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.

Targeted delivery of functionalized PLGA nanoparticles to macrophages by complexation with the yeast Saccharomyces cerevisiae.

Nanoparticles (NPs) are able to deliver a variety of substances into eukaryotic cells. However, their usage is often hampered by a lack of specificity, leading to the undesired uptake of NPs by virtually all cell types. In contrast to this, yeast is known to be specifically taken up into immune cells after entering the body. Therefore, we investigated the interaction of biodegradable surface-modified poly(lactic-co-glycolic acid) (PLGA) particles with yeast cells to overcome the unspecificity of the particulate carriers. Cells of different Saccharomyces cerevisiae strains were characterized regarding their interaction with PLGA-NPs under isotonic and hypotonic conditions. The particles were shown to efficiently interact with yeast cells leading to stable NP/yeast-complexes allowing to associate or even internalize compounds. Notably, applying those complexes to a coculture model of HeLa cells and macrophages, the macrophages were specifically targeted. This novel nano-in-micro carrier system suggests itself as a promising tool for the delivery of biologically active agents into phagocytic cells combining specificity and efficiency.

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.

Transcriptome Kinetics of Saccharomyces cerevisiae in Response to Viral Killer Toxin K1.

The K1 A/B toxin secreted by virus-infected Saccharomyces cerevisiae strains kills sensitive cells via disturbance of cytoplasmic membrane functions. Despite decades of research, the mechanisms underlying K1 toxicity and immunity have not been elucidated yet. In a novel approach, this study aimed to characterize transcriptome changes in K1-treated sensitive yeast cells in a time-dependent manner. Global transcriptional profiling revealed substantial cellular adaptations in target cells resulting in 1,189 differentially expressed genes in total. Killer toxin K1 induced oxidative, cell wall and hyperosmotic stress responses as well as rapid down-regulation of transcription and translation. Essential pathways regulating energy metabolism were also significantly affected by the toxin. Remarkably, a futile cycle of the osmolytes trehalose and glycogen was identified probably representing a critical feature of K1 intoxication. In silico analysis suggested several transcription factors involved in toxin-triggered signal transduction. The identified transcriptome changes provide valuable hints to illuminate the still unknown molecular events leading to K1 toxicity and immunity implicating an evolutionarily conserved response at least initially counteracting ionophoric toxin action.

Adding phosphorylation events to the core oscillator driving the cell cycle of fission yeast.

Much is known about the regulatory elements controlling the cell cycle in fission yeast (Schizosaccharomyces pombe). This regulation is mainly done by the (cyclin-dependent kinase/cyclin) complex (Cdc2/Cdc13) that activates specific target genes and proteins via phosphorylation events during the cell cycle in a time-dependent manner. However, more work is still needed to complement the existing gaps in the current fission yeast gene regulatory network to be able to overcome abnormalities in its growth, repair and development, i.e. explain many phenomena including mitotic catastrophe. In this work we complement the previously presented core oscillator of the cell cycle of fission yeast by selected phosphorylation events and study their effects on the temporal evolution of the core oscillator based Boolean network. Thereby, we attempt to establish a regulatory link between the autonomous cell cycle oscillator and the remainder of the cell. We suggest the unclear yet regulatory effect of phosphorylation on the added components, and discuss many unreported points regarding the temporal evolution of the cell cycle and its components. To better visualize the results regardless of the programming background we developed an Android application that can be used to run the core and extended model of the fission yeast cell cycle step by step.

Maturation and cytokine pattern of human dendritic cells in response to different yeasts.

Activated dendritic cells (DC) induce and polarize T-cell responses by expression of distinct maturation markers and cytokines. This study systematically investigated the capacity of different biotechnically relevant yeast species and strains including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia pastoris, Hansenula polymorpha, Yarrowia lipolytica, and Candida glabrata to initiate maturation of human DC. As important prerequisite for T-cell activation, all yeasts were shown to effectively induce, though to a different extent, the expression of the activation marker CD83, the co-stimulatory molecules CD80, CD86, CD54, CD58, and CD40, as well as the antigen-presenting molecules MHCs I and II. Furthermore, yeast-activated DC secreted various cytokines including inflammatory TNF-α, IL-6, IL-8, and IL-1ß or T-cell polarizing IL-12, IL-10, IL-23, and IL-27. Variability was observed in the expression of TNF-α, IL-6, IL-8, IL-1ß, and IL-10 in response to the tested yeasts, whereas expression levels of IL-12, IL-23, and IL-27 were similar. Interestingly, maturation marker expression and cytokine secretion were not negatively affected after application of yeast mutants with altered cell wall mannoprotein structure (Δmnn11) or defective in protein N-glycosylation (Δost3), indicating that elongated cell wall mannoproteins at the outer yeast cell surface are not a prerequisite for the observed yeast-mediated DC maturation. Thus, our data provide a valuable basic knowledge for the future design of effective yeast-based delivery approaches.

Expression of K1 Toxin Derivatives in Saccharomyces cerevisiae Mimics Treatment with Exogenous Toxin and Provides a Useful Tool for Elucidating K1 Mechanisms of Action and Immunity.

Killer toxin K1 is a heterodimeric protein toxin secreted by Saccharomyces cerevisiae strains infected with the M1 double-stranded RNA 'killer' virus. After binding to a primary receptor at the level of the cell wall, K1 interacts with its secondary plasma membrane receptor Kre1p, eventually leading to an ionophoric disruption of membrane function. Although it has been under investigation for decades, neither the particular mechanisms leading to toxicity nor those leading to immunity have been elucidated. In this study, we constructed derivatives of the K1α subunit and expressed them in sensitive yeast cells. We show that these derivatives are able to mimic the action of externally applied K1 toxin in terms of growth inhibition and pore formation within the membrane, leading to a suicidal phenotype that could be abolished by co-expression of the toxin precursor, confirming a mechanistic similarity of external and internal toxin action. The derivatives were successfully used to investigate a null mutant completely resistant to externally applied toxin. They provide a valuable tool for the identification of so far unknown gene products involved in K1 toxin action and/or immunity.

Yeast-mediated mRNA delivery polarizes immuno-suppressive macrophages towards an immuno-stimulatory phenotype.

Macrophages have increasingly gained interest as a therapeutic target since they represent an integral component of the tumor microenvironment. In fact, M2 macrophage accumulation in solid tumors is associated with poor prognosis and therapy failure. Therefore, reprogramming M2 macrophages towards an M1 phenotype with anti-tumor activity by gene therapy represents a promising therapeutic approach. Herein, we describe recombinant Saccharomyces cerevisiae as a novel gene delivery vehicle for primary human macrophages. Opsonized S. cerevisiae was taken up efficiently by M2 macrophages and initiated the expression of pro-inflammatory cytokines. Recombinant yeast delivered functional nucleic acids to macrophages, especially when constitutively biosynthesized mRNA was used as cargo. Interestingly, expression of the protein encoded for by the delivered nucleic acid was higher in M2 cells when compared to M1 macrophages. Finally, the delivery of mRNA coding for the pro-inflammatory regulators MYD88 and TNF to M2 macrophages induced a prolonged upregulation of pro-inflammatory and cytotoxic cytokines in these cells, suggesting their successful re-education towards an anti-tumor M1 phenotype. Our results suggest the use of yeast-based gene delivery as a promising approach for the treatment of pathologic conditions that may benefit from the presence of M1-polarized macrophages, such as cancer.

H/KDEL receptors mediate host cell intoxication by a viral A/B toxin in yeast.

A/B toxins such as cholera toxin, Pseudomonas exotoxin and killer toxin K28 contain a KDEL-like amino acid motif at one of their subunits which ensures retrograde toxin transport through the secretory pathway of a target cell. As key step in host cell invasion, each toxin binds to distinct plasma membrane receptors that are utilized for cell entry. Despite intensive efforts, some of these receptors are still unknown. Here we identify the yeast H/KDEL receptor Erd2p as membrane receptor of K28, a viral A/B toxin carrying an HDEL motif at its cell binding ß-subunit. While initial toxin binding to the yeast cell wall is unaffected in cells lacking Erd2p, binding to spheroplasts and in vivo toxicity strongly depend on the presence of Erd2p. Consistently, Erd2p is not restricted to membranes of the early secretory pathway but extends to the plasma membrane where it binds and internalizes HDEL-cargo such as K28 toxin, GFP(HDEL) and Kar2p. Since human KDEL receptors are fully functional in yeast and restore toxin sensitivity in the absence of endogenous Erd2p, toxin uptake by H/KDEL receptors at the cell surface might likewise contribute to the intoxication efficiency of A/B toxins carrying a KDEL-motif at their cytotoxic A-subunit(s).

Yeast (Saccharomyces cerevisiae) Polarizes Both M-CSF- and GM-CSF-Differentiated Macrophages Toward an M1-Like Phenotype.

Macrophages are a heterogeneous and plastic cell population with two main phenotypes: pro-inflammatory classically activated macrophages (M1) and anti-inflammatory alternatively activated macrophages (M2). Saccharomyces cerevisiae is a promising vehicle for the delivery of vaccines. It is well established that S. cerevisiae is taken up by professional phagocytic cells. However, the response of human macrophages to S. cerevisiae is ill-defined. In this study, we characterized the interaction between S. cerevisiae and M1- or M2-like macrophages. M1-like macrophages had a higher yeast uptake capacity than M2-like macrophages, but both cell types internalized opsonized yeast to the same extent. The M1 surface markers HLAII and CD86 were upregulated after yeast uptake in M1- and M2-like macrophages. Moreover, mRNA expression levels of pro-inflammatory cytokines, such as TNF-α, IL-12, and IL-6, increased, whereas the expression of anti-inflammatory mediators did not change. These results demonstrate that S. cerevisiae can target both M1 and M2 macrophages, paralleled by skewing toward an M1 phenotype. Thus, the use of yeast-based delivery systems might be a promising approach for the treatment of pathologic conditions that would benefit from the presence of M1-polarized macrophages, such as cancer.

Surface-modified yeast cells: A novel eukaryotic carrier for oral application.

The effective targeting and subsequent binding of particulate carriers to M cells in Peyer's patches of the gut is a prerequisite for the development of oral delivery systems. We have established a novel carrier system based on cell surface expression of the ß1-integrin binding domain of invasins derived from Yersinia enterocolitica and Yersinia pseudotuberculosis on the yeast Saccharomyces cerevisiae. All invasin derivatives were shown to be effectively expressed on the cell surface and recombinant yeast cells showed improved binding to both human HEp-2 cells and M-like cells in vitro. Among the different derivatives tested, the integrin-binding domain of Y. enterocolitica invasin proved to be the most effective and was able to target Peyer's patches in vivo. In conclusion, cell surface-modified yeasts might provide a novel bioadhesive, eukaryotic carrier system for efficient and targeted delivery of either antigens or drugs via the oral route.

Schizosaccharomyces pombe: a novel transport vehicle of functional DNA and mRNA into mammalian antigen-presenting cells.

Vaccine vehicles based on recombinant yeasts have become promising candidates for the induction of cellular immune responses. In this study, we investigated the capacity of the fission yeast Sz. pombe for the delivery of functional nucleic acids into murine and human antigen-presenting cells. We demonstrate that Sz. pombe cells effectively induce maturation of human dendritic cells (DC), an important prerequisite for T-cell activation. Further, recombinant fission yeast efficiently delivers functional DNA and mRNA into murine macrophages and human DC resulting in the expression of the model antigen eGFP in these cells. Thus, Sz. pombe suggests itself as a promising candidate for a novel live vaccine.

Heat treatment improves antigen-specific T cell activation after protein delivery by several but not all yeast genera.

A central prerequisite in using yeast as antigen carrier in vaccination is its efficient interaction with cellular components of the innate immune system, mainly mediated by cell surface structures. Here, we investigated the distribution of major yeast cell wall components such as mannan, ß-glucan and chitin of four different and likewise biotechnologically relevant yeasts (Saccharomyces, Pichia, Kluyveromyces and Schizosaccharomyces) and analyzed the influence of heat-treatment on ß-1,3-glucan exposure at the outer yeast cell surface as well as the amount of yeast induced reactive oxygen species (ROS) production by antigen presenting cells (APC) in human blood. We found that yeasts significantly differ in the distribution of their cell wall components and that heat-treatment affected both, cell wall composition and yeast-induced ROS production by human APCs. We further show that heat-treatment modulates the activation of antigen specific memory T cells after yeast-mediated protein delivery in different ways and thus provide additional support of using yeast as vehicle for the development of novel T cell vaccines.

mRNA delivery to human dendritic cells by recombinant yeast and activation of antigen-specific memory T cells.

The import of functional nucleic acids like messenger RNA into mammalian cells has proven to be a powerful tool in cell biology and several delivery systems have been described. However, as targeting of particular cell types is a major challenge and RNA vaccination represents a promising means for the induction of cellular immune responses, there is a need for novel delivery systems that permit the introduction of functional messenger RNA to the cytosol of immune cells. Here, we describe a delivery system based on the yeast Saccharomyces cerevisiae that allows the delivery of functional messenger RNA to mammalian antigen-presenting cells such as human dendritic cells. Further, we present a method to prove antigen processing and presentation by stimulation of human autologous T lymphocytes.

Human yeast-specific CD8 T lymphocytes show a nonclassical effector molecule profile.

Pathogenic yeast and fungi represent a major group of human pathogens. The consequences of infections are diverse and range from local, clinically uncomplicated mycosis of the skin to systemic, life-threatening sepsis. Despite extensive MHC class I-restricted frequencies of yeast-specific CD8 T lymphocytes in healthy individuals and the essential role of the cell-mediated immunity in controlling infections, the characteristics and defense mechanisms of antifungal effector cells are still unclear. Here, we describe the direct analysis of yeast-specific CD8 T lymphocytes in whole blood from healthy individuals. They show a unique, nonclassical phenotype expressing granulysin and granzyme K in lytic granules instead of the major effector molecules perforin and granzyme B. After stimulation in whole blood, yeast-specific CD8 T cells degranulated and, upon cultivation in the presence of IL-2, their granula were refilled with granulysin rather than with perforin and granzyme B. Moreover, yeast-specific stimulation through dendritic cells but not by yeast cells alone led to degranulation of the effector cells. As granulysin is the only effector molecule in lytic granules known to have antifungal properties, our data suggest yeast-specific CD8 T cells to be a nonclassical effector population whose antimicrobial effector machinery seems to be tailor-made for the efficient elimination of fungi as pathogens.

Uptake of various yeast genera by antigen-presenting cells and influence of subcellular antigen localization on the activation of ovalbumin-specific CD8 T lymphocytes.

Yeasts of the genus Saccharomyces expressing recombinant antigens are currently evaluated as candidate T cell vaccines. Here, we compared the interaction kinetics between four biotechnologically relevant yeast genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Pichia pastoris) and human dendritic cells as well as the involvement of Dectin-1 and mannose receptor in phagocytosis. Further, we analyzed the activation capacity of recombinant yeasts expressing ovalbumin (OVA) either intracellular, extracellular or surface-displayed by OVA-specific CD8 T lymphocytes. We found that the kinetic patterns of yeast uptake by phagocytic cells varied between the tested yeast genera and that both genus and subcellular OVA antigen localization influenced the strength of T cell activation. In particular, in S. cerevisiae, a secreted antigen was less effectively delivered than its cytosolic variant, whereas most efficient antigen delivery with P. pastoris was obtained by cell surface bound antigen. Our data indicate that protein secretion might not be an effective delivery pathway in yeast.

Yeast-based protein delivery to mammalian phagocytic cells is increased by coexpression of bacterial listeriolysin.

Yeast-mediated protein delivery to mammalian antigen-presenting cells is a powerful approach for inducing cell-mediated immune responses. We show that coexpression of the pore-forming protein listeriolysin O from Listeria monocytogenes leads to improved translocation of a proteinaceous antigen and subsequent activation of specific T lymphocytes. As the resulting yeast carrier is self-attenuated and killed after antigen delivery without exhibiting any toxic effect on antigen-presenting cells, this novel carrier system suggests itself as promising approach for the development of yeast-based live vaccines.

RNA-directed DNA methylation and plant development require an IWR1-type transcription factor.

RNA-directed DNA methylation (RdDM) in plants requires two RNA polymerase (Pol) II-related RNA polymerases, namely Pol IV and Pol V. A genetic screen designed to reveal factors that are important for RdDM in a developmental context in Arabidopsis identified DEFECTIVE IN MERISTEM SILENCING 4 (DMS4). Unlike other mutants defective in RdDM, dms4 mutants have a pleiotropic developmental phenotype. The DMS4 protein is similar to yeast IWR1 (interacts with RNA polymerase II), a conserved putative transcription factor that interacts with Pol II subunits. The DMS4 complementary DNA partly complements the K1 killer toxin hypersensitivity of a yeast iwr1 mutant, suggesting some functional conservation. In the transgenic system studied, mutations in DMS4 directly or indirectly affect Pol IV-dependent secondary short interfering RNAs, Pol V-mediated RdDM, Pol V-dependent synthesis of intergenic non-coding RNA and expression of many Pol II-driven genes. These data suggest that DMS4 might be a regulatory factor for several RNA polymerases, thus explaining its diverse roles in the plant.

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD.

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.