Inhibitory action of a truncated derivative of the amphibian skin peptide dermaseptin s3 on Saccharomyces cerevisiae.

The inhibitory activity of a truncated derivative of the natural amphibian skin peptide dermaseptin s3-(1-16)-NH2 [DS s3 (1-16)] against Saccharomyces cerevisiae was studied. Significant growth inhibition was observed after exposure to 3.45 microgram of the peptide per ml at pH 6.0 and 7.0, with complete growth inhibition occurring at 8.63 microgram of peptide per ml for all pH values tested. Using confocal scanning laser microscopy, we have shown that DS s3 (1-16) disrupted the yeast cell membrane resulting in the gross permeabilization of the cell to the nuclear stain ethidium bromide. However, the principal inhibitory action of the peptide was not due to disruption of intracellular pH homeostasis. Instead, growth inhibition by the peptide correlated with the efflux of important cellular constituents such as ADP, ATP, RNA, and DNA into the surrounding medium. The combination of DS s3 (1-16) with mild heating temperatures as low as 35 degreesC significantly enhanced the inhibitory effect of the peptide (8.63 microgram/ml), and at 45 degreesC greater than 99% of the population was killed in 10 min. In summary, a derivative of a natural antimicrobial peptide has potential, either alone or in combination with mild heating, to prevent the growth of or kill spoilage yeast.

Activity of the plasma membrane H(+)-ATPase is a key physiological determinant of thermotolerance in Saccharomyces cerevisiae.

The role of membrane integrity and the membrane ATPase in the mechanism of thermotolerance in Saccharomyces cerevisiae was investigated. The resistance to lethal heat of a mutant strain with reduced expression of the membrane ATPase was significantly less than that of the wild-type parent. However, prior exposure to sub-lethal temperatures resulted in the induction of similar levels of thermotolerance in the mutant compared to the parent strain, suggesting that the mechanism of sub-lethal heat-induced thermotolerance is independent of ATPase activity. Supporting this, exposure to sub-lethal heat stress did not result in increased levels of glucose-induced acid efflux at lethal temperatures and there was little correlation between levels of acid efflux and levels of heat resistance. ATPase activity in crude membrane preparations from sub-lethally heat-stressed cells was similar to that in preparations from unstressed cells. Study of net acid flux during heating revealed that pre-stressed cells were able to protect the proton gradient for longer. This may confer an 'advantage' to these cells that results in increased thermotolerance. This was supported by the observation that prior exposure to sub-lethal heat resulted in a transient protection against the large increase in membrane permeability that occurs at lethal temperatures. However, no protection against the large drop in intracellular pH was detected. Sub-lethal heat-induced protection of membrane integrity also occurred to the same extent in the reduced-expression membrane ATPase mutant, further implying that the mechanism of induced thermotolerance is independent of ATPase activity. To conclude, although the membrane ATPase is essential for basal heat resistance, thermotolerance induced by prior exposure to stress is largely conferred by a mechanism that is independent of the enzyme.