Paulo Jorge Dias

MFS multidrug transporters in pathogenic fungi: do they have real clinical impact?

Infections caused by opportunistic fungal pathogens have reached concerning numbers due to the increase of the immunocrompromised human population and to the development of antifungal resistance. This resistance is often attributed to the action of multidrug efflux pumps, belonging to the ATP-binding cassette (ABC) superfamily and the major facilitator superfamily (MFS). Although many studies have focused on the role of ABC multidrug efflux transporters, little is still known on the part played by the Drug:H+ Antiporter (DHA) family of the MFS in this context. This review summarizes current knowledge on the role in antifungal drug resistance, mode of action and phylogenetic relations of DHA transporters, from the model yeast S. cerevisiae to pathogenic yeasts and filamentous fungi. Through the compilation of the predicted DHA transporters in the medically relevant Candida albicans, C. glabrata, C. parapsilosis, C. lusitaniae, C. tropicalis, C. guilliermondii, Cryptococcus neoformans, and Aspergillus fumigatus species, the fact that only 5% of the DHA transporters from these organisms have been characterized so far is evidenced. The role of these transporters in antifungal drug resistance and in pathogen-host interaction is described and their clinical relevance discussed. Given the knowledge gathered for these few DHA transporters, the need to carry out a systematic characterization of the DHA multidrug efflux pumps in fungal pathogens, with emphasis on their clinical relevance, is highlighted.

Insights into the Mechanisms of Toxicity and Tolerance to the Agricultural Fungicide Mancozeb in Yeast, as Suggested by a Chemogenomic Approach

Abstract Saccharomyces cerevisiae was used to uncover the mechanisms underlying tolerance and toxicity of the agricultural fungicide mancozeb, linked to cancer and Parkinsons disease development. Chemogenomics screening of a yeast deletion mutant collection revealed 286 genes that provide protection against mancozeb toxicity. The most significant Gene Ontology (GO) terms enriched in this dataset are associated to transcriptional machinery, vacuolar organization and biogenesis, intracellular trafficking, and cellular pH regulation. Clustering based on physical and genetic interactions further highlighted the role of oxidative stress response, protein degradation and carbohydrate/energy metabolism in mancozeb stress tolerance. Mancozeb was found to act in yeast as a thiol-reactive compound, but not as a free radical or reative oxygen species (ROS) inducer, leading to massive oxidation of protein cysteins, consistent with the requirement of genes involved in glutathione biosynthesis and reduction and in protein degradation to provide mancozeb resistance. The identification of Botrytis cinerea homologues of yeast mancozeb tolerance determinants is expected to guide studies on mancozeb mechanisms of action and tolerance in phytopathogenic fungi. The generated networks of protein-protein associations of yeast mancozeb tolerance determinants and their human orthologues share a high degree of similarity. This toxicogenomics analysis may, thus, increase the understanding of mancozeb toxicity and adaptation mechanisms in humans.

MFS transporters required for multidrug/multixenobiotic (MD/MX) resistance in the model yeast: understanding their physiological function through post-genomic approaches

Multidrug/Multixenobiotic resistance (MDR/MXR) is a widespread phenomenon with clinical, agricultural and biotechnological implications, where MDR/MXR transporters that are presumably able to catalyze the efflux of multiple cytotoxic compounds play a key role in the acquisition of resistance. However, although these proteins have been traditionally considered drug exporters, the physiological function of MDR/MXR transporters and the exact mechanism of their involvement in resistance to cytotoxic compounds are still open to debate. In fact, the wide range of structurally and functionally unrelated substrates that these transporters are presumably able to export has puzzled researchers for years. The discussion has now shifted toward the possibility of at least some MDR/MXR transporters exerting their effect as the result of a natural physiological role in the cell, rather than through the direct export of cytotoxic compounds, while the hypothesis that MDR/MXR transporters may have evolved in nature for other purposes than conferring chemoprotection has been gaining momentum in recent years. This review focuses on the drug transporters of the Major Facilitator Superfamily (MFS; drug:H+ antiporters) in the model yeast Saccharomyces cerevisiae. New insights into the natural roles of these transporters are described and discussed, focusing on the knowledge obtained or suggested by post-genomic research. The new information reviewed here provides clues into the unexpectedly complex roles of these transporters, including a proposed indirect regulation of the stress response machinery and control of membrane potential and/or internal pH, with a special emphasis on a genome-wide view of the regulation and evolution of MDR/MXR-MFS transporters.

The drug:H + antiporters of family 2 (DHA2), siderophore transporters (ARN) and glutathione:H + antiporters (GEX) have a common evolutionary origin in hemiascomycete yeasts

BackgroundThe Saccharomyces cerevisiae 14-spanner Drug:H+ Antiporter family 2 (DHA2) are transporters of the Major Facilitator Superfamily (MFS) involved in multidrug resistance (MDR). Although poorly characterized, DHA2 family members were found to participate in the export of structurally and functionally unrelated compounds or in the uptake of amino acids into the vacuole or the cell. In S. cerevisiae, the four ARN/SIT family members encode siderophore transporters and the two GEX family members encode glutathione extrusion pumps. The evolutionary history of DHA2, ARN and GEX genes, encoding 14-spanner MFS transporters, is reconstructed in this study.ResultsThe translated ORFs of 31 strains from 25 hemiascomycetous species, including 10 pathogenic Candida species, were compared using a local sequence similarity algorithm. The constraining and traversing of a network representing the pairwise similarity data gathered 355 full size proteins and retrieved ARN and GEX family members together with DHA2 transporters, suggesting the existence of a close phylogenetic relationship among these 14-spanner major facilitators. Gene neighbourhood analysis was combined with tree construction methodologies to reconstruct their evolutionary history and 7 DHA2 gene lineages, 5 ARN gene lineages, and 1 GEX gene lineage, were identified. The S. cerevisiae DHA2 proteins Sge1, Azr1, Vba3 and Vba5 co-clustered in a large phylogenetic branch, the ATR1 and YMR279C genes were proposed to be paralogs formed during the Whole Genome Duplication (WGD) whereas the closely related ORF YOR378W resides in its own lineage. Homologs of S. cerevisiae DHA2 vacuolar proteins Vba1, Vba2 and Vba4 occur widespread in the Hemiascomycetes. Arn1/Arn2 homologs were only found in species belonging to the Saccharomyces complex and are more abundant in the pre-WGD species. Arn4 homologs were only found in sub-telomeric regions of species belonging to the Sacharomyces sensu strictu group (SSSG). Arn3 type siderophore transporters are abundant in the Hemiascomycetes and form an ancient gene lineage extending to the filamentous fungi.ConclusionsThe evolutionary history of DHA2, ARN and GEX genes was reconstructed and a common evolutionary root shared by the encoded proteins is hypothesized. A new protein family, denominated DAG, is proposed to span these three phylogenetic subfamilies of 14-spanner MFS transporters.

Evolution of the 12-spanner drug : H+ antiporter DHA1 family in hemiascomycetous yeasts.

Frequently, although not exclusively, multidrug resistance (MDR) results from the action of drug-efflux pumps, which are thought to be able to catalyze the active expulsion of several unrelated cytotoxic compounds out of the cell or their intracellular partitioning. The transporters of the major facilitator superfamily (MFS) presumably involved in MDR belong to the 12-spanner drug:H(+) antiporter DHA1 or to the 14- spanner drug:H(+) antiporter DHA2 families. The expression of most Saccharomyces cerevisiae DHA1 family members was found to confer broad chemoprotection. The evolution of the hemiascomycetous DHA1 proteins, belonging to the Génolevures GL3C007 family, was studied using a combined phylogenetic and gene neighborhood approach. The phylogenetic analysis of 189 DHA1 proteins belonging to the genome of 13 hemiascomycetous species identified 20 clusters. Eleven clusters contained no S. cerevisiae members. The phylogenetic clusters were analyzed by the IONS method developed for Identification of Orthologues by Neighborhood and Similarity. This allowed reconstructing the evolutionary history of most DHA1 members within 10 main gene lineages, spanning the whole hemiascomycetes clade, encompassing an evolutionary history of about 350 million years. In addition, five other more species specific lineages, spanning only two hemiascomycetous species, were identified. It is concluded that 57 out of the 143 members of the DHA1 hemiascomycetous members originated from gene duplication events. In half of these duplicates, the two members belong to different phylogenetic clusters, indicating that at least one of them has sufficiently differentiated to provide potential novel functions to this complex family from which most physiological substrates remain unknown.

The Zygosaccharomyces bailii transcription factor Haa1 is required for acetic acid and copper stress responses suggesting subfunctionalization of the ancestral bifunctional protein Haa1/Cup2.

BackgroundThe food spoilage yeast species Zygosaccharomyces bailii exhibits an extraordinary capacity to tolerate weak acids, in particular acetic acid. In Saccharomyces cerevisiae, the transcription factor Haa1 (ScHaa1) is considered the main player in genomic expression reprogramming in response to acetic acid stress, but the role of its homologue in Z. bailii (ZbHaa1) is unknown.ResultsIn this study it is demonstrated that ZbHaa1 is a ScHaa1 functional homologue by rescuing the acetic acid susceptibility phenotype of S. cerevisiae haa1Δ. The disruption of ZbHAA1 in Z. bailii IST302 and the expression of an extra ZbHAA1 copy confirmed ZbHAA1 as a determinant of acetic acid tolerance. ZbHaa1 was found to be required for acetic acid stress-induced transcriptional activation of Z. bailii genes homologous to ScHaa1-target genes. An evolutionary analysis of the Haa1 homologues identified in 28 Saccharomycetaceae species genome sequences, including Z bailii, was carried out using phylogenetic and gene neighbourhood approaches. Consistent with previous studies, this analysis revealed a group containing pre-whole genome duplication species Haa1/Cup2 single orthologues, including ZbHaa1, and two groups containing either Haa1 or Cup2 orthologues from post-whole genome duplication species. S. cerevisiae Cup2 (alias Ace1) is a transcription factor involved in response and tolerance to copper stress. Taken together, these observations led us to hypothesize and demonstrate that ZbHaa1 is also involved in copper-induced transcriptional regulation and copper tolerance.ConclusionsThe transcription factor ZbHaa1 is required for adaptive response and tolerance to both acetic acid and copper stresses. The subfunctionalization of the single ancestral Haa1/Cup2 orthologue that originated Haa1 and Cup2 paralogues after whole genome duplication is proposed.

Phylogenetic and syntenic analyses of the 12-spanner drug:H(+) antiporter family 1 (DHA1) in pathogenic Candida species: evolution of MDR1 and FLU1 genes.

Candida albicans and other pathogenic Candida species can develop resistance to clinical fungicides through active drug export mediated by multidrug efflux pumps, in particular by members of the drug:H(+) antiporter family 1 (DHA1). The DHA1 proteins encoded in the genomes of 31 hemiascomycetous strains from 25 species were identified and homology relationships between these proteins and the functionally characterised DHA1 in the model yeast Saccharomyces cerevisiae were established. Gene neighbourhood analysis allowed the reconstruction of sixteen DHA1 lineages conserved during the CTG complex species evolution. The evolutionary history of C. albicans MDR1 and FLU1 genes and Candida dubliniensis, Candida tropicalis and Candida parapsilosis MDR1 genes was detailed. Candida genomes show an abundant number of MDR1 and FLU1 homologues but the chromosome environment where MDR1 homologues reside was poorly conserved during evolution. Gene duplication and loss are major mechanisms underlying the evolution of the DHA1 genes in Candida species.

Using systems biology approaches to study a multidrug resistance network

Multidrug resistance (MDR), a phenomenon with impact in Human Health and in Agro-Food and Environmental Biotechnology, often results from the activation of drug efflux pumps, many times controlled at the transcriptional level. The complex transcriptional control of these genes has been on the focus of our research, guided by the information gathered in the YEASTRACT database. In this paper, the approach used to elucidate the transcriptional control of FLR1, encoding a Saccharomyces cerevisiae Drug:H+ Antiporter, in response to stress induced by the fungicide mancozeb is explained. The transcription regulatory network underlying FLR1 activation was defined based on experimental data. Subsequently, a mathematical model describing this network was built and its response to mancozeb stress in different genetic backgrounds was simulated, using the Genetic Network Analyzer (GNA) software. This approach allowed the identification of essential features of the transition from unstressed to fungicide stressed cells and to make new predictions on the dynamical behavior of the system, which were validated experimentally. This work provides a good example of the successful combination of experimental and computational approaches in a systems biology perspective.

The Paralogous Genes PDR18 and SNQ2, Encoding Multidrug Resistance ABC Transporters, Derive From a Recent Duplication Event, PDR18 Being Specific to the Saccharomyces Genus

Pleiotropic drug resistance (PDR) family of ATP-binding cassette (ABC) transporters play a key role in the simultaneous acquisition of resistance to a wide range of structurally and functionally unrelated cytotoxic compounds in yeasts. Saccharomyces cerevisiae Pdr18 was proposed to transport ergosterol at the plasma membrane, contributing to the maintenance of adequate ergosterol content and decreased levels of stress-induced membrane disorganization and permeabilization under multistress challenge leading to resistance to ethanol, acetic acid and the herbicide 2,4-D, among other compounds. PDR18 is a paralog of SNQ2, first described as a determinant of resistance to the chemical mutagen 4-NQO. The phylogenetic and neighborhood analysis performed in this work to reconstruct the evolutionary history of ScPDR18 gene in Saccharomycetaceae yeasts was focused on the 214 Pdr18/Snq2 homologs from the genomes of 117 strains belonging to 29 yeast species across that family. Results support the idea that a single duplication event occurring in the common ancestor of the Saccharomyces genus yeasts was at the origin of PDR18 and SNQ2, and that by chromosome translocation PDR18 gained a subtelomeric region location in chromosome XIV. The multidrug/multixenobiotic phenotypic profiles of S. cerevisiae pdr18Δ and snq2Δ deletion mutants were compared, as well as the susceptibility profile for Candida glabrata snq2Δ deletion mutant, given that this yeast species has diverged previously to the duplication event on the origin of PDR18 and SNQ2 genes and encode only one Pdr18/Snq2 homolog. Results show a significant overlap between ScSnq2 and CgSnq2 roles in multidrug/multixenobiotic resistance (MDR/MXR) as well as some overlap in azole resistance between ScPdr18 and CgSnq2. The fact that ScSnq2 and ScPdr18 confer resistance to different sets of chemical compounds with little overlapping is consistent with the subfunctionalization and neofunctionalization of these gene copies. The elucidation of the real biological role of ScSNQ2 will enlighten this issue. Remarkably, PDR18 is only found in Saccharomyces genus genomes and is present in almost all the recently available 1,000 deep coverage genomes of natural S. cerevisiae isolates, consistent with the relevant encoded physiological function.

Reconstruction of the evolutionary history of the DHA1 family of yeast multidrug resistance transporters of the major facilitator superfamily

Multidrug resistance (MDR) is frequently mediated by the action of drug-efflux pumps, which are able to catalyze the active expulsion of several unrelated cytotoxic compounds out of the cell or their intracellular partitioning. A group of transporters involved in MDR belongs to the major facilitator superfamily (MFS) which, based on protein topology, phylogeny and function, divides into two families: 12-spanner drug:H+ antiporter-1 (DHA1) and 14-spanner drug:H+ antiporter-2 (DHA2). The evolutionary history of 422 DHA1 proteins identified in twenty-four hemiascomycetous genomes was reconstructed. The phylogenetic tree divided the DHA1 genes into 21 clusters, twelve containing no Saccharomyces cerevisiae members. Inclusion of gene neighbourhood data allowed to scrutinize the orthologous, paralogous, and ohnologous status of these genes, and also to identify ten main DHA1 gene lineages, spanning the whole hemiascomycetes clade. The DHA1 proteins of seven pathogenic Candida spp. were phylogenetically characterized.