Scent of a killer: How 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.

Genome Organization of a New Double-Stranded RNA LA Helper Virus From Wine Torulaspora delbrueckii Killer Yeast as Compared With Its Saccharomyces Counterparts

Wine killer yeasts such as killer strains of Torulaspora delbrueckii and Saccharomyces cerevisiae contain helper large-size (4.6 kb) dsRNA viruses (V-LA) required for the stable maintenance and replication of killer medium-size dsRNA viruses (V-M) which bear the genes that encode for the killer toxin. The genome of the new V-LA dsRNA from the T. delbrueckii Kbarr1 killer yeast (TdV-LAbarr1) was characterized by high-throughput sequencing (HTS). The canonical genome of TdV-LAbarr1 shares a high sequence identity and similar genome organization with its Saccharomyces counterparts. It contains all the known conserved motifs predicted to be necessary for virus translation, packaging, and replication. Similarly, the Gag-Pol amino-acid sequence of this virus contains all the features required for cap-snatching and RNA polymerase activity, as well as the expected regional variables previously found in other LA viruses. Sequence comparison showed that two main clusters (99.2–100% and 96.3–98.8% identity) include most LA viruses from Saccharomyces, with TdV-LAbarr1 being the most distant from all these viruses (61.5–62.5% identity). Viral co-evolution and cross transmission between different yeast species are discussed based on this sequence comparison. Additional 5′ and 3′ sequences were found in the TdV-LAbarr1 genome as well as in some newly sequenced V-LA genomes from S. cerevisiae. A stretch involving the 5′ extra sequence of TdV-LAbarr1 is identical to a homologous stretch close to the 5′ end of the canonical sequence of the same virus (self-identity). Our modeling suggests that these stretches can form single-strand stem loops, whose unpaired nucleotides could anneal to create an intramolecular kissing complex. Similar stem loops are also found in the 3′ extra sequence of the same virus as well as in the extra sequences of some LA viruses from S. cerevisiae. A possible origin of these extra sequences as well as their function in obviating ssRNA degradation and allowing RNA transcription and replication are discussed.

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 β-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

ABSTRACT 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.

Joint use of Ca – Modified Double-Strained RNA as an Immunostimulator with a Vaccine for the Prevention of Food andMouth Disease (FMD)

The purpose of this work is to study the effect of combined use of the immunostimulant preparation which is double-stranded Ca-modified RNA with inactivated FMD vaccine for cattle against A, O and Asia-1 subtypes of FMD. Preparation have been received from sodium nucleinate or allocated from a killer yeast of Saccharomices cerewisiae. The modification of Ca ions assayed by the addition of sterile solution of calcium chloride. Based on received data it can be concluded that the using of immunomodulator in cattle induces more early and high immune response. The FMD vaccine intramuscular administration together with the preparation provides high enough of an immune response and high titer of antibody maintained for a period of 90 days (time of observation).

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

ABSTRACT 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.

Killer Activity of Hanseniaspora uvarum Isolated from Dates Vinegar: Partially Purification and Characterization of Killer Toxin

This study was conducted to isolate and identify killer yeast Hanseniaspora uvarum from dates vinegar and measurement the ability of this yeast to produce killer toxin. The antimicrobial activity of the concentrated supernatant containing partially purified concentrated killer toxin was also detected against several pathogenic bacteria and yeast species, which includes two types of yeast Rhodotorula mucilaginosa and Candida tropicalis and four human pathogenic bacteria Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeurginosa. In addition, the antagonistic activity of examined yeast have been studied toward four types of fungi, where two are pathogenic for human Trichophyton mentagrophytes and Trichophyton rubrum and two are plant pathogens Fusarium solani and Sclerotinia sclerotiorum. The results of killer toxin production experiments revealed the ability of yeast to produce killer toxin with molecular weight at 18 kDa by 12 % SDS electrophoresis. The optimal conditions for killer toxin production were studied, and their antimicrobial activity was determined. The results revealed that killer toxin production was increased at 4 % NaCl, the highest inhibtion zone was 20 mm for S. aureus, while the lowest inhibition zone was 7 mm for E. coli. Killer activity was increased at pH 4 and the best inhibtion zone obtained was about 16 mm for K. pneumoniae, while 8 mm for E. coli and C. tropicalis. The temperature was also affect the production of killer toxin, where 25 °C is the best temperature for toxin production of examined yeast, The best killer activity was 21 mm for C. tropicalis. The antagonistic activity of killer yeast H. uvarum toward pathogenic fungal growth was determined and showed killer activity about 61.11, 44.44, 33.33 and 24.44 % against T. mentagrophytes, T. rubrum, F. solani and S. sclerotiorum in comparison to the control.

Killer Activity of Hanseniaspora uvarum Isolated from Dates Vinegar: Partially Purification and Characterization of Killer Toxin

This study was conducted to isolate and identify killer yeast Hanseniaspora uvarum from dates vinegar and measurement the ability of this yeast to produce killer toxin. The antimicrobial activity of the concentrated supernatant containing partially purified concentrated killer toxin was also detected against several pathogenic bacteria and yeast species, which includes two types of yeast Rhodotorula mucilaginosa and Candida tropicalis and four human pathogenic bacteria Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeurginosa. In addition, the antagonistic activity of examined yeast have been studied toward four types of fungi, where two are pathogenic for human Trichophyton mentagrophytes and Trichophyton rubrum and two are plant pathogens Fusarium solani and Sclerotinia sclerotiorum. The results of killer toxin production experiments revealed the ability of yeast to produce killer toxin with molecular weight at 18 kDa by 12 % SDS electrophoresis. The optimal conditions for killer toxin production were studied, and their antimicrobial activity was determined. The results revealed that killer toxin production was increased at 4 % NaCl, the highest inhibtion zone was 20 mm for S. aureus, while the lowest inhibition zone was 7 mm for E. coli. Killer activity was increased at pH 4 and the best inhibtion zone obtained was about 16 mm for K. pneumoniae, while 8 mm for E. coli and C. tropicalis. The temperature was also affect the production of killer toxin, where 25 °C is the best temperature for toxin production of examined yeast, The best killer activity was 21 mm for C. tropicalis. The antagonistic activity of killer yeast H. uvarum toward pathogenic fungal growth was determined and showed killer activity about 61.11, 44.44, 33.33 and 24.44 % against T. mentagrophytes, T. rubrum, F. solani and S. sclerotiorum in comparison to the control.