The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells.

The measurement of internal pH in microorganisms, in yeast cells and in cells in general, has been studied for many years. Several mechanisms are involved in the regulation of the internal pH of the cell, many cellular processes are regulated by the internal pH, and many transport processes depend on the H ϩ cycle. In yeast cells, very crude procedures were initially used, with disruption of the cells by boiling or freezing and thawing, after which the pH of the resulting sap was measured. In 1950, the group of Conway used the distribution of weak acids, such as carbonic or propionic acid, to measure the internal pH of yeast cells (5,6). By these methods, the pH was probably obtained as an average of that of the entire cell interior, including all internal compartments. More recently, other methods have been used; among them, the shift of the P i peak in nuclear magnetic resonance spectra has been useful but complicated and expensive (1, 3).In yeast cells, the use of ionizable fluorescent probes capable of crossing the membrane and distributing between the cells, organelles, or vesicles depending on the internal and external pH (16) has proven useless. Slavík (17) first introduced indicators into yeast cells whose fluorescence depends on the surrounding pH; some of these, which are available commercially, can be introduced into the cells as acetoxymethyl esters (the permeant form), which are cleaved inside by esterases, transforming them into an impermeant form and preventing their efflux. A recent report on the use of one of these dyes to measure the internal pH of yeast cells has appeared (9). However, Slayman et al. (18) have pointed out some of the drawbacks of these dyes; because of the concentration of esterases in some intracellular compartments, they are preferentially hydrolyzed and accumulated in vacuoles or other internal compartments which accumulate hydrolytic enzymes.Kano and Fendler (10) introduced the use of pyranine (8-hydroxy-1,3,6-pyrene-trisulfonic acid), a fluorescent dye with a ionizable -OH group, which shows remarkable pH dependence in fluorescence. The dye could be trapped in liposomes and used to estimate their internal pH (see also reference 4), the most important advantage being the relatively low interaction with the bilayer or proteins, because of its hydrophilic nature.Unfortunately, this property makes its use with whole cells difficult, because of problems with entry. However, a controlled electric shock of high intensity and short duration appears to produce transient openings of the cell membrane that are closed in a short time after the treatment (11). This phenomenon has been used to introduce even substances with high molecular weights into cells by a procedure known as electroporation. This method has been successfully used in yeast cells, and great improvements have been made recently (2,8).The present communication deals with the use of pyranine to measure the internal pH of yeast cells. MATERIALS AND METHODSCells from an isolated colony of commercial yeast cells (La Azt...

Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.

Different methods to estimate the plasma membrane potential difference (PMP) of yeast cells with fluorescent monitors were compared. The validity of the methods was tested by the fluorescence difference with or without glucose, and its decrease by the addition of 10 mM KCl. Low CaCl₂ concentrations avoid binding of the dye to the cell surface, and low CCCP concentrations avoid its accumulation by mitochondria. Lower concentrations of Ba²+ produce a similar effect as Ca²+, without producing the fluorescence changes derived from its transport. Fluorescence changes without considering binding of the dyes to the cells and accumulation by mitochondria are overshadowed by their distribution between this organelle and the cytoplasm. Other factors, such as yeast starvation, dye used, parameters of the fluorescence changes, as well as buffers and incubation times were analyzed. An additional approach to measure the actual or relative values of PMP, determining the accumulation of the dye, is presented.

The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.