RESUMEN
The enzymatic hydrolysis of the non-reducing disaccharide trehalose in yeasts is carried out by trehalase, a highly specific α-glucosidase. Two types of such trehalase activity are present in yeasts, and are referred to as neutral and acid enzymes. They are encoded by distinct genes (NTH1 and ATH1, respectively) and exhibit strong differences in their biochemical and physiological properties as well as different subcellular location and regulatory mechanisms. Whereas a single gene ATH1 codes for acid trehalase, the genome of some yeasts appears to predict the existence of a second redundant neutral trehalase, encoded by the NTH2 gene, a paralog of NTH1. In S. cerevisiae the corresponding two proteins share 77% amino acid identity, leading to the suggestion that NTH2 codes for a functional trehalase activity. However, Nth2p lacks any measurable neutral trehalase activity and disruption of NTH2 gene has no effect on this activity compared to a parental strain. Likewise, single nth1Δ and double nth1Δ/nth2Δ null mutants display no detectable neutral activity. Furthermore, disruption of NTH2 does not cause any apparent phenotype apart from a slight involvement in thermotolerance. To date, no evidence of a duplicated NTH gene has been recorded in other archetypical yeasts, like C. albicans or C. parapsilosis, and a possible regulatory mechanism of Nth2p remains unknown. Therefore, although genomic analysis points to the existence, in some yeasts, of two distinct genes encoding trehalase activities, the large body of biochemical and physiological evidence gathered from NTH2 gene does not support this proposal. Indeed, much more experimental evidence would be necessary to firmly validate this hypothesis.
RESUMEN
Fungi are eukaryotic organisms with relatively few pathogenic members dangerous for humans, usually acting as opportunistic infections. In the last decades, several life-threatening fungal infections have risen mostly associated with the worldwide extension of chronic diseases and immunosuppression. The available antifungal therapies cannot combat this challenge because the arsenal of compounds is scarce and displays low selective action, significant adverse effects, and increasing resistance. A growing isolation of outbreaks triggered by fungal species formerly considered innocuous is being recorded. From ancient times, natural substances harvested from plants have been applied to folk medicine and some of them recently emerged as promising antifungals. The most used are briefly revised herein. Combinations of chemotherapeutic drugs with natural products to obtain more efficient and gentle treatments are also revised. Nevertheless, considerable research work is still necessary before their clinical use can be generally accepted. Many natural products have a highly complex chemical composition, with the active principles still partially unknown. Here, we survey the field underlying lights and shadows of both groups. More studies involving clinical strains are necessary, but we illustrate this matter by discussing the potential clinical applications of combined carnosic acid plus propolis formulations.
RESUMEN
Fungi have traditionally been considered opportunistic pathogens in primary infections caused by virulent bacteria, protozoan, or viruses. Consequently, antimycotic chemotherapy is clearly less developed in comparison to its bacterial counterpart. Currently, the three main families of antifungals (polyenes, echinocandins, and azoles) are not sufficient to control the enormous increase in life-threatening fungal infections recorded in recent decades. Natural substances harvested from plants have traditionally been utilized as a successful alternative. After a wide screening of natural agents, we have recently obtained promising results with distinct formulations of carnosic acid and propolis on the prevalent fungal pathogens Candida albicans and Cryptococcus neoformans. Here, we extended their use to the treatment against the emerging pathogenic yeast Candida glabrata, which displayed lower susceptibility in comparison to the fungi mentioned above. Taking into account the moderate antifungal activity of both natural agents, the antifungal value of these combinations has been improved through the obtention of the hydroethanolic fractions of propolis. In addition, we have demonstrated the potential clinical application of new therapeutical designs based on sequential pre-treatments with carnosic/propolis mixtures, followed by exposure to amphotericin B. This approach increased the toxic effect induced by this polyene.
RESUMEN
In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.
Asunto(s)
Antifúngicos , Itraconazol , Antifúngicos/farmacología , Itraconazol/farmacología , Itraconazol/metabolismo , Candida parapsilosis/genética , Candida parapsilosis/metabolismo , Catalasa/genética , Catalasa/metabolismo , Catalasa/farmacología , Trehalasa/genética , Trehalasa/metabolismo , Trehalasa/farmacología , Rotenona/farmacología , Rotenona/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Estrés Oxidativo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/farmacología , Mitocondrias/metabolismo , Pruebas de Sensibilidad MicrobianaRESUMEN
Central metabolic pathways may play a major role in the virulence of pathogenic fungi. Here, we have investigated the susceptibility of a Candida parapsilosis mutant deficient in trehalase activity (atc1Δ/ntc1Δ strain) to the azolic compounds fluconazole and itraconazole. A time-course exposure to itraconazole but not fluconazole induced a significant degree of cell killing in mutant cells compared to the parental strain. Flow cytometry determinations indicated that itraconazole was able to induce a marked production of endogenous ROS together with a simultaneous increase in membrane potential, these effects being irrelevant after fluconazole addition. Furthermore, only itraconazole induced a significant synthesis of endogenous trehalose. The recorded impaired capacity of mutant cells to produce structured biofilms was further increased in the presence of both azoles, with itraconazole being more effective than fluconazole. Our results in the opportunistic pathogen yeast C. parapsilosis reinforce the study of trehalose metabolism as an attractive therapeutic target and allow extending the hypothesis that the generation of internal oxidative stress may be a component of the antifungal action exerted by the compounds currently available in medical practice.
RESUMEN
Current antifungal chemotherapy against the prevalent basidiomycete Cryptococcus neoformans displays some drawbacks. This pathogenic fungus is refractory to echinocandins, whereas conventional treatment with amphotericin B plus 5-fluorocytosine has a limited efficacy. In this study, we explored the potential cryptococcal activity of some natural agents. After conducting a screening test with a set of propolis from different geographical areas, we selected an extract from China, which displayed a certain cytotoxic activity against C. neoformans, due to this extract being cheap and easily available in large amounts. The combination of this kind of propolis with carnosic acid in a 1:4 ratio induced a stronger fungicidal effect, which occurred following a synergistic pattern, without visible alterations in external cell morphology. Furthermore, several carnosic acid-propolis formulations applied onto preformed biofilms decreased the metabolic activity of the sessile cells forming biofilms. These data support the potential application of mixtures containing these two natural extracts in the design of new antifungal strategies in order to combat opportunistic infections caused by prevalent pathogenic fungi.
RESUMEN
In fungi, the Mitogen-Activated Protein kinase (MAPK) pathways sense a wide variety of environmental stimuli, leading to cell adaptation and survival. The HOG pathway plays an essential role in the pathobiology of Candida albicans, including the colonization of the gastrointestinal tract in a mouse model, virulence, and response to stress. Here, we examined the role of Hog1 in the C. albicans response to the clinically relevant antifungal Micafungin (MF), whose minimum inhibitory concentration (MIC) was identical in the parental strain (RM100) and in the isogenic homozygous mutant hog1 (0.016 mg/L). The cell viability was impaired without significant differences between the parental strain, the isogenic hog1 mutant, and the Hog1+ reintegrant. This phenotype was quite similar in a collection of hog1 mutants constructed in a different C. albicans background. MF-treated cells failed to induce a relevant increase of both reactive oxygen species (ROS) formation and activation of the mitochondrial membrane potential in parental and hog1 cells. MF was also unable to trigger any significant activation of the genes coding for the antioxidant activities catalase (CAT1) and superoxide dismutase (SOD2), as well as on the corresponding enzymatic activities, whereas a clear induction was observed in the presence of Amphotericin B (AMB), introduced as a positive control of Hog1 signaling. Furthermore, Hog1 was not phosphorylated by the addition of MF, but, notably, this echinocandin caused Mkc1 phosphorylation. Our results strongly suggest that the toxic effect of MF on C. albicans cells is not mediated by the Hog1 MAPK and is independent of the generation of an internal oxidative stress in C. albicans.
RESUMEN
Scientific journals have played an essential role in the diffusion of research breakthroughs. For many years there was no competition between journals, but, in recent decades they have become categorized by a careful assessment of their published contents based on several metric parameters. Of greater note, the 'prestige index' has become an essential tool used by public and private institutions to develop their scientific policy. Thus, the evaluation of research staffs, the concession of grants or fellowships and even the scholarly reputation and academic positions are mainly founded on a given journal's 'quality'. As a consequence, the prestige of some journals has gone up, based on the assumption that they publish cutting-edge science, while the reputation of others has gone down. Within the field of Microbiology, we have carried out a direct analysis by monitoring several representative classic journals according to customary metric parameters over 20 years. This analysis also covers another set of journals of recent appearance (novel journals). Although a direct comparison between both groups is not possible, this approach serves to perceive the trends of publication among microbiologists. Our preliminary conclusion is that the continued existence of many so-termed classic journals devoted to Microbiology is seriously threatened.
Asunto(s)
Microbiología/tendencias , Publicaciones Seriadas/tendencias , Publicaciones Seriadas/estadística & datos numéricosRESUMEN
The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees' panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C. albicans strain caused a moderate degree of cell death at relatively high concentrations. However, the combination of both extracts, especially in a 1:4 ratio, induced a potent synergistic pattern, leading to a drastic reduction in cell survival even at much lower concentrations. The result of a mathematical analysis by isobologram was consistent with synergistic action of the combined extracts rather than a merely additive effect. In turn, the capacity of SC5314 cells to form in vitro biofilms was also impaired by the simultaneous presence of both agents, supporting the potential application of carnosic acid and propolis mixtures in the prevention and treatment of clinical infections as an alternative to antibiotics and other antifungal agents endowed with reduced toxic side effects.
RESUMEN
The HOG MAP kinase pathway plays a crucial role in the response to different stresses in the opportunistic pathogen Candida albicans. The polyene amphotericin B (AMB) has been reported to trigger oxidative stress in several pathogenic fungi, including C. albicans. In the present work, we have analyzed the role of the MAPK Hog1 in sensing and survival to AMB treatment. Mutants lacking Hog1 are more susceptible to AMB than their parental strains and Hog1 became phosphorylated in the presence of this polyene. A set of mutated versions of Hog1 revealed that both the kinase activity and phosphorylation of Hog1 are required to cope with AMB treatment. Flow cytometry analysis showed that AMB induced intracellular ROS accumulation in both parental and hog1 null mutant strains. In addition, AMB triggered a Hog1-independent synthesis of trehalose. The addition of rotenone to AMB-treated cells improved cell viability, decreased intracellular ROS and prevented intracellular trehalose accumulation, suggesting that AMB-induced ROS is associated to a functional electron transport chain but the presence of rotenone did not impair Hog1 phosphorylation in AMB-treated cells. Our results indicate that Hog1 is necessary during AMB treatment to increase its survival.
Asunto(s)
Anfotericina B/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Especies Reactivas de Oxígeno/metabolismo , Trehalosa/metabolismo , Antifúngicos/farmacología , Candida albicans/enzimología , Proteínas Fúngicas/genética , Mutación , Fosforilación/efectos de los fármacosRESUMEN
Purpose. Fungal infections have increased in recent decades, with Candida albicans being the fourth most common aetiological agent of nosocomial infections. Disaccharide trehalose has been proposed as a target for the development of new antifungals. In C. albicans we have examined the susceptibility shown by two mutants deficient in trehalose biosynthesis, namely tps1Δ and tps2Δ, to amphotericin B (AmB) and micafungin (MF).Methodology. Minimum inhibitory concentrations (MICs) were calculated according to the Clinical and Laboratory Standards Institute (CLSI) criteria. Cell viability was assessed by cell counting. Intracellular reactive oxygen species (ROS) and the mitochondrial membrane potential were measured by flow cytometry, while the trehalose content and biofilm formation were determined by enzymatic assays.Results. While the tps1Δ mutant was highly sensitive to AmB exposure, its resistance to MF was similar to that of the wild-type. Notably, the opposite phenotype was recorded in the tps2Δ mutant. In turn, MF induced a significant level of endogenous ROS production in the parental SC5314 and tps2Δ cells, whereas the ROS formation in tps1Δ cells was virtually undetectable. The level of endogenous ROS correlated positively with the rise in mitochondrial activity. Only AmB was able to promote intracellular synthesis of trehalose in the parental strain; it was absent from tps1Δ cells and showed low levels in tps2Δ, confirming the unspecific dephosphorylation of trehalose-6P in C. albicans. Furthermore, the capacity of both tps1Δ and tps2Δ mutants to form biofilms was drastically reduced after AmB exposure, whereas it increased in tps1Δ cells treated with MF.Conclusion. Our data lend weight to the idea of using trehalose biosynthesis as a potential target for antifungal therapy.
Asunto(s)
Anfotericina B/farmacología , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Proteínas Fúngicas/genética , Glucosiltransferasas/genética , Micafungina/farmacología , Trehalosa/biosíntesis , Biopelículas/efectos de los fármacos , Candida albicans/genética , Candida albicans/fisiología , Candidiasis/microbiología , Proteínas Fúngicas/metabolismo , Glucosiltransferasas/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Especies Reactivas de Oxígeno/metabolismo , Eliminación de SecuenciaRESUMEN
Micafungin belongs to the antifungal family of echinocandins, which act as noncompetitive inhibitors of the fungal cell wall ß-1,3-d-glucan synthase. Since Candida albicans is the most prevalent pathogenic fungus in humans, we study the involvement of micafungin in the modulation of the inflammatory response developed by human tissue macrophages against C. albicans The MIC for micafungin was 0.016 µg/ml on the C. albicans SC5314 standard strain. Micafungin induced a drastic reduction in the number of exponential SC5314 viable cells, with the fungicidal effect being dependent on the cellular metabolic activity. Notably, micafungin also caused a structural remodelling of the cell wall, leading to exposure of the ß-glucan and chitin content on the external surface. At the higher doses used (0.05 µg/ml), the antifungal also induced the blowing up of budding yeasts. In addition, preincubation with micafungin before exposure to human tissue macrophages enhanced the secretion of tumor necrosis factor alpha (TNF-α), interleukin-17A (IL-17A), and IL-10 cytokines. Our results strongly suggest that in C. albicans treatment with micafungin, in addition to having the expected toxic antifungal effect, it potentiates the immune response, improving the interaction and activation of human macrophages, probably through the unmasking of ß-glucans on the cell wall surface.
Asunto(s)
Antifúngicos/uso terapéutico , Candida albicans/efectos de los fármacos , Candida albicans/inmunología , Candidiasis/tratamiento farmacológico , Macrófagos/inmunología , Micafungina/uso terapéutico , Pared Celular/efectos de los fármacos , Glucosiltransferasas/antagonistas & inhibidores , Humanos , Inflamación/tratamiento farmacológico , Interleucina-10/metabolismo , Interleucina-17/metabolismo , Pruebas de Sensibilidad Microbiana , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The hypothetical role played by the intracellular formation of reactive oxygen species (ROS) in the fungicidal action carried out by Amphotericin B (AmB) and Micafungin (MF) was examined in Candida albicans, which remains the most prevalent fungal pathogen. The clinical MICs for MF and AmB were 0.016 and 0.12µg/ml, respectively. Whereas AmB (0.5-1.0×MIC) induced a marked production of intracellular ROS accompanied by a high degree of cell killing in the C. albicans SC5314 strain, the fungicidal effect of MF was still operative, but ROS generation was slight. Preincubation with thiourea suppressed the formation of ROS and caused a marked increase in cell viability, regardless of the antifungal used. Simultaneous measurement of several well established antioxidant enzymes (catalase, glutathione reductase and superoxide dismutase) revealed strong AmB-induced activation of the three enzymatic activities, whereas MF only had a weak stimulating effect. Likewise, AmB but not MF promoted a conspicuous rise in the mitochondrial membrane potential together with the intracellular synthesis of trehalose, the non-reducing disaccharide which acts as a specific protector against oxidative stress in C. albicans. Optical and electronic microscopy analysis revealed a significant damage to cell integrity and structural alterations caused by both antifungals. Taken together, our results strongly suggest that the induction of an internal oxidative stress in C. albicans through the accumulation of ROS is a preferential contributory factor to the antifungal action of a widely used polyene (AmB) but not of MF (echinocandin).
Asunto(s)
Anfotericina B/farmacología , Candida albicans/efectos de los fármacos , Equinocandinas/farmacología , Lipopéptidos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Antifúngicos/farmacología , Antioxidantes/farmacología , Candida albicans/metabolismo , Micafungina , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo/efectos de los fármacosRESUMEN
A double homozygous atc1Δ/atc1Δ/ntc1Δ/ntc1Δ mutant (atc1Δ/ntc1Δ KO) was constructed in the pathogen opportunistic yeast Candida parapsilosis by disruption of the two chromosomal alleles coding for NTC1 gene (encoding a neutral trehalase) in a Cpatc1Δ/atc1Δ background (atc1Δ KO strain, deficient in acid trehalase). The Cpatc1Δ/ntc1Δ KO mutant failed to counteract the inability of Cpatc1Δ cells to metabolize exogenous trehalose and showed a similar growth pattern on several monosaccharides and disaccharides. However, upon prolonged incubation in either rich medium (YPD) or nutrient-starved medium the viability of Cpatc1Δ cells exhibited a sensitive phenotype, which was augmented by further CpNTC1/NTC1 disruption. Furthermore, Cpatc1Δ/ntc1Δ KO cells had difficulty in resuming active growth in fresh YPD. This homozygous mutant also lacked any in vitro measurable trehalase activity, whether acid or neutral, suggesting that a single gene codes for each enzyme. By contrast, in Cpatc1Δ/ntc1Δ KO strain the resistance to oxidative and heat stress displayed by atc1Δ mutant was suppressed. Cpatc1Δ/ntc1Δ KO cells showed a significant decrease in virulence as well as in the capacity to form biofilms. These results point to a major role for acid trehalase (Atc1p) in the pathobiology of C. parapsilosis, whereas the activity of neutral trehalase can only partially counteract Atc1p deficiency. They also support the use of ATC1 and NTC1 genes as interesting antifungal targets.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Candida/genética , Proteínas Fúngicas/genética , Trehalasa/metabolismo , Candida/crecimiento & desarrollo , Candida/fisiología , Metabolismo de los Hidratos de Carbono , Proteínas Fúngicas/metabolismo , Estrés Oxidativo , Eliminación de Secuencia , Estrés Fisiológico , Trehalasa/genética , Trehalosa/metabolismo , VirulenciaRESUMEN
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.
Asunto(s)
Anfotericina B/farmacología , Antifúngicos/farmacología , Candida/efectos de los fármacos , Cryptococcus/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Candida/genética , Candidiasis/tratamiento farmacológico , Candidiasis/genética , Candidiasis/microbiología , Catalasa/metabolismo , Membrana Celular/efectos de los fármacos , Criptococosis/tratamiento farmacológico , Criptococosis/microbiología , Farmacorresistencia Fúngica , Transporte de Electrón/efectos de los fármacos , Ergosterol/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estrés Oxidativo/efectos de los fármacos , Consumo de Oxígeno/efectos de los fármacos , Rotenona/farmacología , Desacopladores/farmacologíaRESUMEN
An ORF named CPAR2-208980 on contig 005809 was identified by screening a Candida parapsilosis genome data base. Its 67% identity with the acid trehalase sequence from C. albicans (ATC1) led us to designate it CpATC1. Homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two CpATC1 chromosomal alleles. Phenotypic characterization showed that atc1Δ null cells were unable to grow on exogenous trehalose as carbon source, and also displayed higher resistance to environmental challenges, such as saline exposure (1.2 M NaCl), heat shock (42°C) and both mild and severe oxidative stress (5 and 50 mM H2O2). Significant amounts of intracellular trehalose were specifically stored in response to the thermal upshift in both wild type and mutant strains. Analysis of their antioxidant activities revealed that catalase was only triggered in response to heat shock in atc1Δ cells, whereas glutathione reductase was activated upon mild oxidative stress in wild type and reintegrant strains, and in response to the whole set of stress treatments in the homozygous mutant. Furthermore, yeast cells with double CpATC1 deletion were significantly attenuated in non-mammalian infection models, suggesting that CpATC1 is required for the pathobiology of the fungus. Our results demonstrate the involvement of CpAtc1 protein in the physiological hydrolysis of external trehalose in C. parapsilosis, where it also plays a major role in stress resistance and virulence.
Asunto(s)
Candida/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Respuesta al Choque Térmico/genética , Tolerancia a la Sal/genética , Trehalasa/metabolismo , Secuencia de Aminoácidos , Candida/metabolismo , Candida/patogenicidad , Candida/fisiología , Catalasa/metabolismo , Proteínas Fúngicas/genética , Datos de Secuencia Molecular , Estrés Oxidativo , Proteolisis , Trehalasa/genética , Trehalosa/metabolismo , Virulencia/genéticaRESUMEN
Validamycin A has been successfully applied in the fight against phytopathogenic fungi. Here, the putative antifungal effect of this pseudooligosaccharide against the prevalent human pathogen Candida albicans was examined. Validamycin A acts as a potent competitive inhibitor of the cell-wall-linked acid trehalase (Atc1p). The estimated MIC50 for the C. albicans parental strain CEY.1 was 500 mg/l. The addition of doses below MIC50 to exponentially growing CEY.1 cells caused a slight reduction in cell growth. A concentration of 1 mg/ml was required to achieve a significant degree of cell killing. The compound was stable as evidenced by the increased reduction of cell growth with increasing incubation time. A homozygous atc1delta/atc1delta mutant lacking functional Atc1p activity showed greater resistance to the drug. The antifungal power of validamycin A was limited compared with the drastic lethal action caused by exposure to amphotericin B. The endogenous content of trehalose rose significantly upon validamycin and amphotericin B addition. Neither serum-induced hypha formation nor the level of myceliation recorded in macroscopic colonies were affected by exposure to validamycin A. Our results suggest that, although validamycin A cannot be considered a clinically useful antifungal against C. albicans, its mechanism of action and antifungal properties provide the basis for designing new, clinically interesting, antifungal-related compounds.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Inositol/análogos & derivados , Candida albicans/enzimología , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Candidiasis/microbiología , Pared Celular/efectos de los fármacos , Pared Celular/enzimología , Pared Celular/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Inositol/farmacología , Pruebas de Sensibilidad Microbiana , Trehalasa/genética , Trehalasa/metabolismoRESUMEN
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.
Asunto(s)
Candida albicans/metabolismo , Glicerol/metabolismo , Estrés Oxidativo , Alcoholes del Azúcar/metabolismo , Trehalosa/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Presión Osmótica , Oxidación-Reducción , Trehalosa/biosíntesisRESUMEN
The putative candicidal activity of resveratrol is currently a matter of controversy. Here, the antifungal activity as well as the antioxidant response of resveratrol against Candida albicans, have been tested in a set of strains with a well-established genetic background At the doses usually employed in antifungal tests (10-40 µg/ml), resveratrol has no effect on the exponential growth of the C. albicans CAI.4 strain, a tenfold increase (400 µg/ml) was required in order to record a certain degree of cell killing, which was negligible in comparison with the strong antifungal effect caused by the addition of amphotericin B (5 µg/ml). An identical pattern was recorded in the prototrophic strains of C. albicans SC5314 and RM-100, whereas the oxidative sensitive trehalose-deficient mutant (tps1/tps1 strain) was totally refractory to the presence of resveratrol. In turn, the serum-induced yeast-to-hypha transition remained unaffected upon addition of different concentrations of resveratrol. Determination of endogenous trehalose and catalase activity, two antioxidant markers in C. albicans; revealed no significant changes in their basal contents induced by resveratrol. Collectively, our results seem to dismiss a main antifungal role as well as the therapeutic application of resveratrol against the infections caused by C. albicans.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Estilbenos/farmacología , ResveratrolRESUMEN
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.