Strong correlation between the antifungal effect of amphotericin B and its inhibitory action on germ-tube formation in a Candida albicans URA⁺ strain.

The hypothetical capacity of amphotericin B to suppress the formation of germ-tubes, which is the first step of yeast-to-hypha conversion in Candida albicans, has been investigated in the wild-type strain CEY.1 (CAI.4-URA⁺). Exponential cells exposed to concentrations of amphotericin B below or around the MIC90, exhibited a weak reduction in the percentage of human serum-induced germ-tube formation at 37ºC compared with a non-exposed control. However, the dimorphic transition was drastically suppressed after addition of potentially lethal doses of amphotericin B, which also caused severe cell killing. In contrast, an identical experimental approach carried out with the fungistatic compound 5-fluorocytosine had no significant effect on the level of the germ-tube formation. Together, these results strongly point to a close correlation between the fungicidal action of amphotericin B and its ability to impair morphogenetic conversion in C. albicans.

Strong correlation between the antifungal effect of amphotericin B and its inhibitory action on germ-tube formation in a Candida albicans URA+ strain

The hypothetical capacity of amphotericin B to suppress the formation of germ-tubes, which is the first step of yeast-to-hypha conversion in Candida albicans, has been investigated in the wild-type strain CEY.1 (CAI.4-URA+). Exponential cells exposed to concentrations of amphotericin B below or around the MIC90, exhibited a weak reduction in the percentage of human serum-induced germ-tube formation at 37ºC compared with a non-exposed control. However, the dimorphic transition was drastically suppressed after addition of potentially lethal doses of amphotericin B, which also caused severe cell killing. In contrast, an identical experimental approach carried out with the fungistatic compound 5-fluorocytosine had no significant effect on the level of the germ-tube formation. Together, these results strongly point to a close correlation between the fungicidal action of amphotericin B and its ability to impair morphogenetic conversion in C. albicans (AU)

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Specific stress-induced storage of trehalose, glycerol and D-arabitol in response to oxidative and osmotic stress in Candida albicans.

Candida albicans exponential yeast cells are able to face environmental challenges by mounting a rapid and efficient "general stress response". Here we show that one of the main components of this response consists of the intracellular protective accumulation of the non-reducing disaccharide trehalose and two polyols, glycerol and D-arabitol, an accumulation that occurs in a stress-specific dependent manner. Thus, oxidative exposures promoted a marked increase in both trehalose and D-arabitol in the wild type strain, RM-100, whereas the glycerol content remained virtually unaffected with respect to basal levels. In contrast, osmotic challenges induced the significant storage of glycerol accompanied by minor changes, or even a slight drop, in the intracellular content of trehalose and D-arabitol. We examined the hypothetical role in this process of the MAP kinase Hog1, which regulates the protective responses in C. albicans against both oxidative and osmotic stress. Interestingly, unlike glycerol synthesis, the stress-induced trehalose accumulation was always Hog1-independent, whereas the ability to synthesize D-arabitol was only partially dependent on a functional Hog1 pathway.

Amphotericin B induces trehalose synthesis and simultaneously activates an antioxidant enzymatic response in Candida albicans.

BACKGROUND: Enzymes involved in trehalose metabolism have been proposed as potential targets for new antifungals. To analyse this proposal, the susceptibility to Amphotericin B (AmB) of the C. albicans trehalose-deficient mutant tps1Δ/tps1Δ, was examined. METHODS: Determination of endogenous trehalose and antioxidant enzymatic activities as well as RT-PCR analysis in cells subjected to AmB treatments was performed. RESULTS: Exponential tps1Δ null cultures showed high degree of cell killing upon exposure to increasing AmB doses respect to CAI.4 parental strain. Reintroduction of the TPS1 gene restored the percentage of cell viability. AmB induced significant synthesis of endogenous trehalose in parental cells, due to the transitory accumulation of TPS1 mRNA or to the moderate activation of trehalose synthase (Tps1p) with the simultaneous deactivation of neutral trehalase (Ntc1p). Since tps1Δ/tps1Δ mutant cells are highly susceptible to acute oxidative stress, the putative antioxidant response to AmB was also measured. A conspicuous activation of catalase and glutathione reductase (GR), but not of superoxide dismutase (SOD), was observed when the two cell types were exposed to high concentrations of AmB (5µg/ml). However, no significant differences were detected between parental and tps1Δ null strains as regards the level of activities. CONCLUSIONS: The protective intracellular accumulation of trehalose together with the induction of antioxidant enzymatic defences are worthy mechanisms involved in the resistance of C. albicans to the fungicidal action of AmB. GENERAL SIGNIFICANCE: The potential usefulness of trehalose synthesis proteins as an interesting antifungal target is reinforced. More importantly, AmB elicits a complex defensive response in C. albicans.

Adaptive tolerance to oxidative stress and the induction of antioxidant enzymatic activities in Candida albicans are independent of the Hog1 and Cap1-mediated pathways.

In the pathogenic yeast Candida albicans, the MAP-kinase Hog1 mediates an essential protective role against oxidative stress, a feature shared with the transcription factor Cap1. We analysed the adaptive oxidative response of strains with both elements altered. Pretreatment with gentle doses of oxidants or thermal upshifts (28-->37 and 37-->42 degrees C) improved survival in the face of high concentrations of oxidants (50 mM H(2)O(2) or 40 mM menadione), pointing to a functional cross-protective mechanism in the mutants. The oxidative challenge promoted a marked intracellular synthesis of trehalose, although hog1 (but not cap1) cells always displayed high basal trehalose levels. Hydrogen peroxide (H(2)O(2)) induced mRNA expression of the trehalose biosynthetic genes (TPS1 and TPS2) in the tested strains. Furthermore, oxidative stress also triggered a differential activation of various antioxidant activities, whose intensity was greater after HOG1 and CAP1 deletion. The pattern of activity was dependent on the oxidant dosage applied: low concentrations of H(2)O(2) (0.5-5 mM) clearly induced catalase and glutathione reductase (GR), whereas drastic H(2)O(2) exposure (50 mM) increased Mn-superoxide dismutase (SOD) isozyme-mediated SOD activity. These results firmly support the existence in C. albicans of both Hog1- and Cap1-independent mechanisms against oxidative stress.

On the biochemical classification of yeast trehalases: Candida albicans contains two enzymes with mixed features of neutral and acid trehalase activities.

Two enzymes endowed with trehalase activity are present in Candida albicans. The cytosolic trehalase (Ntc1p), displayed high activity in exponential phase regardless of the carbon source (glucose, trehalose or glycerol). Ntc1p activity was similar in neutral (pH 7.1) or acid (pH 4.5) conditions, strongly inhibited by ATP, weakly stimulated by divalent cations (Ca(2+)or Mn(2+)) and unaffected in the presence of cyclic AMP. The Ntc1p activity decreased in stationary phase, except in glycerol-grown cultures, but the catalytic properties did not change. In turn, the cell wall-linked trehalase (Atc1p) showed elevated activity in resting cells or in cultures growing on trehalose or glycerol. Although Atc1p is subjected to glucose repression, exhaustion of glucose in itself did not increased the activity. Significant Atc1p values could also be measured at neutral or acid pH, but Atc1p was insensitive to ATP, cyclic AMP and divalent cations. These results are in direct contrast with the current classification of yeast trehalases based on their optimum pH. They are also relevant in the light of the proposed use of trehalase inhibitors for the treatment of candidiasis.

Role of trehalose-6P phosphatase (TPS2) in stress tolerance and resistance to macrophage killing in Candida albicans.

Disruption of the TPS2 gene encoding the only trehalose-6P phosphatase activity in Candida albicans caused a pleiotropic defective phenotype, maintaining the cell wall integrity and the ability to form chlamydospores. A homozygous tps2Delta/tps2Delta showed reduced growth at high temperatures and a marked sensitivity to heat shock (42 degrees C) and severe oxidative exposure (50mM H(2)O(2)). Reintroduction of the TPS2 gene reversed these alterations. A more detailed study of the antioxidant response showed that exponential tps2Delta null cells displayed an adaptive response to oxidative stress as well as cross-tolerance between temperature and oxidative stress. Differential measurement of trehalose and trehalose-6P, using reliable new HPLC methodology, revealed a significant accumulation of trehalose-6P in tps2Delta cells, which was enhanced after oxidative exposure. In contrast, the level of trehalose-6P in parental cells was virtually undetectable, and oxidative treatment only induced the synthesis of free trehalose. A transitory increase in the expression of TPS2 and TPS1 genes was promoted in wild-type cells in response to acute (50mM) but not gentle (5mM) oxidative exposure. TPS1 and TPS2 oxidative-induced transcriptions were completely absent from the tps2Delta mutant. Exponential blastoconidia from both parental and tps2Delta/tps2Delta strains were completely phagocytosed by murine and human macrophages, triggering a subsequent proinflammatory response manifested by the release of TNF-alpha. Reflecting the lower resistance to oxidative stress displayed by the tps2Delta mutant, intracellular survival in resting and IFN-gamma and LPS-stimulated macrophages was also diminished. Taken together, our results confirm the mainly protective role played by the trehalose biosynthetic pathway in the cellular response to oxidative stress and subsequently in the resistance to phagocytosis in C. albicans, a defensive mechanism in which TPS2 would be involved.

Stress responses in yeasts: what rules apply?

Living organisms have evolved a complex network of mechanisms to face the unforeseen nutritional and environmental circumstances imposed on their natural habitats, commonly termed "stress". To learn more about these mechanisms, several challenges are usually applied in the laboratory, namely nutrient starvation, heat shock, dehydration, oxidative exposures, etc. Yeasts are chosen as convenient models for studying stress phenomena because of their simple cellular organization and the amenability to genetic analysis. A vast scientific literature has recently appeared on the defensive cellular responses to stress. However, this plethora of studies covers quite different experimental conditions, making any conclusions open to dispute. In fact, the term "yeast stress" is rather confusing, since the same treatment may be very stressful or irrelevant, depending on the yeast. Customary expressions such as "gentle stress" (non-lethal) or "severe stress" (potentially lethal) should be precisely clarified. In turn, although prototypic yeasts share a common repertoire of signalling responsive pathways to stress, these are adapted to the specific ecological niche and biological activity of each particular species. What does "stress" really mean? Before we go any deeper, we have to define this uncertain meaning along with a proper explanation concerning the terms and conditions used in research on yeast stress.

Disruption of the Candida albicans ATC1 gene encoding a cell-linked acid trehalase decreases hypha formation and infectivity without affecting resistance to oxidative stress.

In Candida albicans, the ATC1 gene, encoding a cell wall-associated acid trehalase, has been considered as a potentially interesting target in the search for new antifungal compounds. A phenotypic characterization of the double disruptant atc1Delta/atc1Delta mutant showed that it was unable to grow on exogenous trehalose as sole carbon source. Unlike actively growing cells from the parental strain (CAI4), the atc1Delta null mutant displayed higher resistance to environmental insults, such as heat shock (42 degrees C) or saline exposure (0.5 M NaCl), and to both mild and severe oxidative stress (5 and 50 mM H(2)O(2)), which are relevant during in vivo infections. Parallel measurements of intracellular trehalose and trehalose-metabolizing enzymes revealed that significant amounts of the disaccharide were stored in response to thermal and oxidative challenge in the two cell types. The antioxidant activities of catalase and glutathione reductase were triggered by moderate oxidative exposure (5 mM H(2)O(2)), whereas superoxide dismutase was inhibited dramatically by H(2)O(2), where a more marked decrease was observed in atc1Delta cells. In turn, the atc1Delta mutant exhibited a decreased capacity of hypha and pseudohypha formation tested in different media. Finally, the homozygous null mutant in a mouse model of systemic candidiasis displayed strongly reduced pathogenicity compared with parental or heterozygous strains. These results suggest not only a novel role for the ATC1 gene in dimorphism and infectivity, but also that an interconnection between stress resistance, dimorphic conversion and virulence in C. albicans may be reconsidered. They also support the hypothesis that Atc1p is not involved in the physiological hydrolysis of endogenous trehalose.

The cellular resistance against oxidative stress (H2O2) is independent of neutral trehalase (Ntc1p) activity in Candida albicans.

The protective role of trehalose against oxidative stress caused by hydrogen peroxide in Candida albicans has been investigated in the homozygous mutant ntc1Delta/ntc1Delta, disrupted in the NTC1 gene, which encodes the neutral (cytosolic) trehalase (Ntc1p). After a severe oxidative exposure (50 mM H(2)O(2)), both parental (CAI-4) and ntc1Delta/ntc1Delta exponential-phase cells stored large amounts of intracellular trehalose. In turn, the degree of cell survival was roughly equivalent in both strains, although slightly higher in ntc1Delta/ntc1Delta cultures. The mechanism of 'adaptive tolerance' was functional in the two strains. Thus, a gently oxidative pretreatment (5 mM H(2)O(2)) increased the recovery of cellular viability when it was followed by a severe challenge (50 mM H(2)O(2)); this phenomenon was accompanied by a significant elevation of the endogenous trehalose content. Oxidative stress also induced specific activation of the antioxidant enzymes catalase and glutathione reductase upon gentle oxidative treatment (5 mM H(2)O(2)), whereas superoxide dismutase activity was only activated upon prolonged exposure. Taken together, these results strongly suggest that in C. albicans neutral trehalase activity does not play an essential role in the protective response against oxidative stress. They also suggest that a diminished Ntc1p activity might favour the growth of C. albicans cells subjected to a strong oxidative exposure.

Correlation between the intracellular content of glutathione and the formation of germ-tubes induced by human serum in Candida albicans.

The physiological role of the tripeptide glutathione (GSH) and its oxidized form (GSSG) was investigated during the initial steps of dimorphism (formation of germ-tubes), which is induced by human serum in exponential yeast-like cells (blastoconidia) of the Candida albicans strain CAI-4 (wild type) and its congenic tps1/tps1 mutant, deficient in trehalose synthesis. The content of glutathione, measured both as GSH and the ratio GSH/GSSG, underwent a moderate drop in parallel with the induction of a significant degree of germ-tube emergence. Whereas the supply of exogenous glutathione did not affect the degree of dimorphic transition, depletion of intracellular glutathione by addition of 1-chloro-2,4 dinitrobenzene (CDNB) caused a clear reduction in the percentage of hyphae formation; although this effect must be due to the severe cell mortality produced by CDNB. Simultaneous measurements of GSH-metabolizing activities revealed a moderate decrease of glutathione reductase concomitant with the activation of glutathione peroxidase. In turn, catalase activity did not show noticeable changes. The putative correlation between the redox status of glutathione and the dimorphic conversion in C. albicans is discussed.

Role of antioxidant enzymatic defences against oxidative stress H(2)O(2) and the acquisition of oxidative tolerance in Candida albicans.

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 Mn- and 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.

Trehalose accumulation induced during the oxidative stress response is independent of TPS1 mRNA levels in Candida albicans.

Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex.

Trehalose accumulation induced during oxidative stress response is independent of TPS1 mRNA levels in Candida albicans / La acumulación de trehalosa inducida durante la respuesta al estrés oxidativo es independiente de los niveles de mRNA de TPS1 en Candida albicans

Growing cells of the Candida albicans trehalose-deficient mutant tps1/tps1 were extremely sensitive to severe oxidative stress exposure (H2O2). However, their viability was not affected after saline stress or heat-shock treatments, being roughly equivalent to that of the parental strain. In wild-type cells, these adverse conditions induced the intracellular accumulation of trehalose together with activation of trehalose-6P synthase, whereas the endogenous trehalose content and the corresponding biosynthetic activity were barely detectable in the tps1/tps1 mutant. The addition of cycloheximide did not prevent the marked induction of trehalose-6P synthase activity. Furthermore, the presence of H2O2 decreased the level of TPS1 mRNA expression. Hence, the conspicuous trehalose accumulation in response to oxidative stress is not induced by increased transcription of TPS1. Our results are consistent with a specific requirement of trehalose in order to withstand a severe oxidative stress in C. albicans, and suggest that trehalose accumulation observed under these conditions is a complex process that most probably involves post-translational modifications of the trehalose synthase complex (AU)

Células en crecimiento de Candida albicans tps1/tps1, mutantes deficientes en trehalosa, demostraron ser muy sensibles a un intenso estrés oxidativo (H2O2). No obstante, su viabilidad no se vio afectada tras un estrés salino ni por tratamientos de choque térmico, siendo equivalente a la de la cepa parental. Estas condiciones adversas inducen en las células salvajes la acumulación intracelular de trehalosa junto con la activación del enzima trehalosa-6P sintasa, mientras que en los mutantes tps1/tps1 el contenido de trehalosa y la activación de este enzima eran prácticamente indetectables. El añadido de cicloheximida no impedía una marcada inducción de la actividad trehalosa-6P sintasa. Asimismo, la presencia de H2O2 hacía disminuir el nivel de expresión de mRNA de TPS1. Por lo tanto, la acumulación abundante de trehalosa en respuesta al estrés oxidativo no es inducida por un aumento de la transcripción del gen TPS1. Nuestros resultados concuerdan con una demanda específica de trehalosa que necesita Candida albicans para resistir el estrés oxidativo severo, y sugieren que la acumulación de trehalosa observada en estas condiciones es un proceso complejo que muy probablemente implica modificaciones postraduccionales del sistema de trehalosa sintasa (AU)