The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

Abstract

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.Killer phenomena are widespread in yeasts. Killer toxins are proteins or glycoproteins that are lethal to sensitive strains of the same species and a different variety of other yeast genera. In this line, attention has focused mainly on the characterization of killer toxins from Saccharomyces cerevisiae (K1, K2, and K28) followed more recently by the investigation of yeasts such as Kluyveromyces lactis, Zygosaccharomyces bailii, Hanseniaspora uvarum, Pichia membranifaciens, Debaryomyces hansenii, Schwanniomyces occidentalis,.P. membranifaciens CYC 1106 is a strain originally isolated from fermenting olive brines with pronounced killer activity against a variety of yeast species (8) and fungi (9). Once the protein nature of the toxin produced was established, the secreted protein was purified from the supernatant of growing cultures of P. membranifaciens in the early stationary phase. Previous biochemical studies on the PMKT 2 mechanism of killing of sensitive yeast cells indicated that PMKT is an 18-kDa protein that interacts with the (1 3 6)-␤-D-glucans of the cell wall of sensitive yeasts (6, 10). Recently the killing mechanism of this killer toxin has been elucidated (10). Regardless of certain possible additional effects, the killer toxin of P. membranifaciens CYC 1106 acts by disrupting plasma membrane electrochemical gradients. The death of sensitive cells in the presence of killer toxin is characterized by a leakage of common physiological ions through non-regulated ion channels in the plasma membrane causing a discharge of cellular membrane potential and changes in ionic homeostasis in a way comparable to that of certain killer toxins (K1) (11). Non-selective channel formation has been suggested to be the cytotoxic mechanism of action of PMKT (10).Yeasts must cope with different adverse environmental conditions, including hea...

The Biology of Pichia membranifaciens Killer Toxins

“…In order to confirm the physiological events described for PMKT toxicity [41], the transcriptional response of the sensitive cells was studied with a view to obtain information of the mechanisms of killing action. In addition, the results were confirmed by studying the response of deleted mutants in all those genes that were observed to be significantly up- or down-regulated when sensitive cells were treated with PMKT [73]. When the global gene expression response of the yeast S. cerevisiae to PMKT was explored, 146 genes showed an altered expression in response to PMKT.…”

“…Both, the phosphatases and dehydrogenases implicated in the glycerol production pathway are also up-regulated in response to PMKT. In addition, many similarities are found when the group of up-regulated genes in response to the toxin [73] and those induced by osmotic stress [129] are compared. In fact, most PMKT-induced genes encode proteins involved in counteracting the physiological changes induced by an osmotic shock, thus indicating the existence of an osmotic-related stimulus inside the cells when treated with the toxin.…”

“…Genes that were related to salt tolerance in response to PMKT were CTT1 , HSP12 , GPD1 , GPP2 , TRK2 , PDR12 , ENA1 , SCH9 , HAL9 , YAP1 , XBP1 , and STL1 (Figure 1). Furthermore, ENA1 , TRK2 , PHO89 , ZRT1 and PDR12 are a group of genes that surely are found to be up-regulated because of the leakage of ions and small metabolites induced by the toxin due to the generation of non-specific channels in a cellular response to compensate the leakage over a short period of time [41,73]. …”

“…Several transcription factors have been found to be among the substrates of Hog1p [132], so the phosphorylation and the consequent activation of Hog1p also regulates gene expression profiles [133]. Hog1p phosphorylation has been studied in order to determine whether the observed transcriptional response in the presence of PMKT was connected with the HOG pathway or not [73]. Hog1p phosphorylation is observed rapidly in PMKT-intoxicated cells.…”

“…Deletion mutants of up-regulated genes in response to PMKT are also observed to show hypersensitive phenotypes to PMKT, direct evidence that they are surely involved in the mechanism of action of PMKT [73], an effect also observed in HOG signaling-defective cells of S. cerevisiae when exposed to K1 [134], suggesting a response coincident to other hyperosmotic stresses being effective to counteract K1 toxicity. However, GPD1 Δ mutants, and others defective in intracellular glycerol production, are hypersensitive to PMKT, but this is not the case for K1 and no downstream effectors of Hog1p relevant for K1 resistance could be identified [134].…”