The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28.

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

Expression of the K74 Killer Toxin from Saccharomyces paradoxus Is Modulated by the Toxin-Encoding M74 Double-Stranded RNA 5' Untranslated Terminal Region.

Yeast killer toxins are widely distributed in nature, conferring a competitive advantage to the producer yeasts over nonkiller ones when nutrients are scarce. Most of these toxins are encoded on double-stranded RNAs (dsRNAs) generically called M. L-A members of the viral family Totiviridae act as helper viruses to maintain M, providing the virion proteins that separately encapsidate and replicate L-A and M genomes. M genomes are organized in three regions, a 5' region coding the preprotoxin, followed by an internal poly(A) stretch and a 3' noncoding region. In this work, we report the characterization of K74 toxin encoded on M74 dsRNA from Saccharomyces paradoxus Q74.4. In M74, there is a 5' upstream sequence of 141 nucleotides (nt), which contains regulatory signals for internal translation of the preprotoxin open reading frame (ORF) at the second AUG codon. The first AUG close to the 5' end is not functional. For K74 analysis, M74 viruses were first introduced into laboratory strains of Saccharomyces cerevisiae. We show here that the mature toxin is an α/ß heterodimer linked by disulfide bonds. Though the toxin (or preprotoxin) confers immunity to the carrier, cells with increased K74 loads have a sick phenotype that may lead to cell death. Thus, a fine-tuning of K74 production by the upstream regulatory sequence is essential for the host cell to benefit from the toxin it produces and, at the same time, to safely avoid damage by an excess of toxin. IMPORTANCE Killer yeasts produce toxins to which they are immune by mechanisms not well understood. This self-immunity, however, is compromised in certain strains, which secrete an excess of toxin, leading to sick cells or suicidal phenotypes. Thus, a fine-tuning of toxin production has to be achieved to reach a balance between the beneficial effect of toxin production and the stress imposed on the host metabolism. K74 toxin from S. paradoxus is very active against Saccharomyces uvarum, among other yeasts, but an excess of toxin production is deleterious for the host. Here, we report that the presence of a 5' 141-nt upstream sequence downregulates K74 toxin precursor translation, decreasing toxin levels 3- to 5-fold. Thus, this is a special case of translation regulation performed by sequences on the M74 genome itself, which have been presumably incorporated into the viral RNA during evolution for that purpose.

A Simple Multiplex Reverse Transcription-PCR Method for the Diagnosis of L-A and M Totiviruses in Saccharomyces cerevisiae.

Killer yeasts and their toxins have many potential applications in environmental, medical, and industrial biotechnology. The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two dsRNA viruses, L-A and M. M encodes the toxin, and L-A provides proteins for expression, replication, and capsids for both viruses. Yeast screening and characterization of this trait are usually performed phenotypically based on their toxin production and immunity. In this study, we describe a simple and specific reverse transcription (RT) multiplex PCR assay for direct diagnosis of the dsRNA totivirus genomes associated with the killer trait in the S. cerevisiae yeast. This method obviates RNA purification steps and primer addition to the RT reaction. Using a mixture of specific primers at the PCR step, this multiplex RT-PCR protocol provided an accurate diagnosis of both L-A and M totivirus in all its known variants, L-A-1/M1, L-A-2/M2, L-A-28/M28, and L-A-lus/Mlus, found in infected killer yeasts. Using this method, the expected L-A-2/M2 totivirus associations in natural wine yeasts cells were identified but, importantly, asymptomatic L-A-2/M2 infected cells were found in addition to unexpected L-A-lus/M2 totiviral associations. IMPORTANCE The killer phenomenon in S. cerevisiae yeast cells provides the opportunity to study host-virus interactions in a eukaryotic model. Therefore, the development of simple methods for their detection significantly facilitates their study. The simplified multiplex RT-PCR protocol described here provides a useful and accurate tool for the genotypic characterization of yeast totiviruses in killer yeast cells. The killer trait depended on two dsRNA totiviruses, L-A and M. Each M dsRNA depends on a specific helper L-A virus. Thus, direct genotyping by the described method also provided valuable insights into L-A/M viral associations and their coadaptational events in nature.

Wickerhamomyces Yeast Killer Toxins' Medical Applications.

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described.

The forgotten role of food cultures.

Fermentation is one of if not the oldest food processing technique, yet it is still an emerging field when it comes to its numerous mechanisms of action and potential applications. The effect of microbial activity on the taste, bioavailability and preservation of the nutrients and the different food matrices has been deciphered by the insights of molecular microbiology. Among those roles of fermentation in the food chain, biopreservation remains the one most debated. Presumably because it has been underestimated for quite a while, and only considered - based on a food safety and technological approach - from the toxicological and chemical perspective. Biopreservation is not considered as a traditional use, where it has been by design - but forgotten - as the initial goal of fermentation. The 'modern' use of biopreservation is also slightly different from the traditional use, due mainly to changes in cooling of food and other ways of preservation, Extending shelf life is considered to be one of the properties of food additives, classifying - from our perspective - biopreservation wrongly and forgetting the role of fermentation and food cultures. The present review will summarize the current approaches of fermentation as a way to preserve and protect the food, considering the different way in which food cultures and this application could help tackle food waste as an additional control measure to ensure the safety of the food.

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

Production of a lyophilized ready-to-use yeast killer toxin with possible applications in the wine and food industries.

Kpkt is a yeast killer toxin, naturally produced by Tetrapisispora phaffii, with possible applications in winemaking due to its antimicrobial activity on wine-related yeasts including Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. Here, Kpkt coding gene was expressed in Komagataella phaffii (formerly Pichia pastoris) and the bioreactor production of the recombinant toxin (rKpkt) was obtained. Moreover, to produce a ready-to-use preparation of rKpkt, the cell-free supernatant of the K. phaffii recombinant killer clone was 80-fold concentrated and lyophilized. The resulting preparation could be easily solubilized in sterile distilled water and maintained its killer activity for up to six months at 4 °C. When applied to grape must, it exerted an extensive killer activity on wild wine-related yeasts while proving compatible with the fermentative activity of actively growing Saccharomyces cerevisiae starter strains. Moreover, it displayed a strong microbicidal effect on a variety of bacterial species including lactic acid bacteria and food-borne pathogens. On the contrary it showed no lethal effect on filamentous fungi and on Ceratitis capitata and Musca domestica, two insect species that may serve as non-mammalian model for biomedical research. Based on these results, bioreactor production and lyophilization represent an interesting option for the exploitation of this killer toxin that, due to its spectrum of action, may find application in the control of microbial contaminations in the wine and food industries.

Anti-infective nitazoxanide disrupts transcription of ribosome biogenesis-related genes in yeast.

BACKGROUND: Nitazoxanide is a broad-spectrum, anti-parasitic, anti-protozoal, anti-viral drug, whose mechanisms of action have remained elusive. OBJECTIVE: In this study, we aimed to provide insight into the mechanisms of action of nitazoxanide and the related eukaryotic host responses by characterizing transcriptome profiles of Saccharomyces cerevisiae exposed to nitazoxanide. METHODS: RNA-Seq was used to investigate the transcriptome profiles of three strains of S. cerevisiae with dsRNA virus-like elements, including a strain that hosts M28 encoding the toxic protein K28. From the strain with M28, an additional sub-strain was prepared by excluding M28 using a nitazoxanide treatment. RESULTS: Our transcriptome analysis revealed the effects of nitazoxanide on ribosome biogenesis. Many genes related to the UTP A, UTP B, Mpp10-Imp3-Imp4, and Box C/D snoRNP complexes were differentially regulated by nitazoxanide exposure in all of the four tested strains/sub-strains. Examples of the differentially regulated genes included UTP14, UTP4, NOP4, UTP21, UTP6, and IMP3. The comparison between the M28-laden and non-M28-laden sub-strains showed that the mitotic cell cycle was more significantly affected by nitazoxanide exposure in the non-M28-laden sub-strain. CONCLUSIONS: Overall, our study reveals that nitazoxanide disrupts regulation of ribosome biogenesis-related genes in yeast.

Fungal Kti12 proteins display unusual linker regions and unique ATPase p-loops.

Kti12 (Kluyveromyces lactis toxin insensitive 12) is an evolutionary highly conserved ATPase, crucial for the tRNA-modification activity of the eukaryotic Elongator complex. The protein consists of an N-terminal ATPase and a C-terminal tRNA-binding domain, which are connected by a flexible linker. The precise role of the linker region and its involvement in the communication between the two domains and their activities remain elusive. Here, we analyzed all available Kti12 protein sequences and report the discovery of a subset of Kti12 proteins with abnormally long linker regions. These Kti12 proteins are characterized by a co-occurring lysine to leucine substitution in their Walker A motif, previously thought to be invariable. We show that the K14L substitution lowers the affinity to ATP, but does not affect the catalytic activity of Kti12 at high ATP concentrations. We compare the activity of mutated variants of Kti12 in vitro with complementation assays in vivo in yeast. Ultimately, we compared Kti12 to other known p-loop ATPase family members known to carry a similar deviant Walker A motif. Our data establish Kti12 of Eurotiomycetes as an example of eukaryotic ATPase harboring a significantly deviating but still functional Walker A motif.

The killer yeast Wickerhamomyces anomalus Cf20 exerts a broad anti-Candida activity through the production of killer toxins and volatile compounds.

Candidiasis is a group of opportunistic infections caused by yeast of the genus Candida. The appearance of drug resistance and the adverse effects of current antifungal therapies require the search for new, more efficient therapeutic alternatives. Killer yeasts have aroused as suitable candidates for mining new antifungal compounds. Killer strains secrete antimicrobial proteins named killer toxins, with promissory antifungal activity. Here we found that the killer yeast Wickerhamomyces anomalus Cf20 and its cell-free supernatant (CFS) inhibited six pathogenic strains and one collection strain of Candida spp. The inhibition is mainly mediated by secreted killer toxins and, to a lesser extent, by volatile compounds such as acetic acid and ethyl acetate. A new large killer toxin (>180 kDa) was purified, which exerted 70-74% of the total CFS anti-Candida activity, and the previously described glucanase KTCf20 was inhibitory in a lesser extent as well. In addition, we demonstrated that Cf20 possesses the genes encoding for the ß-1,3-glucanases WaExg1 and WaExg2, proteins with extensively studied antifungal activity, particularly WaExg2. Finally, the 10-fold concentrated CFS exerted a high candidacidal effect at 37°C, completely inhibiting the fungal growth, although the nonconcentrated CFS (RCF 1) had very limited fungistatic activity at this temperature. In conclusion, W. anomalus Cf20 produces different low and high molecular weight compounds with anti-Candida activity that could be used to design new therapies for candidiasis and as a source for novel antimicrobial compounds as well.

Analysis of Yeast Killer Toxin K1 Precursor Processing via Site-Directed Mutagenesis: Implications for Toxicity and Immunity.

K1 represents a heterodimeric A/B toxin secreted by virus-infected Saccharomyces cerevisiae strains. In a two-staged receptor-mediated process, the ionophoric activity of K1 leads to an uncontrolled influx of protons, culminating in the breakdown of the cellular transmembrane potential of sensitive cells. K1 killer yeast necessitate not only an immunity mechanism saving the toxin-producing cell from its own toxin but, additionally, a molecular system inactivating the toxic α subunit within the secretory pathway. In this study, different derivatives of the K1 precursor were constructed to analyze the biological function of particular structural components and their influence on toxin activity as well as the formation of protective immunity. Our data implicate an inactivation of the α subunit during toxin maturation and provide the basis for an updated model of K1 maturation within the host cell's secretory pathway.IMPORTANCE The killer phenotype in the baker's yeast Saccharomyces cerevisiae relies on two double-stranded RNA viruses that are persistently present in the cytoplasm. As they carry the same receptor populations as sensitive cells, killer yeast cells need-in contrast to various bacterial toxin producers-a specialized immunity mechanism. The ionophoric killer toxin K1 leads to the formation of cation-specific pores in the plasma membrane of sensitive yeast cells. Based on the data generated in this study, we were able to update the current model of toxin processing, validating the temporary inactivation of the toxic α subunit during maturation in the secretory pathway of the killer yeast.

Yeast Viral Killer Toxin K1 Induces Specific Host Cell Adaptions via Intrinsic Selection Pressure.

The killer phenomenon in yeast (Saccharomyces cerevisiae) not only provides the opportunity to study host-virus interactions in a eukaryotic model but also represents a powerful tool to analyze potential coadaptional events and the role of killer yeast in biological diversity. Although undoubtedly having a crucial impact on the abundance and expression of the killer phenotype in killer-yeast harboring communities, the influence of a particular toxin on its producing host cell has not been addressed sufficiently. In this study, we describe a model system of two K1 killer yeast strains with distinct phenotypical differences pointing to substantial selection pressure in response to the toxin secretion level. Transcriptome and lipidome analyses revealed specific and intrinsic host cell adaptions dependent on the amount of K1 toxin produced. High basal expression of genes coding for osmoprotectants and stress-responsive proteins in a killer yeast strain secreting larger amounts of active K1 toxin implies a generally increased stress tolerance. Moreover, the data suggest that immunity of the host cell against its own toxin is essential for the balanced virus-host interplay providing valuable hints to elucidate the molecular mechanisms underlying K1 immunity and implicating an evolutionarily conserved role for toxin immunity in natural yeast populations.IMPORTANCE The killer phenotype in Saccharomyces cerevisiae relies on the cytoplasmic persistence of two RNA viruses. In contrast to bacterial toxin producers, killer yeasts necessitate a specific immunity mechanism against their own toxin because they bear the same receptor populations as sensitive cells. Although the killer phenomenon is highly abundant and has a crucial impact on the structure of yeast communities, the influence of a particular toxin on its host cell has been barely addressed. In our study, we used two derivatives secreting different amount of the killer toxin K1 to analyze potential coadaptional events in this particular host/virus system. Our data underline the dependency of the host cell's ability to cope with extracellular toxin molecules and intracellular K1 molecules provided by the virus. Therefore, this research significantly advances the current understanding of the evolutionarily conserved role of this molecular machinery as an intrinsic selection pressure in yeast populations.

Substitution of cysteines in the yeast viral killer toxin K1 precursor reveals novel insights in heterodimer formation and immunity.

The killer toxin K1 is a virally encoded fungal A/B toxin acting by disrupting plasma membrane integrity. The connection of α and ß constitutes a critical feature for toxin biology and for decades the formation of three disulphide bonds linking the major toxin subunits was accepted as status quo. Due to the absence of experimental evidence, the involvement of each cysteine in heterodimer formation, K1 lethality and immunity was systematically analysed. Substitution of any cysteine in α led to a complete loss of toxin dimer secretion and toxicity, whereas K1 toxin derivatives carrying mutations of C248, C312 or the double mutation C248-312 were active against spheroplasted cells. Importantly, substitution of the C95 and C107 in the toxin precursor completely abolished the mediation of functional immunity. In contrast, K1 toxicity, i.e. its ionophoric effect, does not depend on the cysteine residues at all. In contrast to the literature, our data imply the formation of a single disulphide bond involving C92 in α and C239 in ß. This finding not only refines the current model stated for decades but also provides new opportunities to elucidate the mechanisms underlying K1 toxicity and immunity at the molecular level.

The ecology of killer yeasts: Interference competition in natural habitats.

Killer yeasts are ubiquitous in the environment: They have been found in diverse habitats ranging from ocean sediment to decaying cacti to insect bodies and on all continents including Antarctica. However, environmental killer yeasts are poorly studied compared with laboratory and domesticated killer yeasts. Killer yeasts secrete so-called killer toxins that inhibit nearby sensitive yeasts, and the toxins are frequently assumed to be tools for interference competition in diverse yeast communities. The diversity and ubiquity of killer yeasts imply that interference competition is crucial for shaping yeast communities. Additionally, these toxins may have ecological functions beyond use in interference competition. This review introduces readers to killer yeasts in environmental systems, with a focus on what is and is not known about their ecology and evolution. It also explores how results from experimental killer systems in laboratories can be extended to understand how competitive strategies shape yeast communities in nature. Overall, killer yeasts are likely to occur everywhere yeasts are found, and the killer phenotype has the potential to radically shape yeast diversity in nature.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.

Dynamic modelling of the killing mechanism of action by virus-infected yeasts.

Killer yeasts are microorganisms, which can produce and secrete proteinaceous toxins, a characteristic gained via infection by a virus. These toxins are able to kill sensitive cells of the same or a related species. From a biotechnological perspective, killer yeasts are beneficial due to their antifungal/antimicrobial activity, but also regarded as problematic for large-scale fermentation processes, whereby those yeasts would kill starter cultures species and lead to stuck fermentations. Here, we propose a mechanistic model of the toxin-binding kinetics pertaining to the killer population coupled with the toxin-induced death kinetics of the sensitive population to study toxic action. The dynamic model captured the transient toxic activity starting from the introduction of killer cells into the culture at the time of inoculation through to induced cell death. The kinetics of K1/K2 activity via its primary pathway of toxicity was 5.5 times faster than its activity at low concentration inducing the apoptotic pathway in sensitive cells. Conversely, we showed that the primary pathway for K28 was approximately three times slower than its equivalent apoptotic pathway, indicating the particular relevance of K28 in biotechnological applications where the toxin concentration is rarely above those limits to trigger the primary pathway of killer activity.

A Rapid Method for Sequencing Double-Stranded RNAs Purified from Yeasts and the Identification of a Potent K1 Killer Toxin Isolated from Saccharomyces cerevisiae.

Mycoviruses infect a large number of diverse fungal species, but considering their prevalence, relatively few high-quality genome sequences have been determined. Many mycoviruses have linear double-stranded RNA genomes, which makes it technically challenging to ascertain their nucleotide sequence using conventional sequencing methods. Different specialist methodologies have been developed for the extraction of double-stranded RNAs from fungi and the subsequent synthesis of cDNAs for cloning and sequencing. However, these methods are often labor-intensive, time-consuming, and can require several days to produce cDNAs from double-stranded RNAs. Here, we describe a comprehensive method for the rapid extraction and sequencing of dsRNAs derived from yeasts, using short-read next generation sequencing. This method optimizes the extraction of high-quality double-stranded RNAs from yeasts and 3' polyadenylation for the initiation of cDNA synthesis for next-generation sequencing. We have used this method to determine the sequence of two mycoviruses and a double-stranded RNA satellite present within a single strain of the model yeast Saccharomyces cerevisiae. The quality and depth of coverage was sufficient to detect fixed and polymorphic mutations within viral populations extracted from a clonal yeast population. This method was also able to identify two fixed mutations within the alpha-domain of a variant K1 killer toxin encoded on a satellite double-stranded RNA. Relative to the canonical K1 toxin, these newly reported mutations increased the cytotoxicity of the K1 toxin against a specific species of yeast.

Production of a novel killer toxin from Saccharomyces eubayanus using agro-industrial waste and its application against wine spoilage yeasts.

The juicing industry generates large amounts of waste that mostly lack commercial value and, in the absence of waste treatment policies, produces environmental pollution. Also, microbiological spoilage is a major concern in the wine industry and control tools are limited. Taking these challenges into account, agro-industrial waste coming from ultrafiltrated apple and pear juice were used to grow Saccharomyces eubayanus and to produce its killer toxin (SeKT). A Plackett-Burman screening was performed in order to optimize SeKT production in ultrafiltrated apple and pear juice. The optimized medium was characterized: 75% v/v WUJ, 0.5% m/v KH2PO4, 0.5% m/v MgSO4, 0.5% m/v (NH4)SO4, 0.5% g/L urea, 10% v/v glycerol and 0.1% v/v Triton X-100. SeKT produced in WUJ optimised medium was used to perform killer assays against wine spoilage yeasts and showed antagonistic activity against Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens. Different inhibition percentages against spoilage species in a wine environment (49-69%) were detected and preserved for at least 48 h. For the first time, this work reports the ability of S. eubayanus to produce a killer toxin with potential use as a biocontrol tool in winemaking. Producing SeKT using agro-industrial waste as an alternative medium to cultivate S. eubayanus would have industrial, economic and ecological benefits.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.