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. [Int Microbiol 2015; 18(1):25-31]Keywords: Candida albicans · amphotericin B · 5-fluorocytosine · germ-tube · cell killin

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

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different () strains isolated from a wide variety of cheeses were identified as producing killer toxins active against and . We have analyzed the killer activity of these toxins in mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of gene showed that both activities were relevant for the survival of to killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing 242 strain (-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to -242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains

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

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’ 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

Image_2_Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.TIF

<p>Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.</p

Image_4_Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.TIF

<p>Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.</p

Image_1_Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.TIF

<p>Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.</p

Image_3_Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.TIF

<p>Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.</p

Image_5_Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.TIF

<p>Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.</p