High-Yield Expression in Escherichia coli, Purification and Application of Budding Yeast K2 Killer Protein

Podoliankaitė, Monika; Lukša, Juliana; Vyšniauskis, Gintautas; Sereikaitė, Jolanta; Melvydas, Vytautas; Serva, Saulius; Servienė, Elena

doi:10.1007/s12033-014-9740-6

Cited by 5 publications

(3 citation statements)

References 22 publications

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“…The AML-15-66 strain possessed the lowest activity against S. cerevisiae K2 toxin-producing strain M437, suggesting relation of both toxins, while K28-bearing S. cerevisiae strain M300 was killed the most efficiently among all killer strains tested. At the same time, AML-15-66 was sensitive to the action of S. cerevisiae K2 toxin, probably due to the high activity of this toxin [ 69 , 70 ], and completely resistant to other known killer types of S. cerevisiae K1, K28, and Klus.…”

Section: Discussionmentioning

confidence: 99%

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

Vepštaitė-Monstavičė

Lukša

Konovalovas

et al. 2018

Viruses

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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.

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Section: Discussionmentioning

confidence: 99%

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

Vepštaitė-Monstavičė

Lukša

Konovalovas

et al. 2018

Viruses

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“…The amount of K2 protein was calculated according to the SDS-PAGE using bovine serum albumin (BSA) as a concentration standard and quantifying the intensity of bands by the densitometric analysis. The presence of K2 toxin was confirmed by Western blotting using K2-specific polyclonal antibodies (23). The correlation between the concentration of K2 killer protein and strength of killing action was analyzed.…”

Section: Methodsmentioning

confidence: 99%

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

et al. 2015

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b Certain Saccharomyces cerevisiae strains 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 ␤-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-␤-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 ␤-1,6-glucan, rather than the plasma membrane-localized receptors. Taken together, our results reveal that ␤-1,6-glucan is a primary target of K2 toxin and is important for the execution of its killing property.T he production of antimycotic killer toxins has been observed in several yeast genera and proved to be a widespread phenomenon (1, 2). Killer strains of Saccharomyces cerevisiae secrete protein toxins derived from a family of double-stranded RNAs (dsRNAs). The toxins have been grouped into four types (K1, K2, K28, and Klus) based on their killing profiles and lack of crossimmunity (3, 4). Such proteins are able to kill the nonkiller yeast, as well as yeast of other killer types, while the toxin-producing cells remain immune to their own or to the same type of killers (4, 5). K1 toxin disrupts the regulated ion flux across the plasma membrane, leading to the death of sensitive yeast strains (6, 7). The killing action of K1 toxin involves at least two steps. During the first step, the toxin binds to the cell wall, whereas the second step leads to the translocation and insertion of the toxin into the plasma membrane (6). Beta-1,6-glucan was originally proposed to be a cell wall receptor for K1 (8). Analysis of several kre mutants demonstrated that decrease of the cell wall ␤-1,6-glucan level leads to K1 resistance, thus confirming the involvement of this type of glucan in toxin binding (9). During the second step, K1 toxin interacts with plasma membrane receptors and disrupts the functional integrity of the plasma membrane either by inducing the formation of new ion channels (7) or through the activation of existing potassium channels (10). Products of TOK1 (protein, forming the potassium ion channel) and KRE1 (glycoprotein, involved in ␤-glucan assembly) have be...

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“…Kabir et al [ 161 ] published a study on the antifungal potential of peptides derived from both anti-idiotypic antibody and its original fungicidal protein; in particular, SP3 or SP6 peptides proved their potential against Candida and Cryptococcus species infections and as a promising adjunct for conventional antibiotics. Furthermore, S. cerevisiae K2 toxin, with a C-terminal truncation, was obtained in E. coli as the host for large-scale production and suitable for polyclonal antibody production [ 171 ].…”

Section: Killer Toxins: From a Competitive Advantage To The Applicmentioning

confidence: 99%

Bioprotective Role of Yeasts

2015

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The yeasts constitute a large group of microorganisms characterized by the ability to grow and survive in different and stressful conditions and then to colonize a wide range of environmental and human ecosystems. The competitive traits against other microorganisms have attracted increasing attention from scientists, who proposed their successful application as bioprotective agents in the agricultural, food and medical sectors. These antagonistic activities rely on the competition for nutrients, production and tolerance of high concentrations of ethanol, as well as the synthesis of a large class of antimicrobial compounds, known as killer toxins, which showed clearly a large spectrum of activity against food spoilage microorganisms, but also against plant, animal and human pathogens. This review describes the antimicrobial mechanisms involved in the antagonistic activity, their applications in the processed and unprocessed food sectors, as well as the future perspectives in the development of new bio-drugs, which may overcome the limitations connected to conventional antimicrobial and drug resistance.

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High-Yield Expression in Escherichia coli, Purification and Application of Budding Yeast K2 Killer Protein

Cited by 5 publications

References 22 publications

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

Saccharomyces paradoxus K66 Killer System Evidences Expanded Assortment of Helper and Satellite Viruses

Yeast β-1,6-Glucan Is a Primary Target for the Saccharomyces cerevisiae K2 Toxin

Bioprotective Role of Yeasts

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