# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action
### Cellular Responses to Killer Toxins
#### 5.3.1 PMKT
Finally, changes in plasma membrane integrity (determined as permeability to propidium iodide) are observed when cellular death was advanced in time, being probably a late consequence of PMKT treatment [41]. Yeasts response to harsh physical chemical factors (oxidative stress, heat, osmotic shock, nutrient availability, extreme pH, toxins, heavy metals, etc.) by activating a wide variety of defenses that include general or specific regulatory pathways, activating multiple MAPK cascades, which transform stimuli into appropriate physiological responses [126; 127; 128]. 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. PMKT-treated cells with a PMKT dosage of 1205 AU/mL (considered to be a high dose as stated below) and untreated cells were selected for mRNA isolation and determination of the transcriptional response. The most important functional group with 31 up-regulated genes is the group related to signal transduction, gene expression, transcription and RNA processing, followed by the group of genes related to ionic homeostasis, transport facilitation, carbohydrate metabolism, and cell stress and rescue. 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_, _YAPI_, _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]. In _S. cerevisiae_, it is known that the response to rapid changes in the osmolarity relies in the activation of the HOG (High Osmolarity Glycerol) pathway, which is manifested by the phosphorylation of Hog1p. Hog1p encodes for the MAPK that is activated by phosphorylation and then translocated to the nucleus to counteract with several environmental stimuli [130]. _HOG1\(\Delta\)_ mutants are hypersensitive to such environmental stresses, which cause Hog1p activation [130; 131]. 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. Phosphorylation is present in the first ten minutes, and during the rest of the exposure to PMKT, indicating constant activation of the signaling pathway. Accordingly, _HOG1\(\Delta\)_ mutants are found to be hypersensitive to PMKT. Glycerol is the main compatible osmolyte that accumulate rapidly as a consequence of activation of Hog1p and the transcriptional response is correlated with known responses to osmotic shocks.