Mitochondrial Ferredoxin Determines Vulnerability of Cells to Copper Excess.

The essential micronutrient copper is tightly regulated in organisms, as environmental exposure or homeostasis defects can cause toxicity and neurodegenerative disease. The principal target(s) of copper toxicity have not been pinpointed, but one key effect is impaired supply of iron-sulfur (FeS) clusters to the essential protein Rli1 (ABCE1). Here, to find upstream FeS biosynthesis/delivery protein(s) responsible for this, we compared copper sensitivity of yeast-overexpressing candidate targets. Overexpression of the mitochondrial ferredoxin Yah1 produced copper hyper-resistance. 55Fe turnover assays revealed that FeS integrity of Yah1 was particularly vulnerable to copper among the test proteins. Furthermore, destabilization of the FeS domain of Yah1 produced copper hypersensitivity, and YAH1 overexpression rescued Rli1 dysfunction. This copper-resistance function was conserved in the human ferredoxin, Fdx2. The data indicate that the essential mitochondrial ferredoxin is an important copper target, determining a tipping point where plentiful copper supply becomes excessive. This knowledge could help in tackling copper-related diseases.

Novel, Synergistic Antifungal Combinations that Target Translation Fidelity.

There is an unmet need for new antifungal or fungicide treatments, as resistance to existing treatments grows. Combination treatments help to combat resistance. Here we develop a novel, effective target for combination antifungal therapy. Different aminoglycoside antibiotics combined with different sulphate-transport inhibitors produced strong, synergistic growth-inhibition of several fungi. Combinations decreased the respective MICs by ≥8-fold. Synergy was suppressed in yeast mutants resistant to effects of sulphate-mimetics (like chromate or molybdate) on sulphate transport. By different mechanisms, aminoglycosides and inhibition of sulphate transport cause errors in mRNA translation. The mistranslation rate was stimulated up to 10-fold when the agents were used in combination, consistent with this being the mode of synergistic action. A range of undesirable fungi were susceptible to synergistic inhibition by the combinations, including the human pathogens Candida albicans, C. glabrata and Cryptococcus neoformans, the food spoilage organism Zygosaccharomyces bailii and the phytopathogens Rhizoctonia solani and Zymoseptoria tritici. There was some specificity as certain fungi were unaffected. There was no synergy against bacterial or mammalian cells. The results indicate that translation fidelity is a promising new target for combinatorial treatment of undesirable fungi, the combinations requiring substantially decreased doses of active components compared to each agent alone.

Phenotypic heterogeneity is a selected trait in natural yeast populations subject to environmental stress.

Populations of genetically uniform microorganisms exhibit phenotypic heterogeneity, where individual cells have varying phenotypes. Such phenotypes include fitness-determining traits. Phenotypic heterogeneity has been linked to increased population-level fitness in laboratory studies, but its adaptive significance for wild microorganisms in the natural environment is unknown. Here, we addressed this by testing heterogeneity in yeast isolates from diverse environmental sites, each polluted with a different principal contaminant, as well as from corresponding control locations. We found that cell-to-cell heterogeneity (in resistance to the appropriate principal pollutant) was prevalent in the wild yeast isolates. Moreover, isolates with the highest heterogeneity were consistently observed in the polluted environments, indicating that heterogeneity is positively related to survival in adverse conditions in the wild. This relationship with survival was stronger than for the property of mean resistance (IC(50)) of an isolate. Therefore, heterogeneity could be the major determinant of microbial survival in adverse conditions. Indeed, growth assays indicated that isolates with high heterogeneities had a significant competitive advantage during stress. Analysis of yeasts after cultivation for ≥ 500 generations additionally showed that high heterogeneity evolved as a heritable trait during stress. The results showed that environmental stress selects for wild microorganisms with high levels of phenotypic heterogeneity.

The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species.

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary "Achilles' heel" of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster-defective Rli1p construct. The protein's FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily (55)FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress-related disease.

Heterogeneous expression of the virulence-related adhesin Epa1 between individual cells and strains of the pathogen Candida glabrata.

We investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen, Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by the EPA gene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ∼95% of adherence to epithelial cells in vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression in C. glabrata BG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference in EPA1 sequence or (sub)telomere length and was overcome by ectopic SIR3 expression. Moreover, differences between strains in the silencing dependence of EPA1 expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains where EPA1 was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties of C. glabrata cell populations, with potential implications for microbial pathogenesis more broadly.

Candida argentea sp. nov., a copper and silver resistant yeast species.

A new yeast species was isolated from the sediment under metal-contaminated effluent from a disused metal mine in mid-Wales, UK. BLAST searching with DNA sequence amplified from the ribosomal 26S D1/D2 and ITS regions did not reveal a close match with any previously described species (≥6 % and 3 % divergence, respectively). Phylogenetic analysis indicated that the species was a member of the Saccharomycetales, but did not group closely with other established species, the nearest relative being Wickerhamia fluorescens although bootstrap support was not strong. In addition to its unusual phylogeny, the species also exhibited notable physiological and morphological traits. Isolates exhibited unusually high resistance to both copper and silver in laboratory assays. These phenotypes appeared to be inherent to the species rather than a transient adaptation to the metal-enriched site in Wales, as the same phenotypes were observed in an identical (according to 26S rDNA sequence) isolate from Sao Domingos, Portugal in the Iberian Pyrite Belt. The species exhibited a multipolar budding-type cell division but, unusually, accumulated as rod-shaped cells following division on solid medium, contrasting with the larger ellipsoidal cells observed in broth. This dimorphism could be discerned readily with flow cytometry. The yeast was tolerant of hyper osmotic stress and grew in acidic media (pH 3). This new species is designated Candida argentea and five independent strains are deposited at the National Collection of Yeast Cultures, UK (NCYC 3753(T), 3754, 3755, 3756, 3757). Because of its unusual morphological variation and metal resistance properties, C. argentea may provide opportunities to gain new insights into the physiological and genetic bases of these phenotypes. Results illustrate novel fungal biodiversity that can occur at polluted sites.

Chromate toxicity and the role of sulfur.

The molecular mode(s)-of-action of the toxic metal chromium has yet to be fully resolved. This Mini review focuses on interactions between chromate and sulfur in biological systems. Cr binds sulfur ligands, with cysteine and glutathione having the capacity to aggravate or ameliorate Cr toxicity. Competition between chromate and sulfate for uptake and in metabolism provokes sulfur starvation, which can be growth limiting. Recent data indicate that sulfur deficiency determines protein damage-related Cr toxicity, due to mRNA mistranslation caused by Cr-induced S limitation. Sulfur deprivation could contribute to additional aspects of Cr toxicity, including oxidative DNA damage and Cr related disease.

Chromate-induced sulfur starvation and mRNA mistranslation in yeast are linked in a common mechanism of Cr toxicity.

Toxicity of the environmental carcinogen chromate is known to involve sulfur starvation and also error-prone mRNA translation. Here we reconcile those facts using the yeast model. We demonstrate that: (i) cysteine and methionine starvation mimic Cr-induced translation errors, (ii) genetic suppression of S starvation suppresses Cr-induced mistranslation, and (iii) mistranslation requires cysteine and methionine biosynthesis. Therefore, Cr-induced S starvation is the cause of mRNA mistranslation. This establishes a single, novel pathway mediating the toxicity of chromate.

Actin-mediated endocytosis limits intracellular Cr accumulation and Cr toxicity during chromate stress.

Chromate toxicity is well documented, but the underlying toxic mechanism(s) has yet to be fully elucidated. Following a Cr toxicity screen against > 6000 heterozygous yeast mutants, here we show that Cr resistance requires normal function of the cortical actin cytoskeleton. Furthermore, Cr-stressed yeast cells exhibited an increased number of actin patches, the sites of endocytosis. This was coincident with a marked stimulation of endocytosis following Cr exposure. Genetic dissection of actin nucleation from endocytosis revealed that endocytosis, specifically, was required for Cr resistance. A series of further endocytosis mutants (sac6Delta, chc1Delta, end3Delta) exhibited elevated Cr sensitivity. These mutants also showed markedly elevated cellular Cr accumulation, explaining their sensitivities. In wild-type cells, an initial endocytosis-independent phase of Cr uptake was followed by an endocytosis-dependent decline in Cr accumulation. The results indicate that actin-mediated endocytosis is required to limit Cr accumulation and toxicity. It is proposed that this involves ubiquitin-dependent endocytic inactivation of a plasma membrane Cr transporter(s). We showed that such an action was not dependent on the transporters that have been characterized to date, the sulfate (and chromate) permeases Sul1p and Sul2p.