Interactions between killer yeasts and pathogenic fungi

Abstract A total of 17 presumptive killer yeast strains were tested in vitro for growth inhibitory and killing activity against a range of fungal pathogens of agronomic, environmental and clinical significance. Several yeasts were identified which displayed significant activity against important pathogenic fungi. For example, isolates of the opportunistic human pathogen, Candida albicans , were generally very sensitive to Williopsis mrakii killer yeast activity, whilst killer strains of Saccharomyces cerevisiae and Pichia anomala markedly inhibited the growth of certain wood decay basidiomycetes and plant pathogenic fungi. Results indicate that such yeasts, together with their killer toxins, may have potential as novel antimycotic biocontrol agents.     

Viral induced yeast apoptosis

In an analogous system to mammals, induction of an apoptotic cell death programme (PCD) in yeast is not only restricted to various exogenous factors and stimuli, but can also be triggered by viral killer toxins and viral pathogens. In yeast, toxin secreting killer strains are frequently infected with double-stranded (ds)RNA viruses that are responsible for killer phenotype expression and toxin secretion in the infected host. In most cases, the viral toxins are either pore-forming proteins (such as K1, K2, and zygocin) that kill non-infected and sensitive yeast cells by disrupting cytoplasmic membrane function, or protein toxins (such as K28) that act in the nucleus by blocking DNA synthesis and subsequently causing a G1/S cell cycle arrest. Interestingly, while all these virus toxins cause necrotic cell death at high concentration, they trigger caspase- and ROS-mediated apoptosis at low-to-moderate concentration, indicating that even low toxin doses are deadly by triggering PCD in enemy cells. Remarkably, viral toxins are not solely responsible for cell death induction in vivo, as killer viruses themselves were shown to trigger apoptosis in non-infected yeast. Thus, as killer virus-infected and toxin secreting yeasts are effectively protected and immune to their own toxin, killer yeasts bear the intrinsic potential to dominate over time in their natural habitat.     

Physiological characteristics of the biocontrol yeast Pichia anomala J121

The yeast Pichia anomala J121 prevents mold spoilage and enhances preservation of moist grain in malfunctioning storage systems. Development of P. anomala J121 as a biocontrol agent requires in-depth knowledge about its physiology. P. anomala J121 grew under strictly anaerobic conditions, at temperatures between 3 degrees C and 37 degrees C, at pH values between 2.0 and 12.4, and at a water activity of 0.92 (NaCl) and 0.85 (glycerol). It could assimilate a wide range of C- and N-sources and produce killer toxin. A selective medium containing starch, nitrate, acetic acid, and chloramphenicol was developed for P. anomala. P. anomala was equally sensitive as Candida albicans to common antifungal compounds. Growth ability at a range of environmental conditions contributes to the competitive ability of the biocontrol yeast P. anomala J121.     

Marine killer yeasts active against a yeast strain pathogenic to crab Portunus trituberculatus

Some marine yeasts have recently been recognised as pathogenic agents in crab mariculture, but may be inhibited or killed by 'killer' yeast strains. We screened multiple yeast strains from seawater, sediments, mud of salterns, guts of marine fish, and marine algae for killer activity against the yeast Metchnikowia bicuspidata WCY (pathogenic to crab Portunus trituberculatus), and found 17 strains which could secrete toxin onto the medium and kill the pathogenic yeast. Of these, 5 strains had significantly higher killing activity than the others; routine identification and molecular methods showed that these were Williopsis saturnus WC91-2, Pichia guilliermondii GZ1, Pichia anomala YF07b, Debaryomyces hansenii hcx-1 and Aureobasidium pullulans HN2.3. We found that the optimal conditions for killer toxin production and action of killer toxin produced by the marine killer yeasts were not all in agreement with those of marine environments and for crab cultivation. We found that the killer toxins produced by the killer yeast strains could kill other yeasts in addition to the pathogenic yeast, and NaCl concentration in the medium could change killing activity spectra. All the crude killer toxins produced could hydrolyze laminarin and the hydrolysis end products were monosaccharides.     

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.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Characterization of the yeast population from traditional corn and rye bread doughs

M.J. ALMEIDA AND C.S. PAIS. 1996. Yeasts were isolated from a variety of home-made bread doughs and identified. A pure culture of Saccharomyces cerevisiae was found in 18% of the doughs. The same species predominated in 80% of the doughs examined whereas Issatchenkia orientalis, Pichia membranaefaciens and Torulaspora delbrueckii were present in about 40% of the samples. About one quarter of the isolates displayed killer activity, strains of P. anomala showing the broadest spectra. Two isolates of S. cerevisiae and three of T. delbrueckii gave biomass values in sucrose medium similar to or higher than those obtained with commercial compressed baker's yeast strains.     

Enzymic activity of the K5-type yeast killer toxin and its characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).     

Biotechnology, physiology and genetics of the yeast Pichia anomala

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.     

嗜杀酵母的生物学功能及其应用

嗜杀酵母能够分泌毒素蛋白,杀死敏感酵母。嗜杀酵母对自身分泌的嗜杀毒素具有免疫力。嗜杀酵母的嗜杀特性与两种双链线状RNA(dsRNA)有关,即编码产生毒素蛋白的M-dsRNA和编码自身和M-dsRNA外壳蛋白的L-dsRNA。嗜杀毒素破坏细胞跨膜化学质子梯度,造成ATP和钾离子泄漏,导致细胞死亡。应用嗜杀酵母可避免野生型酵母污染,净化发酵体系,改善发酵产物品质;嗜杀毒素也可作为抵制病原酵母和类酵母微生物的抗真菌剂。     

The incidence of killer activity of non-Saccharomyces yeasts towards indigenous yeast species of grape must: potential application in wine fermentation

Fourteen killer yeasts were assayed for their ability to kill species of yeast that are commonly associated with fermenting grape must and wine. A total of 147 of a possible 364 killer-sensitive interactions were observed at pH 4.5. Of the killer yeasts studied, Pichia anomala NCYC 434 displayed the broadest killing range. At a pH value comparable with those of wine ferments, pH 3.5, the incidence of killer-sensitive interactions was reduced by 700% across all the yeasts. Williopsis saturnus var. mrakii CBS 1707 exhibited the broadest killing range at the lower pH, killing more than half of the tester strains. Intraspecific variation in sensitivity to killer yeasts was observed in all species where more than one strain was tested. Also, in strains of Pichia anomala, Kluyveromyces lactis and Pichia membranifaciens, the three species in which more than one killer yeast was analysed, intraspecific variation in killer activity was observed.     

Scent of a killer: How could killer yeast boost its dispersal?

Vector‐borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here, we discuss the ecology and evolution of fruit‐colonizing yeast in a tripartite symbiosis—the so‐called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double‐stranded RNA viruses (M satellite dsRNAs, L‐A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non‐killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.     

Yeast killer toxins,molecular mechanisms of their action and their applications

Killer toxins secreted by some yeast strains are the proteins that kill sensitive cells of the same or related yeast genera. In recent years, many new yeast species have been found to be able to produce killer toxins against the pathogenic yeasts, especially Candida albicans. Some of the killer toxins have been purified and characterized, and the genes encoding the killer toxins have been cloned and characterized. Many new targets including different components of cell wall, plasma membrane, tRNA, DNA and others in the sensitive cells for the killer toxin action have been identified so that the new molecular mechanisms of action have been elucidated. However, it is still unknown how some of the newly discovered killer toxins kill the sensitive cells. Studies on the killer phenomenon in yeasts have provided valuable insights into a number of fundamental aspects of eukaryotic cell biology and interactions of different eukaryotic cells. Elucidation of the molecular mechanisms of their action will be helpful to develop the strategies to fight more and more harmful yeasts.     

Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States.

Representative strains (n = 61) of the yeast Pichia kluyveri from across the United States were studied for their ability to kill 71 other strains (representing 25 species) of yeast. This survey showed killing activity in 69% of the P. kluyveri strains tested. More extensive analysis of killer activity of 197 P. kluyveri strains against strains of five tester species showed comparable activity (67% of strains tested). This activity was shown to be equally variable within localities, within regions, and across the continent. The genetic basis of the variability was ascertained by tetrad analysis and is most likely due to alleles segregating at three epistatic loci. Evidence for the idea that killer toxins have a role in excluding other yeasts from particular habitats is discussed.     

Rapid multiple‐level coevolution in experimental populations of yeast killer and nonkiller strains

Coevolution between different biological entities is considered an important evolutionary mechanism at all levels of biological organization. Here, we provide evidence for coevolution of a yeast killer strain (K) carrying cytoplasmic dsRNA viruses coding for anti‐competitor toxins and an isogenic toxin‐sensitive strain (S) during 500 generations of laboratory propagation. Signatures of coevolution developed at two levels. One of them was coadaptation of K and S. Killing ability of K first increased quickly and was followed by the rapid invasion of toxin‐resistant mutants derived from S, after which killing ability declined. High killing ability was shown to be advantageous when sensitive cells were present but costly when they were absent. Toxin resistance evolved via a two‐step process, presumably involving the fitness‐enhancing loss of one chromosome followed by selection of a recessive resistant mutation on the haploid chromosome. The other level of coevolution occurred between cell and killer virus. By swapping the killer viruses between ancestral and evolved strains, we could demonstrate that changes observed in both host and virus were beneficial only when combined, suggesting that they involved reciprocal changes. Together, our results show that the yeast killer system shows a remarkable potential for rapid multiple‐level coevolution.     

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background

Virus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.

Results

In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.

Conclusion

Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

     

Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States.

Representative strains (n = 61) of the yeast Pichia kluyveri from across the United States were studied for their ability to kill 71 other strains (representing 25 species) of yeast. This survey showed killing activity in 69% of the P. kluyveri strains tested. More extensive analysis of killer activity of 197 P. kluyveri strains against strains of five tester species showed comparable activity (67% of strains tested). This activity was shown to be equally variable within localities, within regions, and across the continent. The genetic basis of the variability was ascertained by tetrad analysis and is most likely due to alleles segregating at three epistatic loci. Evidence for the idea that killer toxins have a role in excluding other yeasts from particular habitats is discussed.     

Isolation of a Wickerhamomyces anomalus yeast strain from the sandfly Phlebotomus perniciosus,displaying the killer phenotype

The yeast Wickerhamomyces anomalus has been studied for its wide biotechnological potential, mainly for applications in the food industry. Different strains of W. anomalus have been isolated from diverse habitats and recently from insects, including mosquitoes of medical importance. This paper reports the isolation and phylogenetic characterization of W. anomalus from laboratory‐reared adults and larvae of Phlebotomus perniciosus (Diptera: Psychodidae), a main phlebotomine vector of human and canine leishmaniasis. Of 65 yeast strains isolated from P. perniciosus, 15 strains were identified as W. anomalus; one of these was tested for the killer phenotype and demonstrated inhibitory activity against four yeast sensitive strains, as reported for mosquito‐isolated strains. The association between P. perniciosus and W. anomalus deserves further investigation in order to explore the possibility that this yeast may exert inhibitory/killing activity against Leishmania spp.     

Phenotypic expression of Kluyveromyces lactis killer toxin against Saccharomyces spp

The secretion of killer toxins by some strains of yeasts is a phenomenon of significant industrial importance. The activity of a recently discovered Kluyveromyces lactis killer strain against a sensitive Saccharomyces cerevisiae strain was determined on peptone-yeast extract-nutrient agar plates containing as the carbon source glucose, fructose, galactose, maltose, or glycerol at pH 4.5 or 6.5. Enhanced activity (50 to 90% increase) was found at pH 6.5, particularly on the plates containing galactose, maltose, or glycerol, although production of the toxin in liquid medium was not significantly different with either glucose or galactose as the carbon source. Results indicated that the action of the K. lactis toxin was not mediated by catabolite repression in the sensitive strain. Sensitivities of different haploid and polyploid Saccharomyces yeasts to the two different killer yeasts S. cerevisiae (RNA-plasmid-coded toxin) and K. lactis (DNA-plasmid-coded toxin) were tested. Three industrial polyploid yeasts sensitive to the S. cerevisiae killer yeast were resistant to the K. lactis killer yeast. The S. cerevisiae killer strain itself, however, was sensitive to the K. lactis killer yeast.     

Mold-inhibitory activity of different yeast species during airtight storage of wheat grain

The yeast Pichia anomala J121 inhibits spoilage by Penicillium roqueforti in laboratory and pilot studies with high-moisture wheat in malfunctioning airtight storage. We tested the biocontrol ability of an additional 57 yeast species in a grain mini silo system. Most yeast species grew to CFU levels comparable to that of P. anomala J121 after 14 days of incubation (>10(6) CFU g(-1)). Of the 58 species, 38 (63 strains) had no mold-inhibitory effects (Pen. roqueforti levels >10(5) CFU g(-1)). Among these were 11 species (18 strains) that did not grow on the wheat grain. Several of the non-inhibiting yeast species have previously been reported as biocontrol agents in other postharvest environments. Weak inhibitory activity, reducing Pen. roqueforti levels to between 10(4) and 10(5) CFU g(-1), was observed with 11 species (12 strains). Candida silvicola and Pichia guillermondii reduced Pen. roqueforti to <10(4) CFU g(-1). Candida fennica, Candida pelliculosa, Candida silvicultrix, P. anomala (29 strains), Pichia burtonii, Pichia farinosa and Pichia membranifaciens strongly inhibited Pen. roqueforti (<10(3) CFU g(-1)) in the mini silos, but none had higher biocontrol activity than P. anomala strain J121. This report is the first of biocontrol activity of C. fennica and C. silvicultrix. The ability of 27 yeast species to grow to high CFU values without inhibiting mold growth suggests that nutrient competition may not be the main mode of action of P. anomala J121.