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.

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.

Wickerhamomyces Yeast Killer Toxins’ Medical Applications

Possible implications and applications of the yeast killer phenomenon in the fight against infectious diseases are reviewed, with particular reference to some wide-spectrum killer toxins (KTs) produced by Wickerhamomyces anomalus and other related species. A perspective on the applications of these KTs in the medical field is provided considering (1) a direct use of killer strains, in particular in the symbiotic control of arthropod-borne diseases; (2) a direct use of KTs as experimental therapeutic agents; (3) the production, through the idiotypic network, of immunological derivatives of KTs and their use as potential anti-infective therapeutics. Studies on immunological derivatives of KTs in the context of vaccine development are also described.

New Cytoplasmic Virus-Like Elements (VLEs) in the Yeast Debaryomyces hansenii

Yeasts can have additional genetic information in the form of cytoplasmic linear dsDNA molecules called virus-like elements (VLEs). Some of them encode killer toxins. The aim of this work was to investigate the prevalence of such elements in D. hansenii killer yeast deposited in culture collections as well as in strains freshly isolated from blue cheeses. Possible benefits to the host from harboring such VLEs were analyzed. VLEs occurred frequently among fresh D. hansenii isolates (15/60 strains), as opposed to strains obtained from culture collections (0/75 strains). Eight new different systems were identified: four composed of two elements and four of three elements. Full sequences of three new VLE systems obtained by NGS revealed extremely high conservation among the largest molecules in these systems except for one ORF, probably encoding a protein resembling immunity determinant to killer toxins of VLE origin in other yeast species. ORFs that could be potentially involved in killer activity due to similarity to genes encoding proteins with domains of chitin-binding/digesting and deoxyribonuclease NucA/NucB activity, could be distinguished in smaller molecules. However, the discovered VLEs were not involved in the biocontrol of Yarrowia lipolytica and Penicillium roqueforti present in blue cheeses.

Vaginal Isolates of Candida glabrata are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae.

Compared to other species of Candida yeasts, the growth of Candida glabrata was inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins were broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

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.

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.

Yeast Microbiota during Sauerkraut Fermentation and Its Characteristics

Sauerkraut is the most important fermented vegetable obtained in Europe. It is produced traditionally by spontaneous fermentation of cabbage. The aim of this study was to determine biodiversity of yeasts present during fermentation of eight varieties of cabbages (Ambrosia, Avak, Cabton, Galaxy, Jaguar, Kamienna Głowa, Manama and Ramco), as well as characterize obtained yeast isolates. WL Nutrient Agar with Chloramphenicol was used to enumerate yeast. Isolates were differentiated using RAPD-PCR and identified by sequencing of the 5.8S-ITS rRNA gene region. The volatiles production was analyzed using SPME-GC-TOFMS. Our research confirmed that during sauerkraut fermentation there is an active growth of the yeasts, which begins in the first phases. The maximal number of yeast cells from 1.82 to 4.46 log CFU g−1 occurred after 24 h of fermentation, then decrease in yeast counts was found in all samples. Among the isolates dominated the cultures Debaryomyces hansenii, Clavispora lusitaniae and Rhodotorula mucilaginosa. All isolates could grow at NaCl concentrations higher than 5%, were relatively resistant to low pH and the presence of lactic acid, and most of them were characterized by killer toxins activity. The highest concentration of volatiles (mainly esters and alcohols) were produced by Pichia fermentans and D. hansenii strains.

Production of Bio-Fungicide from Sugarcane Bagasse using Pichia membranifaciens Yeast and its Activity Against Post-Harvest Pathogenic Fungi

Antagonistic yeast Pichia membranifaciens can produce killer toxins and hydrolytic enzymes to degrade pathogenic fungi cell walls. In this study, sugarcane bagasse was hydrolyzed and used as a low-cost carbon source to produce bio-fungicide using P. membranifaciens. In a growth medium obtained from bagasse hydrolysis containing 25 g/l sugar, with optimized amounts of nitrogen sources (2% (w/v)), NaCl and K2HPO4 (0.5% (w/v)) and in the presence of hydrophilic surfactant, Triton X100 (0.02% (w/v)), the yeast was able to produce 3782 mg/l bio-fungicide. The activity and toxicity of the produced bio-fungicide were tested against some post-harvest pathogenic fungi, including Aspergillus niger, Penicillium digitatum, and Phytophthora capsici. Minimum biocidal concentration (MBC) and minimum inhibitory concentration (MIC) were determined to be 378.2 and 37.82 mg/l, respectively. According to the results, the produced bio-fungicide has the potential for preserving agricultural products in the warehouse.

Vaginal isolates of Candida glabrata are Uniquely Susceptible to Killer Toxins Produced by Saccharomyces Yeasts

ABSTRACTKiller toxins have a range of antifungal activities against pathogenic species of yeasts. Candida glabrata was found to be acutely susceptible to seven killer toxins produced by Saccharomyces species of yeasts. Specifically, the ionophoric K1 and K2 killer toxins were broadly inhibitory to a large collection of C. glabrata from patients with recurrent vulvovaginal candidiasis. The sensitivity of these clinical isolates to killer toxins does not correlate with their resistance to major classes of antifungal drugs.