The disaccharide trehalose is widely distributed in nature and can be found in many organisms, including bacteria, fungi, plants, invertebrates and mammals. Due to its particular physical features, trehalose is able to protect the integrity of the cell against a variety of environmental injuries and nutritional limitations. In addition, data available on several species of bacteria and yeast suggest specific functions for trehalose in these organisms. Bacteria can use exogenous trehalose as the sole source of carbon and energy as well as synthesize enormous amounts of the disaccharide as compatible solute. This ability to accumulate trehalose is the result of an elaborate genetic system, which is regulated by osmolarity. Some mycobacteria contain sterified trehalose as a structural component of the cell wall, whereas yeast cells are largely unable to grow on trehalose as carbon source. In these lower eukaryotes, trehalose appears to play a dual function: as a reserve compound, mainly stored in vegetative resting cells and reproductive structures, and as a stress metabolite. Recent findings also point to important biotechnological applications for trehalose.
e Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.
The cellular response to the oxidative stress caused by hydrogen peroxide and its putative correlation with the stress protector trehalose was investigated in Candida albicans CAI.4 and the tps1/tps1 double mutant, which is deficient in trehalose synthesis. When exponential wild-type blastoconidia were exposed to high concentrations of hydrogen peroxide, they displayed a high cell survival, accompanied by a marked rise of intracellular trehalose. The latter is due to a moderate activation of trehalose synthase and the concomitant inactivation of neutral trehalase. Identical challenge in the tps1/tps1 double mutant severely reduced cell viability, a phenotype which was suppressed by overexpression of the TPS1 gene. Pretreatment of growing cultures from both strains with either a low, non-lethal concentration of H 2 O 2 (05 mM) or a preincubation at 37 SC, induced an adaptive response that protected cells from being killed by a subsequent exposure to oxidative stress. During these mild oxidative preincubations, trehalose was not induced in CAI.4 cells and remained undetectable in their tps1/tps1 counterpart. Blastoconidia from the two strains exhibited a similar degree of cell protection during the adaptive response. The induction of trehalose accumulation by H 2 O 2 was not due to an increased expression of TPS1 mRNA. These results are consistent with a mainly protective role of trehalose in C. albicans during direct oxidative stress but not during acquired oxidative tolerance.
Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-mediated cyclic AMP (cAMP) signal that induces a protein phosphorylation cascade. In yeast mutants (tpklwl, tpk2", and tpk3w') containing reduced activity of cAMP-dependent protein kinase, fermentable sugars, as opposed to nonfermentable carbon sources, induced a permanent hyperaccumulation of cAMP. This finding confirms previous conclusions that fermentable sugars are specific stimulators of cAMP synthesis in yeast cells. Despite the huge cAMP levels present in these mutants, deletion of the gene (BCYI) coding for the regulatory subunit of cAMP-dependent protein kinase severely reduced hyperaccumulation of cAMP. Glucose-induced hyperaccumulation of cAMP was also observed in exponential-phase glucose-grown cells of the tpklwl and tpk2w' strains but not the tpk3w' strain even though addition of glucose to glucose-repressed wild-type cells did not induce a cAMP signal. Investigation of mitochondrial respiration by in vivo 31P nuclear magnetic resonance spectroscopy showed the tpklwl and tpk2wl strains, but not the tpk3Wl strain, to be defective in glucose repression. These results are consistent with the idea that the signal transmission pathway from glucose to adenyl cyclase contains a glucose-repressible protein. They also show that a certain level of cAMP-dependent protein phosphorylation is required for glucose repression. Investigation of the glucose-induced cAMP signal and glucose-induced activation of trehalase in derepressed cells of strains containing only one of the wild-type TPK genes indicates that the transient nature of the cAMP signal is due to feedback inhibition by cAMP-dependent protein kinase.Cells of the yeast Saccharomyces cerevisiae contain two RAS genes, RAS] and RAS2, which are homologs of the mammalian ras genes H-ras, K-ras, and N-ras (21). The RAS proteins are plasma membrane-bound G proteins that are generally considered to be signal transmitters. The yeast RAS proteins were shown to act as controlling elements of adenyl cyclase (21). The product of the yeast gene CDC25 is required for RAS activity and hence for cyclic AMP (cAMP) synthesis in vivo (2). Addition of glucose or related fermentable sugars to derepressed yeast cells triggers a transient increase in the cAMP level which is mediated by the CDC25 and RAS proteins (12, 13). The hormonelike effect of glucose on adenyl cyclase, which resembles the well-known hormone effects on adenyl cyclase in mammalian cells, induces a protein phosphorylation cascade that mobilizes the storage sugar trehalose and stimulates the switch of metabolism from gluconeogenic to fermentative (16).In yeast cells, three genes, TPKJ, TPK2, and TPK3, code for the catalytic subunit of cAMP-dependent protein kinase (19). The presence of one of the three is enough for viability. Recently, yeast mutants tpkl"', tpk2"', and tpk3"' that show greatly reduced activity of cAMP-dependent protein kinase were isolated (14). In these strains, two ...
In Candida albicans, trehalose plays an essential role as a protector of cell integrity against oxidative challenge. A double homozygous mutant, tps1/tps1, deficient in trehalose synthesis, displayed severe cell mortality when exposed to high H 2 O 2 concentrations, compared with its congenic parental (CAI-4) strain (Alvarez-Peral et al., 2002). We have examined the putative role of a set of well-known antioxidant enzymes as components of the defence mechanism against oxidative challenges. When exposed to mild non-lethal oxidative treatment (0.5 mM H 2 O 2 ), a significant induction of catalase, glutathione reductase (GR), and Cu,Zn-superoxide dismutase (SOD) was recorded in tps1/tps1 exponential cultures. However, in CAI-4 cells, subjected to the same conditions, there was only a clear activation of catalase, Mn-SOD and Cu,Zn-SOD activities. The degree of activation was always much more pronounced in the trehalose-deficient mutant than in its wild-type counterpart, except for Mn-SOD activity. After exposure to severe oxidative stress (50 mM H 2 O 2 ) only GR and catalase activities increased in tps1/tps1 cultures, whereas in CAI-4 cells GR but not catalase was induced. In both cell strains, 50 mM H 2 O 2 caused inhibition of the Mnand Cu,Zn-SOD isozymes, this inhibition being more pronounced in tps1/tps1 cells. C. albicans is able to acquire adaptive oxidative tolerance by pretreatment with a low non-stressing concentration of H 2 O 2 before exposure to a drastic oxidative challenge. When these antioxidant activities were measured during the adaptive response, a greater degree of enzymatic antioxidant induction was consistently observed in the tps1/tps1 mutant with respect to the CAI-4 strain. Together with a higher intrinsic sensitivity of tps1/tps1 cells, we suggest that this unexpected increase might be explained in terms of a compensatory mechanism to overcome the lack of endogenous trehalose upon drastic oxidative exposure, although this induction was not sufficient to improve the percentage of cell viability.
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