KTD1 is a yeast defense factor against K28 killer toxin

Secreted protein toxins are widely used weapons in conflicts between organisms. Killer yeast produce killer toxins that inhibit the growth of nearby sensitive yeast. We investigated variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population and discovered a novel defense factor, which we named KTD1, that is an important determinant of K28 toxin resistance. KTD1 is a member of the DUP240 gene family of unknown function. We uncovered a putative role of DUP240 proteins in killer toxin defense and identified a region that is undergoing rapid evolution and is critical to KTD1's protective ability. Our findings implicate KTD1 as a key factor in the defense against killer toxin K28.

α-Synuclein inhibits Snx3-retromer retrograde trafficking of the conserved membrane-bound proprotein convertase Kex2 in the secretory pathway of Saccharomyces cerevisiae

We tested the ability of alpha-synuclein (α-syn) to inhibit Snx3-retromer mediated retrograde trafficking of Kex2 and Ste13 between late endosomes and the trans-Golgi (TGN) using a Saccharomyces cerevisiae model of Parkinson’s disease (PD). Kex2 and Ste13 are a conserved, membrane-bound proprotein convertase and dipeptidyl aminopeptidase, respectively, that process pro-α-factor and pro-killer toxin. Each of these proteins contains a cytosolic tail that binds to sorting nexin Snx3. Using a combination of techniques, including fluorescence microscopy, western blotting and a yeast mating assay, we found that α-syn disrupts Snx3-retromer trafficking of Kex2-GFP and GFP-Ste13 from the late endosome to the TGN, resulting in these two proteins transiting to the vacuole by default. Using three α-syn variants (A53T, A30P, and α-synΔC, which lacks residues 101–140), we further found that A53T and α-synΔC, but not A30P, reduce Snx3-retromer trafficking of Kex2-GFP, which is likely to be due to weaker binding of A30P to membranes. Degradation of Kex2 and Ste13 in the vacuole should result in the secretion of unprocessed, inactive forms of α-factor, which will reduce mating efficiency between MATa and MATα cells. We found that wild-type α-syn but not A30P significantly inhibited the secretion of α-factor. Collectively, our results support a model in which the membrane-binding ability of α-syn is necessary to disrupt Snx3-retromer retrograde recycling of these two conserved endopeptidases.

Formulation and Safety Tests of a Wickerhamomyces anomalus–Based Product: Potential Use of Killer Toxins of a Mosquito Symbiotic Yeast to Limit Malaria Transmission

Wickerhamomyces anomalus strain WaF17.12 is a yeast with an antiplasmodial property based on the production of a killer toxin. For its symbiotic association with Anopheles mosquitoes, it has been proposed for the control of malaria. In an applied view, we evaluated the yeast formulation by freeze-drying WaF17.12. The study was carried out by comparing yeast preparations stored at room temperature for different periods, demonstrating that lyophilization is a useful method to obtain a stable product in terms of cell growth reactivation and maintenance of the killer toxin antimicrobial activity. Moreover, cytotoxic assays on human cells were performed, showing no effects on the cell viability and the proinflammatory response. The post-formulation effectiveness of the killer toxin and the safety tests indicate that WaF17.12 is a promising bioreagent able to impair the malaria parasite in vector mosquitoes.

Toward application of biocontrol to inhibit wine spoilage yeasts: The use of statistical designs for screening and optimisation

Spoilage yeasts generate considerable economic losses in the wine industry, and although sulphur dioxide (SO2) is traditionally used for control, its use has become controversial because of its negative effects on health. Biocontrol has emerged as a partial alternative to SO2, and most research has focused on the selection of biocontrol yeasts and/or the mechanisms involved, while little research has been directed to the environmental conditions that make biocontrol effective for application. When there are two or more interacting yeasts, the physicochemical factors that affect their antagonism are many and therefore the application of biocontrol is complex. To reduce SO2, the present study aimed to elucidate biocontrol mechanisms of two yeast interactions and to establish optimal physicochemical conditions for biocontrol of the spoilage yeast during grape must fermentation. Through the use of statistical design, it was possible to find relevant physicochemical factors and optimise them. Wickerhamomyces anomalus “BWa156” developed an active supernatant against ZygoSaccharomyces rouxii “BZr6” while supernatant from Metschnikowia pulcherrima “BMp29” was ineffective. In mixed must fermentations, the first interaction (BWa156 vs. BZr6) showed fewer physicochemical factors impacting biocontrol compared to the second interaction (BMp29 vs. BZr6). However, the fewer factors of the first interaction had a stronger effect on the decline in the spoilage population. Validations showed that the optimal conditions for biocontrol with the first interaction could be predicted. Analysis of the results with BWa156 vs. BZr6 and BMp29 vs. BZr6 suggests that the first interaction is a competition that includes a killer toxin, while the second interaction involves competition for iron resources. Response surface methodology (RSM) allowed a reduction in the number of experiments and permitted to find the optimal biocontrol conditions (SO2: 0 mg mL-1; pH: 3.7; Reducing sugars: 23 °Brix) for the interaction between BWa156 and BZr6.

Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows β-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25–2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.

In Vitro and In Vivo Anti-Candida Activity and Structural Analysis of Killer Peptide (KP)-Derivatives

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems.

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.