Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

Abstract

BackgroundUnderstanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts.Principal FindingsWe conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility.SignificanceOur work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action.

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

“…Distinct sets of host factors affecting resistance and sensitivity toward K1 and K2 killer toxins were identified, revealing functional discrepancies between these toxins as well (9,19). Analysis of the sensitivity of a ⌬kre1 mutant to either K1 or K2 toxin demonstrated total resistance in both cases, suggesting that Kre1p serves as plasma membrane receptor (19,20). Also, it was shown that both K1 and K2 bind less efficiently to a set of mutants featuring a decreased level of ␤-1,6-glucan, implying that it serves as a cell wall receptor for both proteins (9,19,20).…”

“…The optimal pH for the action of the K2 toxin is 4.0 to 4.3 (17), somewhat lower than that of the K1 toxin, which reaches 4.6 (15,16,18). Distinct sets of host factors affecting resistance and sensitivity toward K1 and K2 killer toxins were identified, revealing functional discrepancies between these toxins as well (9,19). Analysis of the sensitivity of a ⌬kre1 mutant to either K1 or K2 toxin demonstrated total resistance in both cases, suggesting that Kre1p serves as plasma membrane receptor (19,20).…”

A quick and reliable method for genome-wide host factor screening of Saccharomyces cerevisiae killer toxins

“…Secondly, the use of both the K2-toxin producing strain and the purified K2 toxin in parallel increases the sensitivity of screening and allows selection of the candidates possessing even a weak phenotype. We found that 85% of mutants demonstrating a strong phenotype, confirmed by the "killer" assay, could be picked using only a K2 toxin-based "survival" assay (Servienė et al, 2012). This demonstrates that in the majority of cases, such strategy of screening could be used for the selection of marked candidates.…”

“…4. The list and analysis of the aforesaid 332 mutants has been published (Servienė et al, 2012). Surprisingly, some 520 of 850 candidates discovered in the "survival" assay were missing in the "killer" assay.…”

“…In contrary, for the detection of weak phenotype possessing candidates, both "survival" assay approaches should be used. Seventy-five percent of weak mutants confirmed by the "killer" assay demonstrated more pronounced phenotype in the K2-producing strainbased "survival" assay than the one using the K2 toxin (Servienė et al, 2012). The former assay is especially useful for testing of slowly growing mutants with altered physiology because of mild conditions achieved by slow but continuous action of the toxin produced by the killer cells.…”