, an SKP1-like protein that is transcriptionally regulated by As(V). Interestingly, ASK18 responsiveness depends on the arsenic detoxification status of plants. Furthermore, we show that plants reduce their endogenous cytokinin (CK) content as an early response to As(V) that leads to increase detoxification capacity. PHT1;1 repression and CKs reduction thus allows plants to coordinately reduce As(V) uptake and increased capacity to detoxify intracellular arsenic. Accordingly, transgenic lines with reduced levels of endogenous CKs shows remarkable arsenic tolerance phenotype. Surprisingly, after 24 hrs of As(V) exposure PHT1;1 repression is abolished and the expression is reactivated even in the presence of As(V). Here we found that PHT1;1 expression recovery is controlled by PHR1 stabilization that is also dependent on plant detoxification capacity. Overall the plants are able to adapt arsenic uptake to their detoxification capacity.El arsenato es un metaloide tóxico para los seres vivos. La incorporación de este elemento tóxico al suelo y al agua por actividades antropogénicas y volcánicas supuso una amenaza continua para los organismos sésiles. Debido a la estrecha similitud química del [As(V)] con el fosfato (Pi), el As(V) usa el transportador de Pi para entrar en las células vegetales, lo que hace a las plantas particularmente vulnerables a este metaloide. La supresión de los transportadores de Pi es una estrategia ampliamente utilizada por las plantas en la naturaleza para prevenir la absorción de arsenato. Sin embargo, el cierre de los transportadores de fosfato compromete la disponibilidad de este elemento y, por tanto, la supervivencia de la planta. Actualmente se desconoce el modo en que las plantas gestionan esta delicada situación, previniendo la absorción de arsenato sin dificultar la adquisición de fosfato. Hemos descubierto que el transportador de As(V)/Pi PHT1;1 reprime rápidamente su expresión en respuesta a arsenato, durante las 2 a 8 horas iniciales de exposición a dicho metaloide. La represión temprana del transportador de Pi es regulada por WRKY6, un represor cuya expresión aumenta en respuesta a As(V). Dicha represión está coordinada por la degradación de PHR1, un activador transcripcional de PHT1;1, y además está acompañada por la deslocalización del transportador de la membrana plasmática. Todas estas respuestas llevan a una supresión inmediata de la absorción de As(V). La degradación de PHR1 está mediada por ASK18, una proteína del tipo SKP1 cuya trascripción está regulada por As(V). Curiosamente, la respuesta de ASK18 depende del estado de detoxificación de arsénico de la planta. Además, hemos demostrado que las plantas reducen su contenido endógeno en citoquininas (CK) como respuesta temprana al As(V) lo cual produce un aumento en la capacidad de detoxificación. Por tanto la represión de PHT1;1 y la reducción de CKs permite a las plantas dar una respuesta coordinada, reduciendo la absorción de As(V) e incrementando la capacidad de detoxificación del arsénico intracelular. Así, líneas transgénicas con niveles reducidos de CKs endógenas muestran un fenotipo significativo de tolerancia a arsénico. Sorprendentemente, tras 24 horas de exposición a As(V), la represión de PHT1;1 se anula, reactivando su expresión incluso en presencia de As(V). Hemos encontrado que la recuperación de la expresión de PHT1;1 está controlada por la estabilización de PHR1, la cual también depende de la capacidad de detoxificación de la planta. En resumen, las plantas son capaces de adaptar la absorción de As(V) a su capacidad de detoxificació

Investigating the origin and transport of methylated arsenic species in plants

Inorganic arsenic is a toxic element known to cause various diseases and cancers in humans. Arsenic contamination is widespread worldwide, particularly in South-East Asia where arsenic-contaminated groundwater is used for drinking and rice cultivation. Unlike other cereals, paddy rice can efficiently accumulate arsenic in the grain. Rice is a staple food for around 50% of the world's population, so arsenic accumulation in rice is of great concern. Arsenite, As(III), is the predominant form of arsenic within plants, but rice grains often contain significant proportions of organic arsenic species. The most common of these are dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA). A series of axenic experiments demonstrated that plants are unable to methylate arsenic, and instead take these species up from soil where they are produced by micro-organisms. The uptake of undissociated MMA by rice roots is predominantly facilitated by OsNIP2;1 (OsLsi1), a member of the NIP-subfamily of aquaporins, which also accounts for 50% of root DMA uptake. Expression of OsNIP1;1 and OsNIP3;3 in Xenopus oocytes demonstrated that these NIP aquaporins are permeable to pentavalent MMA, as well as arsenite, silicon and water. However, uptake of DMA was not observed for oocytes expressing any NIP gene, including OsNIP2;1. MMA and DMA have a pKa1 of 4.19 and 6.14 respectively, and so increasing the pH of the medium increases the proportion of dissociated complexes. In hydroponic culture, rice plants over-expressing the high-affinity phosphate transporter OsPT8, took up significantly more MMA and DMA than wild-type. Additionally, the presence of phosphate in the medium significantly decreased the uptake of both MMA and DMA by OsPT8-overexpression and wild-type rice plants. Therefore we have discovered that methylated arsenic species are not formed within plants, and can be transported by two different classes of transporters depending on the pH of the medium

Role of Yap8 and Yap1 b-ZIP transcription factors in arsenic stress

Arsenic compounds are highly toxic substances; nevertheless they are used in the treatment of acute promyelocytic leukaemia. Therefore it is pressing to gain knowledge on its toxicity and detoxification mechanisms. The cellular entry pathways have been discovered and by transcriptome analysis it is known that arsenic activates the transcription of genes activated by, among others, Rpn4, Met4 and Yap1.(...)Fundação para a Ciência e a Tecnologi

Personalized Medicine: Studies of Pharmacogenomics in Yeast and Cancer

Advances in microarray and sequencing technology enable the era of personalized medicine. With increasing availability of genomic assays, clinicians have started to utilize genetics and gene expression of patients to guide clinical care. Signatures of gene expression and genetic variation in genes have been associated with disease risks and response to clinical treatment. It is therefore not difficult to envision a future where each patient will have clinical care that is optimized based on his or her genetic background and genomic profiles. However, many challenges exist towards the full realization of the potential personalized medicine. The human genome is complex and we have yet to gain a better understanding of how to associate genomic data with phenotype. First, the human genome is very complex: more than 50 million sequence variants and more than 20,000 genes have been reported. Many efforts have been devoted to genome-wide association studies (GWAS) in the last decade, associating common genetic variants with common complex traits and diseases. While many associations have been identified by genome-wide association studies, most of our phenotypic variation remains unexplained, both at the level of the variants involved and the underlying mechanism. Finally, interaction between genetics and environment presents additional layer of complexity governing phenotypic variation. Currently, there is much research developing computational methods to help associate genomic features with phenotypic variation. Modeling techniques such as machine learning have been very useful in uncovering the intricate relationships between genomics and phenotype. Despite some early successes, the performance of most models is disappointing. Many models lack robustness and predictions do not replicate. In addition, many successful models work as a black box, giving good predictions of phenotypic variation but unable to reveal the underlying mechanism. In this thesis I propose two methods addressing this challenge. First, I describe an algorithm that focuses on identifying causal genomic features of phenotype. My approach assumes genomic features predictive of phenotype are more likely to be causal. The algorithm builds models that not only accurately predict the traits, but also uncover molecular mechanisms that are responsible for these traits. . The algorithm gains its power by combining regularized linear regression, causality testing and Bayesian statistics. I demonstrate the application of the algorithm on a yeast dataset, where genotype and gene expression are used to predict drug sensitivity and elucidate the underlying mechanisms. The accuracy and robustness of the algorithm are both evaluated statistically and experimentally validated. The second part of the thesis takes on a much more complicated system: cancer. The availability of genomic and drug sensitivity data of cancer cell lines has recently been made available. The challenge here is not only the increasing complexity of the system (e.g. size of genome), but also the fundamental differences between cancers and tissues. Different cancers or tissues provide different contexts influencing regulatory networks and signaling pathways. In order to account for this, I propose a method to associate contextual genomic features with drug sensitivity. The algorithm is based on information theory, Bayesian statistics, and transfer learning. The algorithm demonstrates the importance of context specificity in predictive modeling of cancer pharmacogenomics. The two complementary algorithms highlight the challenges faced in personalized medicine and the potential solutions. This thesis detailed the results and analysis that demonstrate the importance of causality and context specificity in predictive modeling of drug response, which will be crucial for us towards bringing personalized medicine in practice

Yeast as a model system to study genetic and post-translational regulation of metabolic pathways in mammals

Dissertation presented to obtain the Ph.D degree in BiologyThe work presented in this thesis describes the use of yeast Saccharomyces cerevisiae as a model system to study two different stress response processes and its extrapolation to higher eukaryotes.(...)Financial Support from Fundação para a Ciência e a Tecnologia (No.SFRH/BD/39389/2007)

Links between electrophilic stress and antifungal resistance in pathogenic Candida species

Collectively, Candida species are the most prevalent cause of both superficial and invasive fungal infections worldwide. Invasive Candida infections have a high mortality rate and predominantly affect individuals with underlying diseases, such as diabetes, HIV, or cancer. Unfortunately, many invasive Candida infections are recalcitrant to antifungal treatment, while intrinsically multidrug-resistant pathogens, like Candida auris, are increasing in prevalence. Although the canonical mechanisms of antifungal resistance in Candida species are well established, i.e., overexpression of efflux pumps and overexpression of or mutations in genes encoding drug targets, factors affecting the natural evolution and regulation of resistance mechanisms remain poorly understood. One cause of antifungal resistance in Candida species is the acquisition of gain-of-function mutations in the transcription factor Mrr1, resulting in overexpression of the multidrug transporter Mdr1. However, little is known about the functions of other genes regulated by Mrr1 or how Mrr1 activity is modulated in vivo. In this work, we demonstrate in Candida lusitaniae and in C. auris that Mrr1 contributes to resistance against methylglyoxal (MG), a toxic, electrophilic dicarbonyl derived from natural metabolic processes, and that Mrr1-mediated MG resistance is driven in part by expression of the methylglyoxal reductase genes MGD1 and MGD2 in C. lusitaniae and MGD1 in C. auris. Furthermore, we show that a sublethal concentration of MG induces expression of MDR1 and MG reductase genes in C. lusitaniae and C. auris, and consequently increases fluconazole (FLZ) resistance in C. lusitaniae. Finally, we characterize the complete Mrr1- dependent and independent transcriptional response of C. auris to MG and to the known inducer of Mrr1-regulated gene expression, benomyl, and show that both compounds cause the differential expression of a multitude of genes involved in metabolism and stress response, which could contribute to pathogen survival while colonizing and infecting a mammalian host. Together, the work presented herein provides valuable insight into a potential mechanism for the regulation of Mrr1-dependent transcription in vivo as well as a possible selective pressure for gain-of-function mutations in the MRR1 gene. This is particularly noteworthy because MG is elevated in many of the same human diseases that are considered risk factors for Candida infection, and MG is also produced by activated phagocytes in response to pathogens. Thus, it is conceivable that Candida would encounter biologically significant levels of MG in the context of infection. We propose that MG-mediated induction of Mrr1-dependent transcription in Candida species is one factor that plays a role in antifungal treatment failure

Saccharomyces cerevisiae como biossensor de arsénio na água: caso de estudo da Ribeira do Bodelhão e do Rio Zêzere

Dissertação para obtenção do Grau de Mestre em Tecnologia e Segurança AlimentarO metalóide arsénio encontra-se disperso na crosta terrestre. Porém, em Portugal encontram-se níveis elevados de arsénio na água pontualmente nalgumas cidades situadas a Norte, devido a processos geológicos. O arsénio é altamente tóxico para o organismo, sendo 10 μg/L a concentração máxima permitida por Lei para a água para consumo humano, o principal veículo através do qual o arsénio inorgânico se propaga no ambiente. Os métodos químicos de detecção de arsénio na água são extremamente precisos, mas dispendiosos. A levedura Saccharomyces cerevisiae, amplamente utilizada como organismo modelo, foi geneticamente manipulada com o objectivo de desenvolver um sistema biossensor para detectar arsénio na água. Testaram-se as estirpes mais sensíveis ao arsenito, BY4741yap8 e BY4741acr3yap8, na presença de diferentes fontes de carbono: glucose, sacarose e galactose. Os plasmídeos recombinantes expressando os genes HXT7 e FPS1 (importadores de arsenito), controlados pelo gene promotor GAL1, foram transformados nas respectivas estirpes, desenvolvendo desta forma os sistemas de biodetecção. As amostras de água colhidas no Rio Zezêre e na Ribeira do Bodelhão foram avaliadas físico-quimicamente e com os modelos biológicos construídos. Estes locais sofrem contaminação por águas de lixiviação de duas escombreiras pertencentes às minas da Panasqueira que contêm níveis elevados de arsénio e do efluente proveniente do tratamento deficitário da ETAM da mina. A Ribeira do Bodelhão revelou ser a mais contaminada (ecotoxicidade pouco significativa), com níveis elevados de condutividade, potencial redox e sólidos inorgânicos, entre eles, arsénio, cobre, manganês e zinco. Apenas as estirpes de levedura transformadas com o plasmídeo YEplac181GAL1FPS1 revelaram sensibilidade a um elemento desconhecido nas amostras de água. As estirpes transformadas com o plasmídeo YEplac195GAL1HXT7 revelaram maior sensibilidade nos ensaios laboratoriais. No entanto, o facto de não ter demonstrado o mesmo resultado com as amostras de água revelou que os elementos se encontravam maioritariamente complexados dificultando a entrada através do transportador Hxt7

Estrategias dietéticas para disminuir la biodisponibilidad oral de arsénico inorgánico

La exposición crónica a arsénico (As) inorgánico [As(III) + As(V)] está asociada con una mayor incidencia de varios tipos de cáncer, diabetes tipo II, enfermedades vasculares y déficits neuroconductuales en población infantil. Se considera que en la actualidad alrededor de 200 millones de personas están expuestas a niveles de As inorgánico superiores a los recomendados, fundamentalmente a través del agua de bebida. Esta situación se ha puesto de manifiesto en los estudios realizados en esta tesis donde se muestran ingestas de As inorgánico muy superiores a las recomendadas en poblaciones del norte de Argentina, aportadas por el agua, pero también por los alimentos cocinados con esta agua contaminada. Los estudios de especiación por espectroscopía de absorción de rayos X muestran que el cocinado en presencia de As(V) produce un incremento en el contenido de As(III) en el producto listo para consumo. Esto supone, en algunos casos, un aumento del riesgo asociado a la ingesta del alimento, ya que la forma trivalente es más tóxica. La caracterización de la fracción solubilizada durante la digestión gastrointestinal de estos alimentos (fracción bioaccesible) evidencia que el As(III) permanece en esta forma, incluso que durante la digestión puede haber un aumento de la cantidad de la forma reducida. Estos resultados evidencian el riesgo asociado a la ingesta de agua contaminada y de los alimentos cocinados con esta agua. Debido a la cantidad de personas afectadas y a las patologías que desarrollan, agencias relacionadas con la salud han emitido informes en los que se indica la necesidad de reducir la ingesta de As inorgánico. Teniendo en cuenta la dificultad de eliminar el As de las fuentes de exposición, una alternativa para reducir la exposición es disminuyendo la biodisponibilidad (cantidad del tóxico que llega a la circulación sistémica tras la ingesta). La biodisponibilidad puede reducirse empleando compuestos que formen complejos con el metaloide durante su paso por el tracto digestivo y eviten que éste se encuentre en una forma de fácil absorción o impidiendo que se absorba empleando compuestos que compitan por sus mismas vías de entrada o que simplemente las afecten. En esta tesis se ha planteado este objetivo empleando componentes de la dieta y microorganismos de grado alimentario. Inicialmente se han seleccionado componentes y microorganismos capaces de reducir la cantidad del tóxico que queda soluble tras la digestión y por tanto disponible para la absorción (bioaccesibilidad) empleando un modelo de digestión in vitro. Este ensayo ha evidenciado que las sales de hierro, el tánico, la lignina y algunas celulosas pueden reducir la bioaccesibilidad del As(III) y el As(V) en solución acuosa y también cuando forman parte de los alimentos (algas y arroz). Sin embargo, las bacterias lácticas y las levaduras Saccharomyces cerevisiae no son capaces de interaccionar con las formas inorgánicas de As y reducir su bioaccesibilidad, posiblemente por las características de sus paredes celulares. Además, se ha puesto de manifiesto, empleando células intestinales como modelo de epitelio intestinal, que determinados compuestos alimentarios (hierro, cisteína, glutatión, curcumina, catequinas y quercetina) y suplementos dietéticos (extracto de alcachofa y de uva y suplementos de hierro); así como algunas cepas de Lactobacillus y S. cerevisiae son capaces de reducir el transporte a través del epitelio intestinal de las formas inorgánicas de As en disolución acuosa y en alimentos. Estos compuestos actúan formando complejos de menor absorción o modulando las uniones intercelulares y afectando la vía paracelular, una de las formas de entrada del As en el epitelio intestinal. Finalmente, se ha confirmado que algunos de los componentes dietarios eficaces in vitro (glutatión y sales férricas) son capaces de reducir la biodisponibilidad de As(III) y As(V) y su acumulación tisular en ratones. Además, también se ha puesto de manifiesto que estos componentes no reducen la biodisponibilidad de otros elementos esenciales. Estos datos sugieren que ambos compuestos podrían constituir estrategias válidas para la reducción de la exposición al tóxico en poblaciones de zonas con arsenicismo crónico endémico.Chronic exposure to arsenic (As) inorganic [As (III) + As (V)] is associated with a higher incidence of several types of cancer, type II diabetes, vascular diseases and neurobehavioral deficits in children. It is considered that currently around 200 million people are exposed to levels of inorganic As above those recommended, mainly through drinking water. This situation has been shown in the studies carried out in this thesis, which shows intakes of inorganic As that are much higher than those recommended in populations of northern Argentina, contributed by water, but also by food cooked with this contaminated water. Speciation studies by X-ray absorption spectroscopy show that cooking in the presence of As (V) produces an increase in the content of As (III) in the product ready for consumption. This supposes, in some cases, an increase in the risk associated with food intake, since the trivalent form is more toxic. The characterization of the fraction solubilized during the gastrointestinal digestion of these foods (bioaccessible fraction) shows that As (III) remains in this form, even that during the digestion there may be an increase in the amount of the reduced form. These results show the risk associated with the intake of contaminated water and the food cooked with this water.Due to the number of people affected and the pathologies they develop, health-related agencies have issued reports indicating the need to reduce the intake of inorganic As. Taking into account the difficulty of eliminating the As from the sources of exposure, an alternative to reduce the exposure is decreasing the bioavailability (amount of the toxic that reaches the systemic circulation after the ingestion). The bioavailability can be reduced by using compounds that form complexes with the metalloid during its passage through the digestive tract and prevent it from being in an easily absorbed form or preventing it from being absorbed by using compounds that compete for their same entry routes or that simply affect In this thesis has been raised this objective using dietary components and food grade microorganisms. Initially, components and microorganisms have been selected that can reduce the amount of the toxin that remains soluble after digestion and therefore available for absorption (bioavailability) using an in vitro digestion model. This trial has shown that the iron salts, tannic, lignin and some celluloses can reduce the bioavailability of As (III) and As (V) in aqueous solution and also when they are part of the food (algae and rice). However, lactic acid bacteria and Saccharomyces cerevisiae yeasts are not able to interact with the inorganic forms of As and reduce their bioavailability, possibly due to the characteristics of their cell walls. In addition, it has been shown, using intestinal cells as a model of intestinal epithelium, that certain food compounds (iron, cysteine, glutathione, curcumin, catechins and quercetin) and dietary supplements (artichoke and grape extract and iron supplements); as well as some strains of Lactobacillus and S. cerevisiae are able to reduce the transport through the intestinal epithelium of the inorganic forms of As in aqueous solution and in food. These compounds act by forming lower absorption complexes or by modulating the intercellular junctions and affecting the paracellular pathway, one of the forms of entry of As in the intestinal epithelium. Finally, it has been confirmed that some of the effective dietary components in vitro (glutathione and ferric salts) are capable of reducing the bioavailability of As (III) and As (V) and their tissue accumulation in mice. In addition, it has also been shown that these components do not reduce the bioavailability of other essential elements. These data suggest that both compounds could be valid strategies for the reduction of toxic exposure in populations of areas with chronic endemic arsenicism