Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

Abstract

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM-1, which kills susceptible yeasts through inhibiting 1,3-beta-glucan synthase. By using HM-1 plate assay, we found that eight gene-deletion mutants had higher HM-1-resistance compared with the wild-type. Among these eight genes, five -ALG3, CAX4, MNS1, OST6 and YBL083C -were associated with N -glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM-1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2-alpha-mannosidase showed with a 13-fold higher HM-1 resistance compared with the wild-type. By HM-1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild-type cells. These results indicate that the presence of the terminal 1,3-alpha-linked mannose residue of the B-chain of the N -glycan structure is essential for interaction with HM-1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM-1 resistance. A deletion mutant of osmoregulatory mitogen-activated protein kinase Hog1p was more sensitive to HM-1, suggesting that high-osmolarity glycerol pathways plays an important role in the compensatory response to HM-1 action.

The high‐osmolarity glycerol– and cell wall integrity–MAP kinase pathways of Saccharomyces cerevisiae are involved in adaptation to the action of killer toxin HM‐1

“…The fps1 cells also showed diphospho‐Hog1 and ‐Slt2p inductions (Figure , fps1 ). It was reported that the high HM‐1‐resistance of the alg3 cell is derived from lack of HM‐1 binding to the plasma membrane receptor (Miyamoto et al ., ). Diphospho‐Hog1p and ‐Slt2p were not induced in alg3 cells (Figure A, B, alg3 ).…”

“…These facts are contrary to the believed fungicidal mechanism of HM‐1, an inhibition of 1,3‐ β ‐glucan synthesis. In addition, it was reported that HM‐1 binds with mannoproteins of the yeast cell wall and plasma membrane (Kimura et al ., , ; Miyamoto et al ., ). Thus, we postulate that HM‐1 has a two‐step action on the cell wall‐disturbing mechanism: (a) binding and insertion to cell wall structures (e.g.…”

“…We reported that hog1 and slt2 cells show a HM‐1‐susceptible phenotype (Table ) (Miyamoto et al ., ). These results indicate that activations of both the HOG– and CWI–MAP kinase pathways are involved in the adaptive response to HM‐1 action.…”

“…Previously, we screened a set of S. cerevisiae deletion mutants for sensitivity to HM‐1 and found that several genes of S. cerevisiae were involved in the yeast‐killing action of HM‐1 (Miyamoto et al ., ). We also showed that disruptions of genes involved in N ‐glycan maturation and of a chaperone give strong HM‐1‐resistant phenotypes to yeast cells.…”

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

“…Nowadays, a particular interest in yeast killer strains and corresponding secreted toxins has emerged. Both of those have found application in industry (food protection from spoiling, wine production), agriculture (phytopathogen control), or medicine (creation of new generation vaccines and antibiotics, development of antifungal immunotherapies) (Goretti et Performing the experiments with the W. saturnus, K. lactis, and P. anomala toxins, colonies from the S. cerevisiae libraries were manually arrayed on the agar plates, and the growth of mutants was further tested either in solid or liquid media (Conti et al, 2008;Huang et al, 2008;Miyamoto et al, 2011). In the case of K1 and K28 toxins, a different approach was used.…”