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Yeasts are very versatile, model unicellular eukaryotes that have been extensively used for over a century to explore fundamental aspects of living systems. This collection brings together the latest studies showcasing research on biotechnological applications of yeasts, yeasts as disease models, and pathogenic yeasts.


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    • Identification of a novel gene required for competitive growth at high temperature in the thermotolerant yeast

      It is important to understand the basis of thermotolerance in yeasts to broaden their application in industrial biotechnology. The capacity to run bioprocesses at temperatures above 40 °C is of great interest but this is beyond the growth range of most of the commonly used yeast species. In contrast, some industrial yeasts such as can grow at temperatures of 45 °C or higher. Such species are valuable for direct use in industrial biotechnology and as a vehicle to study the genetic and physiological basis of yeast thermotolerance. In previous work, we reported that evolutionarily young genes disproportionately changed expression when yeast were growing under stressful conditions and postulated that such genes could be important for long-term adaptation to stress. Here, we tested this hypothesis in by identifying and studying species-specific genes that showed increased expression during high-temperature growth. Twelve such genes were identified and 11 were successfully inactivated using CRISPR-mediated mutagenesis. One gene, , is required for competitive growth at high temperature, supporting the hypothesis that evolutionary young genes could play roles in adaptation to harsh environments. is predicted to encode an 83 aa peptide, and RNA sequencing and ribo-sequencing were used to confirm transcription and translation of the gene. The precise function of KLMX_70384 remains unknown but some features are suggestive of RNA-binding activity. The gene is located in what was previously considered an intergenic region of the genome, which lacks homologues in other yeasts or in databases. Overall, the data support the hypothesis that genes that arose in after the speciation event that separated and contribute to some of its unique traits.

    • The regulation of iron homeostasis in the fungal human pathogen

      Iron is an essential element to most microorganisms, yet an excess of iron is toxic. Hence, living cells have to maintain a tight balance between iron uptake and iron consumption and storage. The control of intracellular iron concentrations is particularly challenging for pathogens because mammalian organisms have evolved sophisticated high-affinity systems to sequester iron from microbes and because iron availability fluctuates among the different host niches. In this review, we present the current understanding of iron homeostasis and its regulation in the fungal pathogen . This yeast is an emerging pathogen which has become the second leading cause of candidemia, a life-threatening invasive mycosis. is relatively poorly studied compared to the closely related model yeast or to the pathogenic yeast . Still, several research groups have started to identify the actors of iron homeostasis and its transcriptional and post-transcriptional regulation. These studies have revealed interesting particularities of and have shed new light on the evolution of fungal iron homeostasis.

    • The enigmatic role of fungal annexins: the case of

      Annexins are multifunctional proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins play a myriad of critical and well-characterized roles in mammals, ranging from membrane repair to vesicular secretion. The role of annexins in the kingdoms of bacteria, protozoa and fungi have been largely overlooked. The fact that there is no known homologue of annexins in the yeast model organism may contribute to this gap in knowledge. However, annexins are found in most medically important fungal pathogens, with the notable exception of . In this study we evaluated the function of the one annexin gene in , a causative agent of cryptococcosis. This gene CNAG_02415, is annotated in the genome as a target of calcineurin through its transcription factor Crz1, and we propose to update its name to cryptococcal annexin, AnnexinC1. strains deleted for AnnexinC1 revealed no difference in survival after exposure to various chemical stressors relative to wild-type strain, as well as no major alteration in virulence or mating. The only alteration observed in strains deleted for AnnexinC1 was a small increase in the titan cells' formation . The preservation of annexins in many different fungal species suggests an important function, and therefore the lack of a strong phenotype for annexin-deficient indicates either the presence of redundant genes that can compensate for the absence of AnnexinC1 function or novel functions not revealed by standard assays of cell function and pathogenicity.

    • Molecular epidemiology of otomycosis in Isfahan revealed a large diversity in causative agents

      Purpose. To elucidate the clinical and microbial epidemiology of otomycosis in Isfahan, Iran.

      Methodology. From January 2016 to January 2017 all patients clinically suspected of otomycosis at Al-Zahra Hospital, Isfahan, Iran were recruited. Specimens were taken using sterile swabs by an otorhinolaryngologist and subjected to culture and microscopy using potassium hydroxide and Giemsa stain. Isolated fungi were identified based on morphological and molecular characteristics.

      Results. Otomycosis was confirmed in 97/120 patients (80.8 %). Females (72.2 %) and patients aged 30–39 years (33 %) were more commonly affected than others. Manipulation of ear canal (62.9 %) was the most common predisposing factor. Pruritus was observed in 84.54  % of the patients followed by hearing impairment (81.4 %), and most episodes were detected over the summer (50.5 %). Culture was positive for 81 (83.5  %) of confirmed cases and molds were the most prevalent causative agents (n=51, 63 %) followed by yeasts (n=19, 23.4 %) and yeast/mold mixes (n=11, 13.6 %). For the 16 remaining patients, no growth was seen in culture despite a positive result on direct examination. In total, 92 isolates (63 molds and 29 yeasts) were recovered in culture. Application of molecular methods showed 18 fungal species and the vast majority of them belonged to Aspergillus (n=53, 57.6 %) and Candida genus. Among the species involved, Candida parapsilosis (n=22, 22.7 %) and Aspergillus tubingensis (n=15, 15.5 %) were the most encountered species.

      Conclusion. Outcomes from this study showed a different picture of prevalence, where C. parapsilosis and A. tubingensis but not Aspergillus niger were the most species encountered from patients suffering from otomycosis.

    • The gene and related spliceosome factor genes in respond to nutrients and antifungals

      is a dermatophyte that causes most human superficial mycoses worldwide. The spliceosome, a large ribonucleoprotein complex responsible for pre-mRNA processing, may confer adaptive advantages to deal with different stresses. Here, we assessed the structural aspects of the Prp4 kinase protein and other pre-mRNA-splicing factors (Prps) in grown in different protein sources and exposed to antifungal drugs.

      Quantitative Reverse Transcription PCR (RT-PCR) assessed the modulation of , , and genes after exposure of to sub-lethal doses of amphotericin B, caspofungin and acriflavine, or after growth on keratin sources for 48 and 72 h. We also performed the analysis of the domain organization of Prps orthologues from filamentous fungi and yeasts.

      The gene was modulated in a time-dependent manner. Transcription levels were mostly up-regulated when was grown on keratin for 72 h, while exposure to amphotericin B promoted prp4 gene down-regulation at the same time point. We also observed co-expression of and , and their down-regulation after amphotericin B exposure. analysis revealed a conserved domain organization for most Prps orthologues with slight differences, which were mostly related to structural elements such as repetition domains in Prp1 and complexity in motif assembly for the Prp4 kinase. These differences were mainly observed in dermatophyte species and may alter protein interactions and substrate affinity.

      Our results improve the understanding of spliceosome proteins in fungi as well as their roles in adaptation to different environmental situations.

    • The impact of the absence of Toll-like receptor-2 during Sporothrix brasiliensis infection

      Purpose. Sporothrix brasiliensis, a member of the Sporothrix schenckii complex, is a major cause of epidemic outbreaks of sporotrichosis due to its greater virulence and ability to evade the immune system. The absence of studies about this species led to this study, with the aim to evaluate the importance of Toll-like receptor-2 (TLR-2) during S. brasiliensis infection.

      Methodology. In vitro assays were performed using bone marrow-derived macrophages from both wild-type (C57BL/6) and TLR-2 knockout () mice. In vivo assays were also performed, on which the mice (C57BL/6 and TLR-2) were intraperitoneally infected with S. brasiliensis yeast American Type Culture Collection MYA-4831 and euthanized on days 7, 14 and 28 post infection. The following parameters were then evaluated: fungal burden in spleen, liver, kidney and brain; the production of cytokines TNF-α, IFN-γ, IL-4, IL-2, IL-6 and IL-10.

      Results. The in vitro results showed that the absence of TLR-2 resulted in impaired phagocytosis, microbicide mechanisms utilizing the production of nitric oxide, and the cytokine production (TNF-α, IL-6 and IL-10). The in vivo results demonstrated that the absence of TLR-2 during experimental S. brasiliensis infection promoted increased dissemination after 14 and 28 days and suggests a polarized Th17 response in an attempt to control the infection.

      Conclusions. TLR-2 signalling appears to be important in the innate immune response against S. brasiliensis.

    • Quantifying the parametric sensitivity of ethanol production by Scheffersomyces (Pichia) stipitis: development and verification of a method based on the principles of growth on mixtures of complementary substrates

      Under aerobic conditions, Crabtree-negative yeasts grow but do not ferment, and under anaerobic conditions, they ferment but do not grow. It is therefore believed that fermentation by these yeasts is sensitive to small variations of the operating parameters, e.g. dilution rate , mass transfer coefficient and oxygen solubility . However, this parametric sensitivity has never been quantified. Here, we present a method to quantify the parametric sensitivity of ethanol production in the Crabtree-negative yeast Scheffersomycesstipitis. The method is based on our experimental observation that S. stipitis cultures follow the principles of growth on mixtures of complementary substrates. Specifically, if a chemostat operating at fixed , and is fed with progressively increasing glucose feed concentrations , the culture passes through three regimes. (1) At low , the culture is carbon-limited and no ethanol is produced. (2) At high , the culture is oxygen-limited and ethanol is produced, but unused glucose is lost with the effluent. (3) At intermediate , both glucose and oxygen are limiting, and ethanol is produced without loss of glucose. Ethanol must therefore be produced in this dual-limited regime. The dual-limited regime can be predicted by simple unstructured models. It is characterized by the relation , where and denote the g of glucose consumed per g of oxygen during carbon- and oxygen-limited growth. Hence, the parametric sensitivity of fermentation by Crabtree-negative yeasts can be improved by targeting the yields and .

    • Salmonella infection – prevention and treatment by antibiotics and probiotic yeasts: a review

      Global Salmonella infection, especially in developing countries, is a health and economic burden. The use of antibiotic drugs in treating the infection is proving less effective due to the alarming rise of antibiotic-resistant strains of Salmonella, the effects of antibiotics on normal gut microflora and antibiotic-associated diarrhoea, all of which bring a growing need for alternative treatments, including the use of probiotic micro-organisms. However, there are issues with probiotics, including their potential to be opportunistic pathogens and antibiotic-resistant carriers, and their antibiotic susceptibility if used as complementary therapy. Clinical trials, animal trials and in vitro investigations into the prophylactic and therapeutic efficacies of probiotics have demonstrated antagonistic properties against Salmonella and other enteropathogenic bacteria. Nonetheless, there is a need for further studies into the potential mechanisms, efficacy and mode of delivery of yeast probiotics in Salmonella infections. This review discusses Salmonella infections and treatment using antibiotics and probiotics.

    • Metal induction of two metallothionein genes in the ectomycorrhizal fungus and their role in metal tolerance

      Metallothioneins (MTs) are small proteins with highly conserved cysteine residues and are involved in metal homeostasis and metal detoxification. Two metallothionein genes and from the ectomycorrhizal fungus were characterised for their potential role in heavy metal detoxification. The response of these MTs to the exogenous concentrations of copper and cadmium was studied by qPCR analysis. The exogenous copper but not the cadmium at the tested concentrations induced the expression of the MT genes. The functional role of was validated by expressing the two genes through functional complementation in yeast mutant strain (copper-sensitive), (cadmium- sensitive) and (zinc-sensitive). The mutant strain successfully expressed the two genes resulting in wild-type phenotype restoration of copper, cadmium and zinc tolerance. The present study shows that the ectomycorrhizal fungus encodes two metallothionein genes ( and ) which are more inducible by copper than cadmium and could play an important role in their detoxification.

    • The impact of ERAD on recombinant protein secretion in (syn spp.)

      The yeast (syn. spp.) is a popular cell factory for recombinant protein production. Yeasts in general provide a good starting point for cell factory engineering. They are intrinsically robust and easy to manipulate and cultivate. However, their secretory pathway is not evolutionarily adapted to high loads of secretory protein. In particular, more complex proteins, like the antibody fragment (Fab) used in this study, overwhelm the folding and secretion capacity. This triggers cellular stress responses, which may cause excessive intracellular degradation. Previous results have shown that, in fact, about 60 % of the newly synthesized Fab is intracellularly degraded. Endoplasmic reticulum-associated protein degradation (ERAD) is one possible intracellular degradation pathway for proteins aimed for secretion. We therefore targeted ERAD for cell factory engineering and investigated the impact on recombinant protein secretion in . Three components of the ERAD-L complex, which is involved in the degradation of luminal proteins, and a protein involved in proteasomal degradation, were successfully disrupted in Fab-secreting . Contrary to expectation, the effect on secretion was marginal. In the course of more detailed investigation of the impact of ERAD, we took a closer look at the intracellular variants of the recombinant protein. This enabled us to further zero in on the issue of intracellular Fab degradation and exclude an overshooting ER quality control. We propose that a major fraction of the Fab is actually degraded before entering the secretory pathway.

    • The fission yeast Mtf2 is required for mitochondrial gene expression

      Mitochondrial gene expression is essential for adenosine triphosphate synthesis via oxidative phosphorylation, which is the universal energy currency of cells. Here, we report the identification and characterization of a homologue of Mtf2 (also called Nam1) in . The Δ mutant with the intron-containing mitochondrial DNA (mtDNA) exhibited impaired growth on a rich medium containing the non-fermentable carbon source glycerol, suggesting that is involved in mitochondrial function. deletion in a mitochondrial intron-containing background resulted in a barely detectable level of the mRNA and a reduction in the level of the mRNA, and severely impaired translation. In contrast, deletion in a mitochondrial intron-less background did not affect the levels of and mRNAs. However, Cox1 synthesis could not be restored to the control level in the Δ mutant with intron-less mtDNA. Our results suggest that unlike its counterpart in which plays a general role in synthesis of mtDNA-encoded proteins, Mtf2 primarily functions in translation and the effect of deletion on splicing of introns in mtDNA is likely due to a deficiency in the synthesis of intron-encoded maturases.

    • CAR gene cluster and transcript levels of carotenogenic genes in

      A molecular approach was applied to the study of the carotenoid biosynthetic pathway of . At first, functional annotation of the genome of C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

    • Fusion proteins towards fungi and bacteria in plant protection

      In agriculture, although fungi are considered the foremost problem, infections by bacteria also cause significant economical losses. The presence of different diseases in crops often leads to a misuse of the proper therapeutic, or the combination of different diseases forces the use of more than one pesticide. This work concerns the development of a ‘super-Blad’: a chimeric protein consisting of Blad polypeptide, the active ingredient of a biological fungicide already on the market, and two selected peptides, SP10-5 and Sub5, proven to possess biological potential as antibacterial agents. The resulting chimeric protein obtained from the fusion of Blad with SP10-5 not only maintained strong antibacterial activity, especially against spp. and , but was also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. However, the antibacterial activity of Sub5 was considerably diminished when fused with Blad, which seems to indicate that not all fusion proteins behave equally. These newly designed drugs can be considered promising compounds for use in plant protection. A deeper and focused development of an appropriate formulation may result in a potent biopesticide that can replace, two conventional chemistries with less impact on the environment.

    • Insights into the candidacidal mechanism of Ctn[15–34] – a carboxyl-terminal, crotalicidin-derived peptide related to cathelicidins

      Ctn[15–34], a carboxyl-terminal fragment of crotalicidin (a cathelicidin from the venom gland of a South American rattlesnake), has shown antifungal activity against clinical and standard strains of species. The aim of the present work was to investigate the underlying mechanisms of the candidicidal activity of Ctn[15–34].

      The time-kill profile and drug synergism were evaluated by means of a microdilution assay and multi-parametric flow cytometry. The presumptive interaction of Ctn[15–34] with lipid membranes was estimated with a lipid-mimic compound, the chromogenic substance 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA).

      The absorbance increment (at 425 nm) indicated a concentration- and time-dependent in-solution association between Ctn[15–34] and 4N3OBA. The interaction of Ctn[15-34] with cells was confirmed by flow cytometric measurements with the 5(6)-carboxyfluorescein-labelled peptide (CF-Ctn[15–34]). Analysis of the killing time of exposed to Ctn[15–34] and amphotericin B (AMB) showed that both the peptide and polyene drug reduce the number of c.f.u. but in mechanistically different ways. The Ctn[15–34] peptide alone caused yeast cell membrane disruption, which was confirmed by lactate dehydrogenase leakage and biomarkers of cell death mediated by necrosis.

      Overall, Ctn[15–34] displays a synergistic antifungal activity with AMB, an effect that can be further developed into a multi-target therapeutic option with other antimycotics currently in use.

    • Molecular cloning and overexpression of , an acyl-CoA-dependent diacylglycerol acyltransferase, in the oleaginous yeast DMKU-RK253

      Triacylglycerol (TAG) is a major component of lipid storage in yeast. The acyl CoA: diacylgycerol acyltransferase (DGAT) that catalyzes the final and rate-limiting step in the production of TAG is rather interesting. Consequently, cloning and analysis of the gene-encoding TAG synthase, diacylglycerol acyltransferase gene (), of the oleaginous yeast DMKU-RK253 were undertaken. Analysis of the deduced amino acid sequence of DGA1 from DMKU-RK253 () showed similarity with the acyl-CoA:diacylglycerol acyltransferase 2 (DGAT2) from other organisms. The cDNA of was cloned into the yeast expression vector pYES2 and heterologously overexpressed in . One of the transformants showed a 1.6-fold increase in lipid content compared with the wild-type strain harbouring the pYES2 empty vector. Furthermore, overexpression in DMKU-RK253 resulted in a 2.5-fold increase in lipid content when compared with the wild-type strain, and no significant differences in fatty acid composition were observed between -overexpressed and wild-type strains. Taken together, our results supported our hypothesis that the is a genetic factor that can be used for the development of a strain with improved lipid accumulation capabilities.

    • Outbreak of fungaemia in a neonatal intensive care unit

      Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, (a teleomorph of ). To the best of our knowledge, this is the first report of neonatal sepsis due to .

      Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed.

      Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices.

      This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.

    • Clotrimazole is highly effective against feline isolates

      , the most virulent species in the complex, is responsible for the ongoing epidemics of human and animal sporotrichosis in Brazil. Feline outbreaks are usually driven by and followed by extensive transmission to humans. Itraconazole is the first-line treatment for both feline and human sporotrichosis; however, reduced sensitivity is an emerging issue. Thus, we investigated the effect of the widely used antifungal clotrimazole – alone or in combination with itraconazole – against the pathogenic (yeast) form of feline and human isolates, .

      Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values were determined for treatment with clotrimazole and itraconazole, as monotherapy or in combination. In addition, the effect of the drugs on neutral lipid levels and the yeast ultrastructure were evaluated by flow cytometry and transmission electron microscopy (TEM), respectively.

      The MIC and MFC values show that clotrimazole was more effective than itraconazole against feline isolates, while human isolates were more sensitive to itraconazole. Similarly to itraconazole, treatment with clotrimazole induced statistically significant neutral lipid accumulation in yeasts, and treated yeasts displayed irregularities in the cell membrane and a thicker cell wall when observed by TEM. Clotrimazole increased the antifungal activity of itraconazole in combination assays, with a synergistic effect for two feline isolates.

      The strong activity of clotrimazole against feline isolates suggests that this drug is potentially a new alternative for the treatment of feline sporotrichosis, alone or in combination with itraconazole.

    • The amino-terminal domain of ELL transcription elongation factor is essential for ELL function in

      Transcriptional elongation is a critical step for regulating expression of protein-coding genes. Multiple transcription elongation factors have been identified , but the physiological roles of many of them are still not clearly understood. The ELL (Eleven nineteen Lysine rich Leukemia) family of transcription elongation factors are conserved from fission yeast to humans. contains a single ELL homolog (SpELL) that is not essential for its survival. Therefore to gain insights into the cellular functions of SpELL, we identified phenotypes associated with deletion of in . Our results demonstrate that SpELL is required for normal growth of cells. Furthermore, cells lacking exhibit a decrease in survival when exposed to DNA-damaging conditions, but their growth is not affected under environmental stress conditions. ELL orthologs in different organisms contain three conserved domains, an amino-terminal domain, a middle domain and a carboxyl-terminal domain. We also carried out an functional mapping of these conserved domains within ELL and uncovered a critical role for its amino-terminus in regulating all its cellular functions, including growth under different conditions, transcriptional elongation potential and interaction with EAF. Taken together our results suggest that the domain organization of ELL proteins is conserved across species, but the functions as well as the relationship between the various domains and roles of ELL show species-specific differences.

    • Unravelling the transcriptional regulation of genes: the dual role of transcription factor Leu3

      Yeast cells can use γ-aminobutyric acid (GABA), a non-protein amino acid, as a nitrogen source that is mainly imported by the permease Uga4 and catabolized by the enzymes GABA transaminase and succinate-semialdehyde dehydrogenase, encoded by the and genes, respectively. The three genes are inducible by GABA and subject to nitrogen catabolite repression. Hence, their regulation occurs through two mechanisms, one dependent on the inducer and the other on nitrogen source quality. The aim of this work was to better understand the molecular mechanisms of transcription factors acting on different regulatory elements present in promoters, such as Uga3, Dal81, Leu3 and the GATA factors, and to establish the mechanism of the concerted action between them. We found that Gat1 plays an important role in the induction of transcription by GABA and that Gzf3 has an effect in cells grown in a poor nitrogen source such as proline and that this effect is positive on expression. We also found that Gln3 and Dal80 affect the interaction of Uga3 and Dal81 on promoters. Moreover, our results indicated that the repressing activity of Leu3 on and occurs through Dal80 since we demonstrated that Leu3 facilitates Dal80 interaction with DNA. However, when the expression of GATA factors is null or negligible, Leu3 functions as an activator.

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