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.

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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.

Saccharomyces cerevisiae was used to uncover the mechanisms underlying tolerance and toxicity of the agricultural fungicide mancozeb, linked to cancer and Parkinson's 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.

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.

Multidrug resistance is often the result of the activation of drug efflux pumps able to catalyze the extrusion of the toxic compound to the outer medium, this activation being frequently controlled at the transcriptional level. Transcriptional regulation in the model eukaryote S. cerevisiae is the result of the interaction and cross-talk between networks of transcription factors. This is the case of the transcriptional activation of the FLR1 gene occurring in response to stress induced by the agricultural fungicide mancozeb in yeast. FLR1 up-regulation depends on the integrated action of Yap1, a key regulator of oxidative stress response, Pdr3 and Yrr1, two of the transcription factors controlling multidrug resistance, and Rpn4, a regulator of proteasome gene expression, which interplay to produce the observed transcriptional up-shift. Based on the expression profiles of FLR1, YAP1, PDR3, YRR1 and RPN4 registered during yeast adaptation to stress induced by mancozeb and using a qualitative modeling approach, a model of the FLR1 regulatory network was built, and the response of S. cerevisiae to mancozeb stress was simulated. The use of a qualitative approach is especially useful to overcome the lack of enough quantitative data on kinetic parameters and molecular concentrations, permitting the immediate focus on the qualitative behavior of the system. This Systems Biology approach allowed the identification of essential features of the early yeast response to fungicide stress. The resulting model allowed the formulation of new hypotheses, in a quick and cost effective manner, on the qualitative behavior of the system following mancozeb challenge, some of which were validated experimentally. In particular, Pdr3 and Yrr1 were shown to directly control FLR1 up-regulation in mancozeb-challenged cells, based on the analysis of the effect of the inactivation of their putative binding sites in the FLR1 promoter. Furthermore, the inter-dependent role of Yap1 and Yrr1 in the regulation of PDR3 and RPN4 was brought to light, this joint activity possibly being extensible to eight other genes involved in multidrug resistance. The FLR1 network structure was revised, based on the comparison between simulated and experimental gene expression data in the double deletion mutant strains Δyrr1Δpdr3 and Δyrr1Δrpn4, and an additional, still unidentified, transcription factor was found to be required to fully explain the behavior of the network.

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3443-2) contains supplementary material, which is available to authorized users.