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.

Purification and molecular characterization of a Metschnikowia saccharicola killer toxin lethal to a crab pathogenic yeast.

The marine yeast strain Metschnikowia saccharicola DD21-2, isolated from sediments in the Yalu River, produces a killer toxin with a lethal effect on Metschnikowia bicuspidate strain WCY, a pathogenic yeast strain that infects crabs. In this study, the killer toxin was purified and characterized. After sequential purification, the purity of the killer toxin was increased 72.2-fold over the purity of the yeast cell culture supernatant. The molecular weight of the purified killer toxin was 47.0 kDa. The optimal pH and temperature for killing activity were 5.5°C and 16°C, respectively. The killing activity was stable over a pH range of 4.0-6.5 and temperature range of 0°C-40°C. The purified killer toxin was only effective against toxin-sensitive integral cells and had no killing effect on the protoplasts of toxin-sensitive cells. When exerting the killing effect, the toxin bind to a cell wall receptor of the treated strain, disrupted cell wall integrity and eventually caused death. The amino acid sequence identified by mass spectroscopy indicated that the purified killer toxin might be a protein kinase, but did not show ß-1,3-glucanase activity, consistent with the laminarin hydrolysis results. These findings provide a basis for disease prevention and control in marine aquaculture.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris.

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/ß 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 ß-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.

TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts.

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was N30 kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH 4.2 and 4.8 and inactivated at temperature above 40 °C. Pustulan and chitin ­ but not other cell wall polysaccharides ­ prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3 h treatment and apoptotic cell death after 24 h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts.

Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double-stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co-evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co-evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.

Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

Yeast killer toxin-like candidacidal Ab6 antibodies elicited through the manipulation of the idiotypic cascade.

A mouse anti-anti-anti-idiotypic (Id) IgM monoclonal antibody (mAb K20, Ab4), functionally mimicking a Wyckerhamomyces anomalus (Pichia anomala) killer toxin (KT) characterized by fungicidal activity against yeasts presenting specific cell wall receptors (KTR) mainly constituted by ß-1,3-glucan, was produced from animals presenting anti-KT Abs (Ab3) following immunization with a rat IgM anti-Id KT-like mAb (mAb K10, Ab2). MAb K10 was produced by immunization with a KT-neutralizing mAb (mAb KT4, Ab1) bearing the internal image of KTR. MAb K20, likewise mAb K10, proved to be fungicidal in vitro against KT-sensitive Candida albicans cells, an activity neutralized by mAb KT4, and was capable of binding to ß-1,3-glucan. MAb K20 and mAb K10 competed with each other and with KT for binding to C. albicans KTR. MAb K20 was used to identify peptide mimics of KTR by the selection of phage clones from random peptide phage display libraries. Using this strategy, four peptides (TK 1-4) were selected and used as immunogen in mice in the form of either keyhole limpet hemocyanin (KLH) conjugates or peptide-encoding minigenes. Peptide and DNA immunization could induce serum Abs characterized by candidacidal activity, which was inhibited by laminarin, a soluble ß-1,3-glucan, but not by pustulan, a ß-1,6-glucan. These findings show that the idiotypic cascade can not only overcome the barrier of animal species but also the nature of immunogens and the type of technology adopted.

A novel killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b.

In our previous study, it was found that the killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b has both killing activity and ß-1,3-glucanase activity and the molecular mass of it is 47.0 kDa. In this study, the same yeast strain was found to produce another killer toxin which only had killing activity against some yeast strains, but had no ß-1,3-glucanase activity and the molecular mass of the purified killer toxin was 67.0 kDa. The optimal pH, temperature and NaCl concentration for action of the purified killer toxin were 3.5, 16 °C and 4.0 % (w/v), respectively. The purified killer toxin could be bound by the whole sensitive yeast cells, but was not bound by manann, chitin and ß-1,3-glucan. The purified killer toxin had killing activity against Yarrowia lipolytica, Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis, Candida albicans and Kluyveromyces aestuartii. Lethality of the sensitive cells treated by the newly purified killer toxin from W. anomalus YF07b involved disruption of cellular integrity by permeabilizing cytoplasmic membrane function.

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

Occurrence of killer yeasts in isolates of clinical origin.

A total of 1 025 strains belonging to different Candida species of clinical origin were evaluated for their killer activity against sensitive strains of Saccharomyces cerevisiae. Isolates were identified by standard morphological and biochemical analyses. For the evaluation of the killer activity, potential killer isolates were streaked on plates previously seeded with the sensitive strain. A total of 52 Candida isolates (5%) exhibited killer activity against both sensitive yeast strains. The occurrence of the killer phenomenon was proportionally higher in isolates recovered from closed cavities. Candida glabrata was the species with the most occurrences of killer strains, but a bigger proportion of killer activity was observed in Candida utilis. Secretion of killer toxins could represent at least partially, an advantage against other candida and non-Candida strains in the colonization process, especially for uncommon Candida species.

Characterization of two different toxins of Wickerhamomyces anomalus (Pichia anomala) VKM Y-159.

Wickerhamomyces anomalus VKM Y-159 strain produces two types of toxin designated as WAKT a and WAKT b, encoded by chromosomal genes. The WAKT a toxin is heat-labile, pronase sensitive acting in pH range 3-4 affecting on several yeasts including pathogenic Candida species while the WAKT b toxin is protease- and thermo-resistant, acting in pH range 3-7 on two species, Candida alai and Candida norvegica. The rapid decrease of the number of viable cells after toxin treatment demonstrates that both toxins have cytocidic effect.

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2.

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0 kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3-3.5 and temperature 16 °C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378 bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6 kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.

Peptide derived from anti-idiotypic single-chain antibody is a potent antifungal agent compared to its parent fungicide HM-1 killer toxin peptide.

Based on anti-idiotypic network theory in light of the need for new antifungal drugs, we attempted to identify biologically active fragments from HM-1 yeast killer toxin and its anti-idiotypic antibody and to compare their potency as an antifungal agent. Thirteen overlapping peptides from HM-1 killer toxin and six peptides from its anti-idiotypic single-chain variable fragment (scFv) antibodies representing the complementarity determining regions were synthesized. The binding affinities of these peptides were investigated and measured by Dot blot and surface plasmon resonance analysis and finally their antifungal activities were investigated by inhibition of growth, colony forming unit assay. Peptide P6, containing the potential active site of HM-1 was highly capable of inhibiting the growth of Saccharomyces cerevisiae but was less effective on pathogenic fungi. However, peptide fragments derived from scFv antibody exerted remarkable inhibitory effect on the growth of pathogenic strains of Candida and Cryptococcus species in vitro. One scFv-derived decapeptide (SP6) was selected as the strongest killer peptide for its high binding affinity and antifungal abilities on both Candida and Cryptococcus species with IC(50) values from 2.33 × 10(-7) M to 36.0 × 10(-7) M. SP6 peptide activity was neutralized by laminarin, a ß-1,3-glucan molecule, indicating this peptide derived from scFv anti-idiotypic antibody retains antifungal activity through interaction with cell wall ß-glucan of their target fungal cells. Experimental evidence strongly suggested the possibility of development of anti-idiotypic scFv peptide-based antifungal agents which may lead to improve therapeutics for the management of varieties of fungal infections.

Production, purification, and characterization of a novel killer toxin from Kluyveromyces siamensis against a pathogenic yeast in crab.

The yeast Kluyveromyces siamensis HN12-1 isolated from mangrove ecosystem was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the killer yeast was grown in the medium with pH 4.0 and 0.5% NaCl and at 25 °C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The killing activity of the purified killer toxin against the pathogenic yeast M. bicuspidata WCY was the highest when it was incubated at 25 °C in the assay medium without added NaCl and pH 4.0. The molecular weight of the purified killer toxin was 66.4 kDa. The killer toxin produced by the yeast strain HN12-1 could kill only the whole cells of M. bicuspidata WCY among all the yeast species tested in this study. This is the first time to report that the killer toxin produced by the yeast K. siamensis HN12-1 isolated from the mangrove ecosystem only killed pathogenic yeast M. bicuspidata WCY.

Kluyveromyces wickerhamii killer toxin: purification and activity towards Brettanomyces/Dekkera yeasts in grape must.

Brettanomyces/Dekkera yeasts have been identified as part of the grape yeast flora. They are well known for colonizing the cellar environmental and spoiling wines, causing haze, turbidity and strong off-flavours in wines and enhancing the volatile acidity. As the general practices applied to combat Brettanomyces/Dekkera yeasts are not particularly appropriate during wine ageing and storage, a biological alternative to curtailing their growth would be welcomed in winemaking. In this study, we investigated the Kluyveromyces wickerhamii killer toxin (Kwkt) that is active against Brettanomyces/Dekkera spoilage yeasts. Purification procedures allowed the identification of Kwkt as a protein with an apparent molecular mass of 72 kDa and without any glycosyl residue. Interestingly, purified Kwkt has fungicidal effects at low concentrations under the physicochemical conditions of winemaking. The addition of 40 and 80 mg L(-1) purified Kwkt showed efficient antispoilage effects, controlling both growth and metabolic activity of sensitive spoilage yeasts. At these two killer toxin concentrations, compounds known to contribute to the 'Brett' character of wines, such as ethyl phenols, were not produced. Thus, purified Kwkt appears to be a suitable biological strategy to control Brettanomyces/Dekkera yeasts during fermentation, wine ageing and storage.

Structure-activity relationships in Kluyveromyces lactis gamma-toxin, a eukaryal tRNA anticodon nuclease.

tRNA anticodon damage inflicted by secreted ribotoxins such as Kluyveromyces lactis gamma-toxin and bacterial colicins underlies a rudimentary innate immune system that distinguishes self from nonself species. The intracellular expression of gamma-toxin (a 232-amino acid polypeptide) arrests the growth of Saccharomyces cerevisiae by incising a single RNA phosphodiester 3' of the modified wobble base of tRNA(Glu). Fungal gamma-toxin bears no primary structure similarity to any known nuclease and has no plausible homologs in the protein database. To gain insight to gamma-toxin's mechanism, we tested the effects of alanine mutations at 62 basic, acidic, and polar amino acids on ribotoxin activity in vivo. We thereby identified 22 essential residues, including 10 lysines, seven arginines, three glutamates, one cysteine, and one histidine (His209, the only histidine present in gamma-toxin). Structure-activity relations were gleaned from the effects of 44 conservative substitutions. Recombinant tag-free gamma-toxin, a monomeric protein, incised an oligonucleotide corresponding to the anticodon stem-loop of tRNA(Glu) at a single phosphodiester 3' of the wobble uridine. The anticodon nuclease was metal independent. RNA cleavage was abolished by ribose 2'-H and 2'-F modifications of the wobble uridine. Mutating His209 to alanine, glutamine, or asparagine abolished nuclease activity. We propose that gamma-toxin catalyzes an RNase A-like transesterification reaction that relies on His209 and a second nonhistidine side chain as general acid-base catalysts.

Reversible self-assembly: a key feature for a new class of autodelivering therapeutic peptides.

Effective delivery is a critical issue in the use of conventional free drugs. Studies on the structure-function relationship of a therapeutic antibody-derived candidacidal decapeptide (killer peptide, KP) revealed its ability to spontaneously and reversibly self-assemble in an organized network of fibril-like structures. This process is catalyzed by 1,3-beta-glucans. While the self-assembled state may provide protection against proteases and the slow kinetic of dissociation assures a release of the active dimeric form over time, the beta-glucan affinity is responsible for targeted delivery. Thus, KP represents a novel paradigm of targeted autodelivering drugs.

PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis.

Pichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 degrees C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions.

Therapeutic activity of an anti-idiotypic antibody-derived killer peptide against influenza A virus experimental infection.

The in vitro and in vivo activities of a killer decapeptide (KP) against influenza A virus is described, and the mechanisms of action are suggested. KP represents the functional internal image of a yeast killer toxin that proved to exert antimicrobial and anti-human immunodeficiency virus type 1 (HIV-1) activities. Treatment with KP demonstrated a significant inhibitory activity on the replication of two strains of influenza A virus in different cell lines, as evaluated by hemagglutination, hemadsorption, and plaque assays. The complete inhibition of virus particle production and a marked reduction of the synthesis of viral proteins (membrane protein and hemagglutinin, in particular) were observed at a KP concentration of 4 microg/ml. Moreover, KP administered intraperitoneally at a dose of 100 microg/mice once a day for 10 days to influenza A/NWS/33 (H1N1) virus-infected mice improved the survival of the animals by 40% and significantly decreased the viral titers in their lungs. Overall, KP appears to be the first anti-idiotypic antibody-derived peptide that displays inhibitory activity and that has a potential therapeutic effect against pathogenic microorganisms, HIV-1, and influenza A virus by different mechanisms of action.