# The Biology of _Pichia membranifaciens_ Killer Toxins
## 5 Killing Mechanism of Action
### Cellular Responses to Killer Toxins  
#### 5.3.1 PMKT  
Many targets to the killer toxin action in sensitive cells have been identified and, in consequence, some molecular mechanisms of killing action have been also determined. The first study for determining some aspects of the killing action of PMKT was intended to assess the importance of the addition of salt (which is common in the production of traditional fermented foods) in the expression of the killer phenotype [44]. In preliminary studies, we observed that sodium chloride in concentrations of up to 6% (wt/vol) eventually enhanced the toxicity and enlarged the killer spectra of a few of the yeasts tested. This observation raised the question of whether this higher activity was due to increased killer toxin production or increased sensitivity of the strains affected. In addition, it has been also shown that the killing action of some other killer yeasts isolated from high salinity environments are related on the presence of NaCl. For example, SMK toxin produced by _Pichia farinosa_ KK1 shows its maximum killer activity in the presence of 2.0 M NaCl [70]. Furthermore, CnKT, produced by _Candida nodaensis_, presents a strong salt-dependent phenotype [45] and the same can also be observed for _Mrakia frigida_ 2E00797 and _Williipsis saturnus_ WC91-2 killer toxins [119; 120]. However, so far, little has been studied concerning the reasons why, in these particular cases, the killing activity is dependent on NaCl. PMKT finds its greatest similarity in K1, which share certain biochemical characteristics such as similar isoelectric point and molecular mass. In a similar way to the mode of action in which K1 acts from outside the cell after reaching the cytoplasmic membrane, PMKT exerts its lethal effect by ion channel formation and disruption of cytoplasmic membrane gradients [19; 121; 122; 123]. PMKT binds to the cell wall (1\(\rightarrow\)6)-\(\beta\)-d-glucans in the first 2-3 min after toxin addition, and then a lag phase of approximately one hour is necessary to observe reductions in cell viability, probably reflecting the time necessary for the development of the mechanism of action, gradual intoxication and death. The lag phase observed after cell wall binding and previously to cell death, is supposed to involve several sequential interactions and effects: interaction with secondary receptors, ion channel formation in the plasma membrane, alteration of transmembrane gradients and deenergization of cells and eventually, formation of large unspecific plasma membrane pores that permit the passage of other intracellular molecules [124]. During the first 30 min of incubation with PMKT, the pHi of toxin-treated sensitive cells and control cells is observed to be similar, thereafter, pHi is progressively acidified, reaching values of about 4.6-4.7 after 0.5-1 h incubation with PMKT. Then a leakage of K\({}^{+}\) out of the cells starts (1-1.5 h after exposure to PMKT) and simultaneously, a flow of Na\({}^{+}\) began. In the same way as K\({}^{+}\), the intake of Na\({}^{+}\) is directly related with killing [41]. The disruption of ionic equilibrium across the cytoplasmic membrane may could generate an augmented mortality of the cells in the presence of salt. The increased permeability of the cytoplasmic membrane to protons could consequently generate the influx of ions through the plasma membrane. Any compound accumulated in or outside the cell against its concentration gradient will equilibrate and a flow could occur. These findings are also in agreement with those studies that observed in yeast spheroplasts and artificial liposomes treated with K1 that the ion channels caused by K1 do not distinguish between Na\({}^{+}\) and K\({}^{+}\)[125; 122]. In addition, PMKT causes in liposomes a conductance when added at low pH, showing a characteristic channel activity. Furthermore, all the common physiological ions are observed to flow through the pores caused by PMKT in the selectivity sequence: K\({}^{+}\) > Na\({}^{+}\) > Li\({}^{+}\) > Ca\({}^{2+}\) > Cl\({}^{-}\).