Academic literature on the topic 'K9 type yeast killer toxin'

The strain Saccharomyces cerevisiae Y500-4L, previously selected from the must of alcohol producing plants and showing high fermentative and killer capacities, was characterized according to the interactions between the yeasts and examined for curing and detection of dsRNA plasmids, which code for the killer character. The killer yeast S. cerevisiae Y500-4L showed considerable killer activity against the Fleischmann and Itaiquara commercial brands of yeast and also against the standard killer yeasts K2 (S. diastaticus NCYC 713), K4 (Candida glabrata NCYC 388) and K11 (Torulopsis glabrata ATCC 15126). However S. cerevisiae Y500-4L showed sensitivity to the killer toxin produced by the standard killer yeasts K8 (Hansenula anomala NCYC 435), K9 (Hansenula mrakii NCYC 500), K10 (Kluyveromyces drosophilarum NCYC 575) and K11 (Torulopsis glabrata ATCC 15126). No M-dsRNA plasmid was detected in the S. cerevisiae Y500-4L strain and these results suggest that the genetic basis for toxin production is encoded by chromosomal DNA. The strain S. cerevisiae Y500-4L was more resistant to the loss of the phenotype killer with cycloheximide and incubation at elevated temperatures (40oC) than the standard killer yeast S. cerevisiae K1.

The killer toxin (KT 28), a glycoprotein of Saccharomyces cerevisiae strain 28, was almost completely adsorbed by bentonite, when applied at a concentration of 1 g per liter. No significant differences were found between several types of bentonite. Killer toxin KT 28 is similarly adsorbed by intact yeast cells or by a commercial preparation of yeast cell walls that has been recommended to prevent stuck fermentations. An investigation of the cell wall fractions revealed that the toxin KT 28 was mainly adsorbed by mannan, that removed the toxin completely. The alkali-soluble and the alkali-insoluble β-1,3- and β-1,6-D-glucans lowered the toxin concentration to one tenth of the original amount. The killer toxin of the type K1 of S. cerevisiae was adsorbed much better by glucans than by mannan.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

ABSTRACTCertainSaccharomyces cerevisiaestrains secrete different killer proteins of double-stranded-RNA origin. These proteins confer a growth advantage to their host by increasing its survival. K2 toxin affects the target cell by binding to the cell surface, disrupting the plasma membrane integrity, and inducing ion leakage. In this study, we determined that K2 toxin saturates the yeast cell surface receptors in 10 min. The apparent amount of K2 toxin, bound to a single cell of wild type yeast under saturating conditions, was estimated to be 435 to 460 molecules. It was found that an increased level of β-1,6-glucan directly correlates with the number of toxin molecules bound, thereby impacting the morphology and determining the fate of the yeast cell. We observed that the binding of K2 toxin to the yeast surface receptors proceeds in a similar manner as in case of the related K1 killer protein. It was demonstrated that the externally supplied pustulan, a poly-β-1,6-glucan, but not the glucans bearing other linkage types (such as laminarin, chitin, and pullulan) efficiently inhibits the K2 toxin killing activity. In addition, the analysis of toxin binding to the intact cells and spheroplasts confirmed that majority of K2 protein molecules attach to the β-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that β-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S. cerevisiae BJ1824 rhk1Δ : : URA3 and HM-1-insensitive yeast Candida albicans even after 1 h incubation. H35A HM-1 inhibited the activity of partially purified 1,3-β-glucan synthase from S. cerevisiae A451, and its extent was almost the same as wild-type HM-1. Co-immunoprecipitation experiments showed that wild-type and H35A HM-1 directly interact with the 1,3-β-glucan synthase complex. These results strongly suggest that histidine-35 has an important role in the cytocidal action of HM-1 that participates in the binding process to the HM-1 receptor protein on the cell membrane, but it is not essential for the interaction with, and inhibition of, 1,3-β-glucan synthase.

The Saccharomycetaceae yeast family recently became recognized for expanding of the repertoire of different dsRNA-based viruses, highlighting the need for understanding of their cross-dependence. We isolated the Saccharomyces paradoxus AML-15-66 killer strain from spontaneous fermentation of serviceberries and identified helper and satellite viruses of the family Totiviridae, which are responsible for the killing phenotype. The corresponding full dsRNA genomes of viruses have been cloned and sequenced. Sequence analysis of SpV-LA-66 identified it to be most similar to S. paradoxus LA-28 type viruses, while SpV-M66 was mostly similar to the SpV-M21 virus. Sequence and functional analysis revealed significant differences between the K66 and the K28 toxins. The structural organization of the K66 protein resembled those of the K1/K2 type toxins. The AML-15-66 strain possesses the most expressed killing property towards the K28 toxin-producing strain. A genetic screen performed on S. cerevisiae YKO library strains revealed 125 gene products important for the functioning of the S. paradoxus K66 toxin, with 85% of the discovered modulators shared with S. cerevisiae K2 or K1 toxins. Investigation of the K66 protein binding to cells and different polysaccharides implies the β-1,6 glucans to be the primary receptors of S. paradoxus K66 toxin. For the first time, we demonstrated the coherent habitation of different types of helper and satellite viruses in a wild-type S. paradoxus strain.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on other sensitive yeast genera but are immune to their own toxins. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer phenotypes are classified into 11 typical types (K1-K11). The toxic actions of yeast killer proteins on sensitive cells show differences and one of the most important toxic actions involves the selective functional damage by hydrolyzing major cell wall components. Because mammalian cells lack a cell wall, novel highly selective antifungals tend to be harmless to people by targeting important cell wall components specific to fungi. We have previously characterized the K9 type yeast killer protein isolated from Hansenula mrakii. This protein is stable at pH and temperature values appropriate for its medical usage. Antifungal activity of this protein was tested against 23 human pathogenic yeast and 9 dermathophyte strains. Pathogenic yeast strains found to be susceptible and both the MIC and MFC values ranged from 0.25 to 8 µ
g/ml except C. parapsilosis and C guilliermondii isolates. 9 dermatophyte strains were not susceptible to this protein and MICs were >
64 µ
g/ml. According to the cell killing analysis toxin activity starts within the first 4 hours and complete cell death was observed for the 4, 8 and 16 times the MIC concentrations at 24 hour. The results obtained from this study might make the potential use of this protein possible as a selective antimycotic agent.

Killer yeasts secrete polypeptide toxins which kill sensitive cells of their own species and frequently those of other species and genera of yeasts. These protein compounds are designated as killer toxins. Also killer toxins of certain yeast strains have potential growth inhibitory activity on gram-positive pathogenic bacteria and plant pathogenic fungi. The yeasts are immune to their own killer protein. The killer phenomenon can be utilized for the protection of fermentation process against contaminating yeasts and for biological control of undesirable yeasts in the preservation of foods. The killer trait can also be used to produce large amount of foreign proteins in yeast. In the medical field , it is thought that their anti-microbial and anti-mycotic activity could be exploited in a therapeutic strategy. Yeast killer toxins are classified into 11 types according to their killing spectra and immunity-specificities such as K1, K2, etc. Altough there is considerable amount of published information concerning the applications of yeast killer toxins , among the 11 types , only K1 , K2 and K6 have been characterized. In this study , it was aimed to purify and characterize the K4 type yeast killer toxin secreted by the Hansenula anomala NCYC 432. Gel permeation chromatography was performed to isolate the killer toxin by using a HPLC system. The toxin was shown to be a glycoprotein having a molecular mass of between 49.08 kDa and 47.25 kDa and isoelectric point of between 3.77 and 3.41.

La paroi de la levure Saccharomyces cerevisiae est une structure très dynamique composée de beta-glucanes, de mannanes et de chitine (polymère de N-acétylglucosamine). Elle peut s’adapter à l’état physiologique et aux changements morphologiques des cellules, ainsi qu’aux contraintes environnementales. Cette remarquable plasticité est assurée par l’intervention de différentes voies de régulation et de signalisation dont la voie CWI (Cell Wall Integrity) et la voie de la Calcineurine ou Protein Phosphatase 2B. La toxine killer K9 est une petite protéine sécrétée par la levure Hansenula mrakii. Cette toxine exerce son action létale sur les souches contrôles de S. cerevisiae mais pas sur des mutants du gène KNR4. Elle inhibe in vitro la beta-(1,3)-glucan syntase. Ce travail a dans un premier temps utilisé la Microscope à Force Atomique (AFM) et mis en évidence que la paroi de S. cerevisiae contrôle et mutant knr4 sont affectées de façon similaires par un traitement par la toxine K9. Dans un second temps, nous avons pu démontrer que la localisation cellulaire de Knr4 aux sites de croissance polarisée est nécéssaire pour l’action létale de la toxine K9 sur les cellules de S. cerevisiae.Knr4 fait partie d’une famille de protéines très conservées dans le domaine fongique, impliquées dans le contrôle de l’intégrité pariétale et la morphogenèse. Elle constitue un élément coordinateur pour la voie CWI et la voie de la Calcineurine. Notre travail a mis en évidence que la phosphorylation des résidus serine 200 et serine 203 de Knr4 joue un rôle dans ce mécanisme de coordination
The aim of my thesis was to study the fuction of Knr4 in the cell wall synthesis, morphogenesis, and related signaling pathways. The content of my thesis is mainly divided into three parts: The first part concerns our search to find out unknown partners of Knr4 and to investigate the cellular pathways required for localization of Knr4 protein. To that end, we decided to use a series of deletion mutants interrupted in genes related to morphogenesis and establishment of cellular polarity. We selected candidate genes from the Saccharomyces cerevisiae genome database (SGD, Stanford), using the keywords “Morphogenesis” and “Cell Polarity”. After selection and addition, 25 genes related to the morphogenesis and cell polarity were chosen for our Knr4 localization analysis. Through analysis of the results, we got 10 interesting mutants related to morphogenesis and polarity in which knr4 protein localization was affected: bem2Δ, pcl1Δ, pcl2Δ, rrd1Δ, spa2Δ, tpd3Δ, bem1Δ, bnI1Δ, yck1Δ and bud6Δ, and two additional mutants pph21Δ related to the tpd3Δ and cna1Δ involved in the calcinerin pathway. The second part deals with a mutational analysis of in vivo phosphorylated residues of Knr4 in the function and localization the protein, as well as in the modulation of calcineurin activity and CWI pathway. We found that S200S203 phosphorylation mutants cannot rescue viability of a double mutant bck1Δknr4Δ, while they can rescue slt2Δknr4Δ. In addition, S200S203 phosphorylation mutants behave as the absence of Knr4 towards suppression of lethality caused by an hyperactivated Mkk1 allele. Also we found that the knr4with KNR4S200AS203A mutant can results in hyperactivation of the Calcineurin pathway compared to control situation. So serin 200 and serin 203 may be involved in the cross-talking with the calcineurin pathway and CWI pathway. The third part is the study of K9 killer toxin’s strong cytocidal activity against sensitive yeast strains, including Saccharomyces cerevisiae. Treatment with this toxin results in the formation of pores at the surface of the cells, and more specifically at places where cell wall synthesis is the most active, namely at the tip of growing buds or mating projections. Yeast cells treated with K9 toxin then die by releasing cytoplasm and cellular materials from these pores. In the yeast S. cerevisiae, Knr4 protein localizes at the sites of polarized growth (bud tips, shmoo tips), which are also the sites where the toxin forms pores in the cell wall. Mutants defective in KNR4 gene are remarkably resistant to this toxin. In this study, we analyzed for the first time the biophysical effects of K9 on the yeast cell wall using Atomic Force Microscopy (AFM), a cutting edge technology that allows measuring the nanomechanical properties of living yeast cells, and their alterations by various drugs. To this end, we measured the effects of K9 toxin on the nanomechanical properties of the cell wall of S. cerevisiae wild-type cells and mutants deleted for KNR4 gene, at the short (2 h) and long term (20 h). Our results reveal an important cell wall remodeling occurring in wild-type cells already after 2 hours and only visible in knr4 mutant after 20 hours of treatment. Moreover, we investigated the role of Knr4 protein in the cells sensitivity towards the toxin. We were able to show that the presence of the N-terminal domain of Knr4 protein, which is required for its correct cellular localization at the bud tip during cell cycle, is essential for the toxin K9 wild-type sensitivity. In addition, a series of deletion mutants from the YKO collection in which the Knr4 cellular localization is also lost display a reduced sensitivity to the K9 toxin. Taken together, these results shed light on the importance of the proper localization of Knr4 protein at sites of intensive cell wall growth for the wild-type cells sensitivity to K9 killer toxin

Some yeasts secrete polypeptide toxins, which are lethal to other sensitive yeast cells, gram-positive pathogenic bacteria and pathogenic fungi. Therefore these are designated as killer toxins. Killer toxins are suggested as potent antimicrobial agents especially for the protection of fermentation process against contaminating yeasts, biological control of undesirable yeasts in the preservation of foods. Moreover they are promising antimicrobial agents in the medical field
due to immune system suppressing diseases like AIDS, there is an increase in the incidence of fungal diseases and current antimycotics have low selectivity and severe side effects. In this study our aim was to explain the cytocidal effect and enzymatic properties of K5 type yeast killer protein, which is secreted by Pichia anomala NCYC 434 cells, and known to have a broad range of killing spectrum. Competitive inhibition of the toxin with cell wall polysaccharides showed that primary binding site of toxin is &
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-1,3-glucans of sensitive cells. Toxin showed exo-&
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-1,3-glucanase activity which causes loss of cell wall rigidity leading cell death. Km and Vmax were found to be 0,3 mg/ml and 372,3 µ
mol/min/mg for laminarin hydrolysis. The toxin exerted its cytocidal effect after 2 h contact with the target cells. Toxin production was found to be dependent on &
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-1,3-glucan content of the media. Toxin activity was completely inhibited by Hg+2 ,while several metal ions and DTT increased the activity to different extends. Our findings revealed the characteristics of K5 type killer toxin which will help for its possible uses in near future.

Some yeast strains under certain conditions secrete into the medium polypeptide toxins which are inhibitory to sensitive cells. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component &
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-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. We have previously characterized the K5 type killer protein. This protein is an exo &
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-1,3-glucanase which is stable at pH&rsquo
s and temperatures appropriate for its medical usage. &
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-1,3- glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antimycotic agent. Antifungal activity of the K5 type yeast killer protein was tested against 26 human pathogenic yeast and 9 dermathophyte strains and found to be affective on all of the tested strains. Toxin MIC50, MIC100 and MFC values were found to be between 0.25-4, 0.5-8, 1-8 µ
g/ml respectively except Candida krusei isolates. Cell killing analysis revealed that toxin activity starts within first 2 hours and complete cell death time differs due to the susceptibility of strains to the K5 type yeast killer protein. K5 type yeast killer protein would be used as a novel and selective agents with the results obtained from this study.

Some yeast strains under certain conditions secrete polypeptide toxins which are inhibitory to sensitive fungal cells into the medium. These yeast strains are termed as killer yeasts and their toxins are designated as killer proteins or killer toxins. Killer proteins are classified into 11 typical types (K1-K11). These toxins have different killing mechanisms on sensitive cells. Some of them hydrolyze major cell wall component, beta-1,3- glucans. As mammalian cells lack cell walls research and development of novel highly selective antifungals are mostly focused on the agents which target the components of the fungal cell wall. K5 type killer protein was characterized in our labarotory previously. This protein is an exo beta-1,3-glucanase which is stable at pH&
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s and temperatures appropriate for its biocontrol usage. Beta-1,3-glucan hydrolyzing activity of the K5 type killer protein highlighted the potential use of this protein as a selective antifungal agent. According to CLSI methodology, antifungal activity of the K5 type yeast killer protein was tested against 6 fungal strains causing postharvest spoilage in citrus fruits and found to be effective on Botrytis cinerea, Penicillium digitatum, Penicillium italicum whereas non effective on Colletotrichum gloeosporoides, Phythophythora citrophthora, Alternaria citri. The MIC values of the toxin for B.cinerea, P.digitatum, P.italicum were found to be 16 mikrogram/ml while IC 50 values of the toxin were 2.12, 3.31, 2.57 mikrogram/ml respectively. The results showed that K5 type yeast killer protein would be used as a novel and selective agent against B.cinerea, P.digitatum and P.italicum.

Some yeast strains secrete extracellular polypeptide toxins known to have potential growth inhibitory activity on sensitive yeast cells. These yeast strains are known as killer yeasts and their toxins are named as killer toxins or killer proteins. Yeast killer proteins are found inhibitory to Gram-positive bacteria in several studies which were based on microbial interactions of the producer strains tested with sensitive strains. K5 type yeast killer protein produced by Pichia anomala NCYC 434 was previously purified and characterized in our laboratory. The protein is glycosilated and has a pI value of 3,7 and molecular mass of 49 kDa, with exo &
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-1,3-glucanase activity. Antibacterial activity of the pure K5 type yeast killer protein was tested against 19 clinical isolates of gram-positive bacteria causing skin infections and 2 quality control strains and found to have inhibitory activity on the isolates of Methicillin-sensitive Staphylococcus aureus (MSSA) and Enterococcus faecium. Toxin MIC and MBC ranges were 32 - 256 µ
g/ml and 64 - >
512 µ
g/ml respectively. Cell killing analysis revealed that toxin has a bacteriostatic activity and the inhibitory effect starts between 8. and 12. hours. Regrowth of the bacteria is retarded with the increased dose of the toxin. K5 type yeast killer protein might be used as a topical antibacterial agent with its bacteriostatic activity for skin and wound infections caused by MSSA and Enterococcus faecium with appropriate formulation studies upon the antibacterial spectrum determination of the toxin in this study.