Killer Toxin Activity Research Articles

The Role of Plasma Membrane Pleiotropic Drug Resistance Transporters in the Killer Activity of Debaryomyces hansenii and Wickerhamomyces anomalus Toxins.

The killer strains of Debaryomyces hansenii and Wickerhamomyces anomalus species secrete antimicrobial proteins called killer toxins which are active against selected fungal phytopathogens. In our research, we attempted to investigate the role of plasma membrane pleiotropic drug resistance (PDR) transporters (Pdr5p and Snq2p) in the mechanism of defense against killer toxins. Saccharomyces cerevisiae mutant strains with strengthened or weakened pleiotropic drug resistance due to increased or reduced number of mentioned PDR efflux pumps were tested for killer toxin susceptibility. The present study demonstrates the influence of the Snq2p efflux pump in immunity to W. anomalus BS91 killer toxin. It was also shown that the activity of killer toxins of D. hansenii AII4b, KI2a, MI1a and CBS767 strains is regulated by other transporters than those influencing W. anomalus killer toxin activity. In turn, this might be related to the functioning of the Pdr5p transporter and a complex cross-talk between several regulatory multidrug resistance networks. To the best of our knowledge, this is the first study that reports the involvement of PDR transporters in the cell membrane of susceptible microorganisms in resistance to killer yeasts’ toxins.

Biological activities of melanin pigment extracted from Bombyx mori gut-associated yeast Cryptococcus rajasthanensis KY627764.

Melanin pigment has been produced and extracted from a wide variety of living forms ranging from microorganisms to higher organisms. Owing to the therapeutic nature of the pigment, various microbial populations have been explored for its production. Hence, we isolated a melanin producing yeast from the insect Bombyx mori gut microflora and identified it as Cryptococcus rajasthanensis based on the molecular characterization. The isolated yeast produced enhanced melanin pigment when cultured in the minimal L-tyrosine broth as compared to the Saboraud medium. The pigment was extracted and characterized as melanin based on UV-Visible spectroscopy, FTIR (Fourier-transform infrared) spectroscopy and 1H NMR (Nuclear magnetic resonance). The melanin pigment was evaluated as a potent bioactive molecule with bioactivity like antimicrobial, antioxidant, anti-inflammatory, and anticancer activity that describes the therapeutic nature of the extracted melanin pigment. Distinct from the biologically active role the melanin pigment isolated from the yeast, the Cryptococcus extract also exhibited killer toxin activity against the pathogenic yeast Candida albicans.

English

A marine yeast strain, DD21-2 was isolated from sediments in Dandong, China, which has killer activity against yeast Metschnikowia bicuspidata WCY (pathogenic to crab, Portunus trituberculatus). Routine identification, sequence analysis of 26S rDNA and ITS sequencing showed that the strain was M. saccharicola DD21-2, and has not been previously reported as having killer activity against M. bicuspidata WCY. To optimize the production of the killer toxin by M. saccharicola DD21-2, the interaction effects of fermentation process variables were investigated by Response Surface Methodology (RSM). The following reaction factors were selected in screening experiments: Inoculum concentration (2 to 6%), pH (5.0 to 6.0), temperature (20 to 28°C), and fermentation time (2 to 3 days), and the diameter of the inhibition zone produced by the killer toxin was used as a response variable. A quadratic regression model of killer toxin activity was established by regression analysis and significance testing (P test). The results identified the following parameters as optimal for maximal production of the killer toxin by M. saccharicola DD21-2: Fermentation temperature 28°C, pH 5.5, fermentation time 2.7 days, and inoculum concentration 4.1% (v/v). Thus, RSM was effective in determining the best conditions for killer toxin production, suggesting the practical usage of this marine yeast in the investigation of process variables. Key words: Marine killer yeast, killer toxin, response surface methodology (RSM), fermentation, optimization.

Production of killer toxin from DSMZ70238 Debaryomyces hansenii and test the effectiveness against cancer cell line L20B.

This study was conducted to detect the ability of yeast Debryomyces hansenii DSMZ70238 to produce the killer toxin and testing the optimal conditions for production rather than studying the cytotoxicity toward the cancer cell line L20B. The optimal conditions studied for their effect on yeast growth rates and their ability to increased production of killer toxin included different concentrations of NaCl salt, glucose, different concentrations of pH as well as different temperatures. The results showed that the killer yeast had the ability to produce the killer toxin in the molecular weight of 22 KDa were detected by the using of 12% SDS Polyacryelamide Gel, The optimal conditions of the production was studied and the killing activity of this toxin was determined towards microbes used in the present study, The results showed 8% of salts, pH 4.5, Temperature 25 ͦ C and 10 % from carbon source (glucose) are the best conditions for production and the killing activity. To detect the killing activity of killer toxin toward the cancer cells, The cytotoxicity test of the killer toxin was investigated against the cell line (L20B), by using the partially purified concentrated killer toxin produced under optimal conditions, The results of this experiment showed the killer toxin has a lethal effect of cancer cells, which reached the highest percentage at 8% salts concentration and it was 47.61% and at pH 4.5 reached 23.07%, while the percentage of killing was 21.53% at 25 ͦ C and 47.30% at 10% of glucose concentration.

Isolation of Saccharomyces Cerevisiae from Pineapple and Orange and Study of Metal's Effectiveness on Ethanol Production.

In view of the anticipated shortage of the traditional supplies of fossil fuels, there is a great deal of interest in the production of ethanol as an alternative biofuel in recent years. The main objective of this research work was to isolate and characterize stress tolerant, high potential ethanol producing yeast strains from various fruit peel. Two yeast isolates from pineapple (Pa) and orange (Or) have been isolated, characterized on the basis of morphological and physic-chemical characters and optimized on ethanol producing capability using sugarcane molasses as substrate. Ethanol production percentage was estimated by Conway method. Isolates were thermotolerant, pH tolerant, ethanol tolerant as well as osmotolerant. They were resistant to Chloramphenicol (30 μg/disc) and Nalidixic acid (30 μg/disc). The isolates showed no killer toxin activity against E. coli. The highest production capacity of the yeasts was found to be 7.39% and 5.02% for Pa and Or, respectively, at pH 5.0, 30 °C temperature in media with an initial reducing sugar concentration of 6.5% for Pa and 5.5% for Or (shaking). Addition of metal ions increased the rate of ethanol production highest to 10.61% by KH2PO4. This study revealed that indigenous yeast isolates could be used to benefit the fuel demand and industrial alcohol industries.

Production, Characterization, and Antimicrobial Activity of Mycocin Produced by Debaryomyces hansenii DSMZ70238.

The present study was conducted to estimate the antimicrobial activity and the potential biological control of the killer toxin produced by D. hansenii DSMZ70238 against several pathogenic microorganisms. In this study, the effects of NaCl, pH, and temperature, killer toxin production, and antimicrobial activity were studied. The results showed that the optimum inhibitory effect of killer toxin was at 8% NaCl, and the diameters of clear zones were 20, 22, 22, 21, 14, and 13 mm for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Candida albicans, and Candida neoformans, respectively. The largest inhibition zones were observed at pH 4.5 with inhibition zone of 16, 18, 17, 18, 11, and 12 mm for the same microorganisms. The results also showed that 25°C is the optimal temperature for toxin killing activity against all targeted microorganisms. In addition, the activity of killer toxin significantly inhibited the growth of fungal mycelia for all target pathogenic fungi and the percentages of inhibition were 47.77, 48.88, 52.22, and 61.11% for Trichophyton rubrum, Alternaria alternata, Trichophyton concentricum, and Curvularia lunata, respectively. The results showed the highest growth rate of D. hansenii DSMZ70238 under condition of 8% NaCl concentration, pH 4.5, and 25°C for 72 h.

A yeast strain associated to Anopheles mosquitoes produces a toxin able to kill malaria parasites.

BackgroundMalaria control strategies are focusing on new approaches, such as the symbiotic control, which consists in the use of microbial symbionts to prevent parasite development in the mosquito gut and to block the transmission of the infection to humans. Several microbes, bacteria and fungi, have been proposed for malaria or other mosquito-borne diseases control strategies. Among these, the yeast Wickerhamomyces anomalus has been recently isolated from the gut of Anopheles mosquitoes, where it releases a natural antimicrobial toxin. Interestingly, many environmental strains of W. anomalus exert a wide anti-bacterial/fungal activity and some of these ‘killer’ yeasts are already used in industrial applications as food and feed bio-preservation agents. Since a few studies showed that W. anomalus killer strains have antimicrobial effects also against protozoan parasites, the possible anti-plasmodial activity of the yeast was investigated.MethodsA yeast killer toxin (KT), purified through combined chromatographic techniques from a W. anomalus strain isolated from the malaria vector Anopheles stephensi, was tested as an effector molecule to target the sporogonic stages of the rodent malaria parasite Plasmodium berghei, in vitro. Giemsa staining was used to detect morphological damages in zygotes/ookinetes after treatment with the KT. Furthermore, the possible mechanism of action of the KT was investigated pre-incubating the protein with castanospermine, an inhibitor of β-glucanase activity.ResultsA strong anti-plasmodial effect was observed when the P. berghei sporogonic stages were treated with KT, obtaining an inhibition percentage up to around 90 %. Microscopy analysis revealed several ookinete alterations at morphological and structural level, suggesting the direct implication of the KT-enzymatic activity. Moreover, evidences of the reduction of KT activity upon treatment with castanospermine propose a β-glucanase-mediated activity.ConclusionThe results showed the in vitro killing efficacy of a protein produced by a mosquito strain of W. anomalus against malaria parasites. Further studies are required to test the KT activity against the sporogonic stages in vivo, nevertheless this work opens new perspectives for the possible use of killer strains in innovative strategies to impede the development of the malaria parasite in mosquito vectors by the means of microbial symbionts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-1059-7) contains supplementary material, which is available to authorized users.

16 strains of Debaryomyces hansenii tested against Penicillium digitatum, a post harvest fungus, affecting citrus crops in Puerto Rico

Citrus crops contribute 11 million dollars to the Puerto Rican economy. Unfortunately, Penicillium spp. infects these crops causing a great economic loss. Debaryomyces hansenii, a marine halotolerant yeast, has the ability to produce a killer toxin which seems to be homologous to the KHR (killer toxin) gene of Saccharomyces cerevisiae whose activity in the reduction of the decay of citrus fruit caused by Penicillium spp. has been reported. Microbiology and molecular biology techniques have been used to characterize sixteen D. hansenii strains and isolate their killer toxin. Different assays of the killer toxin activity in vivo were realized to determine its potential as a biological control. Two strains, D. hansenii BCS 006 and YB‐17914, were more efficient against Penicillium digitatum.

Cell cycle arrest and apoptosis, two alternative mechanisms for PMKT2 killer activity

Pichia membranifaciens CYC 1086 secretes a unique 30kDa killer toxin (PMKT2) that inhibits a variety of spoilage yeasts and fungi of agronomical interest. The cytocidal effect of PMKT2 on Saccharomyces cerevisiae cells was studied. Metabolic events associated with the loss of S. cerevisiae viability caused by PMKT2 were qualitatively identical to those reported for K28 killer toxin activity, but different to those reported for PMKT.At higher doses, none of the cellular events accounting for the action of PMKT, the killer toxin secreted by P. membranifaciens CYC 1106, was observed for PMKT2. Potassium leakage, sodium influx and the decrease of intracellular pH were not among the primary effects of PMKT2. We report here that this protein is unable to form ion-permeable channels in liposome membranes, suggesting that channel formation is not the mechanism of cytotoxic action of PMKT2. Nevertheless, flow cytometry studies have revealed a cell cycle arrest at an early S-phase with an immature bud and pre-replicated 1n DNA content. By testing the sensitivity of cells arrested at different stages in the cell cycle, we hoped to identify the execution point for lethality more precisely. Cells arrested at the G1-phase by α-factor or arrested at G2-phase by the spindle poison methyl benzimidazol-2-yl-carbamate (MBC) were protected against the toxin. Cells released from the arrest in both cases were killed by PMKT2 at a similar rate. Nevertheless, cells released from MBC-arrest were able to grow for a short time, and then viability dropped rapidly. These findings suggest that cells released from G2-phase are initially able to divide, but die in the presence of PMKT2 after initiating the S-phase in a new cycle, adopting a terminal phenotype within that cycle.By contrast, low doses of PMKT and PMKT2 were able to generate the same cellular response. The evidence presented here shows that treating yeast with low doses of PMKT2 leads to the typical membranous, cytoplasmic, mitochondrial and nuclear markers of apoptosis, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release.

Disruption of the Gene Encoding β-1, 3-Glucanase in Marine-Derived Williopsis saturnus WC91-2 Enhances its Killer Toxin Activity

As the β-1, 3-glucanase produced by the marine-derived Williopsis saturnus WC91-2 could inhibit the activity of the killer toxin produced by the same yeast, the WsEXG1 gene encoding exo-β-1, 3-glucanase in W. saturnus WC91-2 was disrupted. The disruptant WC91-2-2 only produced a trace amount of β-1, 3-glucanase but had much higher activity of killer toxin than W. saturnus WC91-2. After the disruption of the WsEXG1 gene, the expression of the gene was significantly decreased from 100% in the cells of W. saturnus WC91-2 to 27% in the cells of the disruptant WC91-2-2 while the expression of the killer toxin gene in W. saturnus WC91-2 and the disruptant WC91-2-2 was almost the same. During 2-l fermentation, the disruptant WC91-2-2 could produce the highest amount of killer toxin (the size of the inhibition zone was 22 ± 0.7mm) within 36h when the cell growth reached the middle of the log phase.

Production of a novel and cold-active killer toxin by Mrakia frigida 2E00797 isolated from sea sediment in Antarctica

The psychrotolerant yeast Mrakia frigida 2E00797 isolated from sea sediment in Antarctica was found to be able to produce killer toxin against the pathogenic yeast (Metschnikowia bicuspidata WCY) in crab. When the psychrotolerant yeast was grown in the medium with pH 4.5 and 3.0% (wt/vol) NaCl and at 15°C, it could produce the highest amount of killer toxin against the pathogenic yeast M. bicuspidata WCY. The crude killer toxin activity against the pathogenic yeast M. bicuspidata WCY was the highest when it grew at 15°C in the assay medium with 3.0% (wt/vol) NaCl and pH 4.5. At temperatures higher than 25°C, the killing activity produced by M. frigida 2E00797 was completely lost and after the crude killer toxin was pre-incubated at temperatures higher than 40°C for 4h, the killing activity was also completely lost. The killer toxin produced by M. frigida 2E00797 could kill only M. bicuspidata WCY, Candida tropicalis and Candida albicans among all the fungal species and bacterial species tested in this study.

Williopsis saturnus yeast killer toxin does not kill Streptococcus pneumoniae

Streptococcus pneumoniae is an important human bacterial pathogen, and the increase in antibiotic resistance demands the development of new antimicrobial compounds. Several reports have suggested that yeast killer toxins show activity against bacteria and we therefore investigated the activity of K9 killer toxin from the yeast Williopsis saturnus var. mrakii NCYC 500 against S. pneumoniae. However, no inhibition of bacterial growth was observed with concentrated K9 preparations in agar diffusion assays and in liquid culture. Although cell morphology was slightly affected by K9 treatment, no effect on cellular viability was detectable, and K9 had no stimulatory effect on cell lysis induced by β-lactams or Triton X-100. This indicated that K9 did not contribute to cell wall damage. Moreover, flow cytometry was used as a sensitive assessment of integrity of cells exposed to killer toxin. No significant damage of S. pneumoniae cells was evident, although minor changes in fluorescence suggested that K9 killer toxin may interact with bacterial surface components.

Isolation of Yeasts from Various Food Products and Detection of Killer Toxin Activity <i>In vitro</i>

Yeasts produce killer toxin, which gives them a selective advantage against susceptible yeasts. In this study we isolated 8 various non-pathogenic yeasts from 12 different food products and detected killer toxin activity in them against a clinical isolate of Cryptococcus neoformans by chloroform exposure method. The experiment was performed in two sets that had 3 replicates each. The first set plates and the second set plates were exposed to chloroform once and twice respectively. Among the 8 yeasts, 5 yeasts namely Sachharomyces cerevisiae, Candida pintolopesii, Candida tropicalis, Pichia anomala and Dekkera spp. showed significant amount of killer toxin activity against Cryptococcus neoformans in all the replicates, followed by candida parapsilosis and Trichosporon asahii, which showed killer toxin activity only in two of the three replicates. Geotrichum candidum showed no killer toxin activity. Killer toxin activity was observed in vitro in non-pathogenic yeast strains against medically important Cryptococcus neoformans. Keywords: Yeasts; Killer toxin; Chloroform exposure. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i2.4159 J. Sci. Res. 2 (2), 407-411 (2010)

Purification and molecular characterization of exo-β-1,3-glucanases from the marine yeast Williopsis saturnus WC91-2

The extracellular beta-1,3-glucanases in the supernatant of cell culture of the marine yeast Williopsis saturnus WC91-2 was purified to homogeneity with a 115-fold increase in specific beta-1,3-glucanase activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography, and anion-exchange chromatography. According to the data from sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 47.5 kDa. The purified enzyme could convert laminarin into monosaccharides and disaccharides, but had no killer toxin activity. The optimal pH and temperature of the purified enzyme were 4.0 and 40 degrees C, respectively. The enzyme was significantly stimulated by Li+, Ni2+, and Ba2+. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1,10-phenanthroline. The Km and Vmax values of the purified enzyme for laminarin were 3.07 mg/ml and 4.02 mg/min ml, respectively. Both matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectroscopy and DNA sequencing identified a peptide YIEAQLDAFEKR which is the conserved motif of the beta-1,3-glucanases from other yeasts.

Occurrence of killer yeast strains in industrial and clinical yeast isolates.

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.

From yeast killer toxins to antibiobodies and beyond

Antibiobodies are paradigmatic of yeast killer toxin (KT)-like antibodies (KAbs) mimicking the antimicrobial activity of KTs in the frame of the yeast killer phenomenon. Polyclonal, monoclonal and recombinant anti-idiotypic antibiobodies (anti-idiotypic KAbs), internal images of a wide-spectrum KT produced by the yeast Pichia anomala (PaKT), have been produced by immunization with the idiotype of a PaKT-neutralizing monoclonal antibody. Anti-idiotypic KAbs showed microbicidal activity against eukaryotic and prokaryotic pathogenic agents through the interaction with specific KT receptors (KTRs), putatively constituted by beta-glucans. Natural KAbs have been found in animals and humans experimentally or naturally infected by KTR-bearing microorganisms. Recombinant KAb-derived synthetic killer peptides showed further antiviral and immunomodulatory activities. The perspectives of KAbs and killer peptides as potential sources of novel therapeutic agents, and of KTRs and idiotypes as vaccines against infectious diseases are discussed.

Occurrence of killer yeast strains in industrial and clinical yeast isolates

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.

Enzymic Activity of the K5-Type Yeast Killer Toxin and Its Characterization

K5-type yeast killer toxin secreted by P. anomala NCYC 434 cells has a broad killing spectrum. Competitive inhibiton of killer activity showed that glucans, mainly the beta-1,3 glucan, represent the primary toxin binding site within the cell wall of sensitive cells. Its hydrolytic activity on laminarin in an exo-like fashion revealed that the toxin exerts its killing effect by exo-beta-1,3-glucanase activity. Its specific activity on laminarin was 120 U/mg, and the Michaelis constants K(m) and V(max) for laminarin hydrolysis were 0.25 mg/ml and 370 micromol/min/mg. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Production of the toxin by the cells was induced only when they were grown in culture media rich in beta-glucan sources, and the addition of glucose increased the specific production rate. The enzymic activity of the toxin was fully inhibited by Hg(+2), but increased with some other metal ions, most of all by Pb(+2).

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts

Large-scale screening of selected Candida maltosa, Debaryomyces hansenii and Pichia anomala killer toxin activity against pathogenic yeasts