Analysis of validamycin as a potential antifungal compound against Candida albicans

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 atc1Δ/atc1Δ 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. [Int Microbiol 2013; 16(4):217-225]Keywords: Candida albicans · Rhizoctonia solani · validamycin A · amphotericin B · trehalos

Caracterización del gen ATC1 de Candida parapsilosis : clonación, estudio fenotípico e interacción con el sistema inmunitario.

Candida parapsilosis is the second most frequently isolated pathogenic yeast in blood from SouthAmerica, Asia and Europe. The enzymes involved in the metabolism of trehalose, have been proposed as an antifungal target because this disaccharide acts against various types of stress in fungi but it is not present in mammals. Here, we have cloned the gene encoding acid trehalase (ATC1) in C. parapsilosis and performed the phenotypic analysis of the null mutants. We studied the interaction of the obtained strains with the immune system. The homozygous mutant is unable to grow on trehalose and displays greater resistance to thermal, saline and oxidative stress in vitro. The ATC1 gene disruption increased nitric oxide, TNF-α and IL-10 cytokine production by human macrophages, and decreased cell survival to macrophage killing. These results are consistent with those obtained in vivo, where mutants also showed lower invasiveness in murine model and reduced virulence in Galleria mellonella. Keywords: Candida parapsilosis, ATC1 gene, virulence, Cytokine, trehalose. RESUMEN Candida parapsilosis es la segunda levadura patógena aislada en hemocultivos de Hispanoamérica latina, Asia y Europa. Las enzimas implicadas en el metabolismo de trehalosa pueden servir como dianas antifungicas, ya que dicho disacárido actúa frente a varios tipos de estrés en hongos y además no se encuentra presente en mamíferos. Hemos clonado el gen que codifica para la trehalasa ácida de C. parapsilosis, realizando un análisis fenotípico del mutante nulo y de su interacción con el sistema inmunitario. El mutante homocigótico es incapaz de crecer con trehalosa y presenta elevada resistencia al estrés térmico, salino y oxidativo in vitro. La interrupción del gen ATC1 incrementa los niveles de oxido nítrico y la producción de las citoquinas; TNF-α y IL-10, mientras reduce la supervivencia celular a lisis en macrófagos humanos. Estos resultados coinciden con los obtenidos in vivo, donde los mutantes presentan una menor invasividad en modelo de ratón y pérdida de virulencia en Galleria mellonella. Palabras clave: Candida parapsilosis, gen ATC1, virulencia, citoquinas, trehalosa

Natural Substances as Valuable Alternative for Improving Conventional Antifungal Chemotherapy: Lights and Shadows

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

The Enigma of <i>NTH2</i> Gene in Yeasts

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

Análisis comparativo del efecto de la Validamicina A y la Anfotericina B sobre Candida albicans

RESUMEN La levadura dimórfica Candida albicans es el hongo patógeno oportunista con mayor prevalencia en humanos. Actualmente, los mayores problemas en la práctica clínica provienen del incremento de candidiasis sistémicas nosocomiales, la baja toxicidad selectiva de los antifúngicos disponibles y el aislamiento creciente de cepas resistentes. Todos estos datos hacen necesaria la búsqueda de nuevos blancos antifúngicos. Las rutas implicadas en el metabolismo de la trehalosa se han considerado dianas potenciales para el desarrollo de nuevos compuestos antifúngicos. La validamicina A es un inhibidor competitivo de de la actividad trehalasa y se ha aplicado con éxito para controlar el añublo de la vaina de arroz producida por el hongo fitopatógeno Rhizoctonia solani en Japón y China. Sin embargo, la acción de la validamicina A contra hongos patógenos en humanos no ha sido estudiada. Nuestros datos sugieren que bajas concentraciones de validamicina A (0,1 mg/ml) causan una pérdida parcial de la viabilidad celular en la cepa parental (CAI-4). Es necesario aplicar dosis elevadas, (1 mg/ml) de validamicina A, para lograr un grado significativo de muerte celular. El mutante congénico atc1Δ/atc1Δ carente de actividad trehalasa ácida (Atc1p) funcional fue menos sensible a la presencia de validamicina A. Además, la validamicina A actúa como un potente inhibidor competitivo de esta actividad Atc1p localizada en la pared celular así como de la trehalasa citosólica (Ntc1p). En general, la validamicina A actúa como un compuesto antifúngico débil contra Candida albicans, pero efectivo como inhibidor de trehalasas.ABSTRACT The opportunistic dimorphic yeast Candida albicans is the most prevalent infectious fungus in humans. At present, the most dangerous problems faced in clinical practice are the dramatic increase of nosocomial bloodstream candidiasis, the low selective toxicity of available antifungal therapies and the increase resistant yeast strains. All these data make necessary the search for new antifungal targets. The pathways involved in trehalose metabolism have been proposed as potentially interesting targets for the development of new antibiotics. Validamicin A is a competitive inhibitor of trehalase activity and has been successfully applied in the fight against rice sheath blight caused by the phytopathogen fungus Rhizoctonia solani in Japan and China. However, the action of validamycin A against fungal human pathogens had never been examined so far. Our data suggest that a concentration of 0.1 mg/ml of validamycin A caused a partial loss of cell viability on the parental strain (CAI-4). Higher concentrations (1 mg/ml) of Validamycin A were required in order to achieve a significant degree of cell killing. A congenic atc1Δ/atc1Δ mutant lacking functional Atc1p activity was less sensitive to the presence of validamycin A. In addition, validamycin A acts as a potent competitive inhibitor on the cell-wall linked trehalase (Atc1p) as well as on the cytosolic trehalase (Ntc1p). Collectively, validamycin A behaves as a weak antifungal against Candida albicans, but is an effective compound as inhibitor of trehalases

A Specific Mixture of Propolis and Carnosic Acid Triggers a Strong Fungicidal Action against Cryptococcus neoformans

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

Novel Bi-Factorial Strategy against Candida albicans Viability Using Carnosic Acid and Propolis: Synergistic Antifungal Action

The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees&rsquo; 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

Intracellular content of trehalose following different stress treatments in exponential phase cultures of the parental strain (WT) and its congenic mutant deficient in acid trehalase (KO).

<p>Yeast cells were grown at 30°C in YPD until they reached exponential phase (OD₆₀₀  =  1.0–1.2). The samples were prepared and the trehalose content was measured as described in Methods. The results are the mean ± SD of one representative experiment of two performed in triplicate. The distinction between the treated samples and control values obtained was significant at **P<0.01 and ***P<0.001 according to the Student t-test.</p

Phenotypic analysis of the <i>C. parapsilosis</i> strains.

<p>The growth cycle at 30°C of wild type (WT), heterozygous (HET) and homozygous (KO) and reintegrant (RE) yeast strains was monitored in YPD (<b>A</b>) or YPtrehalose (<b>B</b>). To check the ability to use different carbon sources, cells from the WT and KO strains were cultured in liquid minimal medium (MM) supplemented with: glucose (<b>C</b>), sucrose (<b>D</b>), trehalose (<b>E</b>) and lactose (<b>F</b>) at 30°C for 6 hours; or in solid MM medium supplemented with glucose (<b>G</b>) or trehalose (<b>H</b>) for 24h. The susceptibility to compounds that affect the cell wall architecture (<b>I</b>) was examined by spotting approximately 10⁵ cells and 10-fold dilutions thereof, on YPD plates containing the indicated compounds at the following concentrations: SDS (0.02%, w/v); Calcofluor White (60 µg/ml); Congo Red (100 µg/ml) and Caffeine (50 mM). The plates were incubated at 30°C for 48 h and photographed. Growth in liquid medium was measured by cell density at OD₆₀₀. Results are expressed as mean ± standard deviation of one representative experiment of two performed in triplicate. Growth in solid medium was monitored by visual inspection of plates performed in duplicate and repeated twice with similar results.</p

Level of cell survival after different stress treatments in <i>C. parapsilosis</i> strains.

<p>YPD-grown cultures of exponential <i>C. parapsilosis</i> wild type (WT), its isogenic mutant (KO), deficient in <i>CpATC1</i> gene, and the reintegrant (RE) strains were adjusted to a cell density of 1.0×10⁶ cells/ml and subjected to the following stress challenges for 1h: 5 mM H₂0₂, 50 mM H₂0₂, 1.2 M NaCl or 42°C. Identical, untreated samples were maintained at 30°C as a control. Results are expressed as mean ± standard deviation of one representative experiment of two performed in triplicate. Student t-test: *P<0.05; **P<0.01 between WT and RE or KO strains.</p