Novel physiological roles for glutathione in sequestering acetaldehyde to confer acetaldehyde tolerance in Saccharomyces cerevisiae.

In this work, we identified novel physiological functions of glutathione in acetaldehyde tolerance in Saccharomyces cerevisiae. Strains deleted in the genes encoding the enzymes involved in glutathione synthesis and reduction, GSH1, GSH2 and GLR1, exhibited severe growth defects compared to wild-type under acetaldehyde stress, although strains deleted in the genes encoding glutathione peroxidases or glutathione transferases did not show any growth defects. On the other hand, intracellular levels of reduced glutathione decreased in the presence of acetaldehyde in response to acetaldehyde concentration. Moreover, we show that glutathione can trap a maximum of four acetaldehyde molecules within its molecule in a non-enzymatic manner. Taken together, these findings suggest that glutathione has an important role in acetaldehyde tolerance, as a direct scavenger of acetaldehyde in the cell.

Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders.

The fungicide fludioxonil antagonizes fluconazole activity in the human fungal pathogen Candida albicans.

The fungicide fludioxonil is widely used in agriculture. Residua of this fungicide are occasionally detected in fruits and can therefore be ingested by humans. The human fungal pathogen Candida albicans expresses the target of fludioxonil, Nik1p, a type III histidine kinase involved in stress response. Inhibition of yeast and hyphae growth was hardly observable after treatment of C. albicans SC5314 with fludioxonil. As a side effect, however, we observed a concentration-dependent induction of the expression of the genes CDR1 and CDR2, encoding ATP-binding cassette (ABC) transporters. This was independent of the presence of the target of fludioxonil as induction was also observed in a Δnik1 deletion mutant. Deletion of the CDR1 gene aggravated the inhibition of germ tube formation by fludioxonil, indicating that, in the wild-type, the fungicide was discharged from the cell by Cdr1p. Cdr1p is also known as a resistance factor of C. albicans against the commonly used antimycotic fluconazole. Thus, the effect of concurrent exposure to fludioxonil and known cargoes of ABC transporters on their extrusion and the growth of C. albicans was examined. Pre-incubation with fludioxonil decreased the export rate of rhodamine 6G. The resistance to fluconazole was increased by fludioxonil, independently of Nik1p. Therefore, exposure of C. albicans to fludioxonil may lead to increased resistance to fluconazole treatment.

Fungicidal activity of lysozyme is inhibited in vitro by commercial sinus irrigation solutions.

BACKGROUND: Lysozyme is an innate immune peptide with bactericidal and fungicidal activity (FA). Despite increased expression of lysozyme protein in chronic rhinosinusitis (CRS) sinus mucosa, CRS patients experience repeated bacterial and/or fungal infections. Commercial sinus irrigation solutions are often used to provide symptomatic relief. However, one of the mechanisms of action of lysozyme involves ionic interactions with the microbial cell wall, which may be inhibited by ionic solutions such as commercial sinus irrigation solutions. OBJECTIVE: Determine if the FA of lysozyme is reduced in the presence of solutions with increasing ionic strength and inhibited in the presence of commercial sinus irrigation solutions. METHODS: Using an in vitro colony-forming unit (CFU) assay, the FA of lysozyme (5 µM) was tested against a fungi commonly isolated from CRS patients, Aspergillus fumigatus, in solutions of increasing ionic strength or commercial sinus irrigation solutions. FA was presented as percent of control. RESULTS: FA of lysozyme against A. fumigatus was 95% in a 21-mM ionic strength solution. However, with increasing ionic strength, FA decreased and was abolished in a 46-mM ionic strength solution. Commercial sinus irrigation solutions abolished the FA of lysozyme against A. fumigatus. CONCLUSION: The in vitro FA of lysozyme is dependent on the ionic strength of the solution. The use of sinus irrigation solutions should be further evaluated with regard to maintaining functional activity of cationic antimicrobial peptides involved in sinonasal innate immunity.

Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)3 was also detected and direct transport assays demonstrate that As(GS)3 does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.

Triclosan antagonizes fluconazole activity against Candida albicans.

Triclosan is a broad-spectrum antimicrobial compound commonly used in oral hygiene products. Investigation of its activity against Candida albicans showed that triclosan was fungicidal at concentrations of 16 mg/L. However, at subinhibitory concentrations (0.5-2 mg/L), triclosan antagonized the activity of fluconazole. Although triclosan induced CDR1 expression in C. albicans, antagonism was still observed in cdr1Δ and cdr2Δ strains. Triclosan did not affect fluconazole uptake or alter total membrane sterol content, but did induce the expression of FAS1 and FAS2, indicating that its mode of action may involve inhibition of fatty acid synthesis, as it does in prokaryotes. However, FAS2 mutants did not exhibit increased susceptibility to triclosan, and overexpression of both FAS1 and FAS2 alleles did not alter triclosan susceptibility. Unexpectedly, the antagonistic effect was specific for C. albicans under hypha-inducing conditions and was absent in the non-filamentous efg1Δ strain. This antagonism may be due to the membranotropic activity of triclosan and the unique composition of hyphal membranes.

Efg1 Controls caspofungin-induced cell aggregation of Candida albicans through the adhesin Als1.

Echinocandin drugs such as caspofungin (CASP), micafungin, and anidulafungin inhibit fungal cell wall biogenesis by blocking Fks1-mediated ß-glucan deposition into the cell surface. Candins have become suitable drugs to treat life-threatening diseases caused by several fungal species, including Candida albicans, that are pathogenic for humans. Here, we present the discovery of a novel CASP-induced flocculation phenotype of C. albicans, which formed large cell aggregates in the presence of CASP. High concentrations of sugars such as mannose or glucose inhibit CASP-induced flocculation and improve survival of C. albicans cells exposed to CASP. Notably, exposure of C. albicans cells to CASP triggers Efg1-dependent expression of the adhesin ALS1 and induces invasive growth on agar plates. Indeed, cells lacking either Efg1 or Als1 show strongly diminished CASP-induced flocculation, and the absence of Efg1 leads to marked CASP hypersensitivity. On the other hand, CASP-induced invasive growth is enhanced in cells lacking Efg1. Hence, CASP stress drives an Efg1-dependent response, indicating that this multifunctional transcriptional regulator, which is otherwise involved in filamentation, white-to-opaque switching, and virulence, also modulates cell wall remodeling upon CASP challenge. Taken together, our data suggest that CASP-induced cell wall damage activates Efg1 in parallel with the known cell integrity stress signaling pathway to coordinate cell wall remodeling.

Subterranean termite prophylactic secretions and external antifungal defenses.

Termites exploit environments that make them susceptible to infection and rapid disease transmission. Gram-negative bacteria binding proteins (GNBPs) signal the presence of microbes and in some insects directly damage fungal pathogens with ß-1,3-glucanase activity. The subterranean termites Reticulitermes flavipes and Reticulitermes virginicus encounter soil entomopathogenic fungi such as Metarhizium anisopliae, which can evade host immune responses after penetrating the cuticle. An external defense that prevents invasion of fungal pathogens could be crucial in termites, allowing them to thrive under high pathogenic pressures. We investigated the role of secreted ß-1,3-glucanases in Reticulitermes defenses against M. anisopliae. Our results show that these termites secrete antifungal ß-1,3-glucanases on the cuticle, and the specific inhibition of GNBP associated ß-1,3-glucanase activity with d-δ-gluconolactone (GDL) reduces this activity and can cause significant increases in mortality after exposure to M. anisopliae. Secreted ß-1,3-glucanases appear to be essential in preventing infection by breaking down fungi externally.

Molecular determinants of transient and reversible induced up-regulation of CaCDR1 in azole susceptible clinical isolates of Candida albicans.

The present study examines the molecular mechanism underlying in vitro-induced resistance to FLC (fluconazole), KTC (ketaonazole), MCZ (miconazole) and CHX (cycloheximide) in AS (azole-susceptible) strains of Candida albicans when exposed to CaCDR1/CaCDR2 inducers like FPZ (fluphenazine) and steroids [PRG (progesterone) and ß-EST (ß-oestradiol)]. By employing spot and checkerboard titre assays, we provide evidence of an in vitro-induced antagonism between tested drugs and inducers, which was accompanied with a concomitant increase in CaCDR1 and CaCDR2 transcript levels. Notably, unlike AS isolates, parental WT (wild-type) and Δcdr2 null strains, Δcdr1 as well as Δcdr1/Δcdr2 nulls, when challenged with the inducers could not display antagonism. Our results validated by Northern blotting, reporter gene transcription and TRO (transcription run on) assays show that in vitro-induced antagonism between tested drugs and inducer in AS isolates was mainly due to a transient and reversible transcriptional activation of CaCDR1. Notwithstanding our earlier observation that consistent high transcript levels of CaCDR1 in clinical AR (azole-resistant) isolates were maintained due to the combination of its transcriptional activation and enhanced mRNA stability via elongated poly(A) tails, this study shows that transient and reversible transcriptional activation of CaCDR1 was the major determinant of induced antagonism in AS isolates. The distinct strategies between sustained (in AR isolates) and transiently induced resistance mechanisms (in AS isolates) adopted by Candida should become useful in improving therapeutic approaches.

Anti-Candida-biofilm activity of micafungin is attenuated by voriconazole but restored by pharmacological inhibition of Hsp90-related stress responses.

We have conducted an in vitro evaluation of the efficacy of a voriconazole-micafungin combination against Candida albicans. When used alone, both micafungin and voriconazole decreased the metabolic activity of planktonic cells, but only micafungin displayed potent anti-biofilm activity. Their combination appeared to have an additive effect against planktonic cells. However, voriconazole significantly antagonized the fungicidal effect of micafungin against Candida biofilms. Time-lag experiments showed that pre-treatment with voriconazole induced resistance to micafungin in Candida biofilms. The micafungin-antagonizing effect of voriconazole persisted even when the biofilm was no longer exposed to voriconazole. In contrast, voriconazole addition after 24 h of micafungin treatment did not alter micafungin sensitivity. To investigate the mechanism of antagonism, we used inhibitors of Hsp90 and its effectors because Hsp90 seems to be implicated in the resistance to micafungin. These molecules reversed the voriconazole-induced resistance to micafungin which suggests that Hsp90-related stress responses are involved in the antagonism. Our results may provide clues as to the mechanism of increased drug resistance in Candida biofilms and raises concerns about the use of the voriconazole-micafungin combination in clinical settings.

Differential toxicity of antifungal protein AFP against mutants of Fusarium oxysporum.

Antifungal protein (AFP) from Aspergillus giganteus was assayed for toxicity against the Fusarium oxysporum wild-type strain and mutants in genes involved in cell signaling (DeltapacC, pacCc Deltafmk1) or cell-wall biogenesis (DeltachsV, Deltachs7, Deltagas1). The mutants were classified into two groups according to their sensitivity to AFP: DeltapacC, Deltagas1 and Deltachs7, which were significantly more resistant to AFP than the wild-type, and pacCC, Deltafmk1 and DeltachsV, which were more sensitive. Western blot analysis revealed increased binding of AFP to the three resistant mutants, DeltapacC, Deltagas1 and Deltachs7, but also to DeltachsV, indicating that differential binding may not be a key determinant for sensitivity. Addition of Ca2+ or K+ dramatically reduced antifungal activity and binding of AFP, suggesting that these cations compete for the same targets as AFP at the surface of the fungal cell.

Candidosis en pacientes HIV o con otras inmunodeficiencias secundarias en hospitales de la ciudad de Tucumán (Argentina) / Candidosis in HIV patients or with other secondary immunodeficiencies detected in hospitals of the city of Tucumán (Argentina)

Con el objeto de conocer las especies causantes de candidosis humanas en pacientes HIV positivos o con otras inmunodeficiencias secundarias y la incidencia de especies con capacidad de resistencia a antifúngicos, se estudiaron 76 aislamientos de Candida procedentes de 61 casos de candidosis superficiales y profundas de niños y adultos. Obtenidas desde piel, anexos, mucosas, abscesos, catéteres y secreciones diversas, entre otras. La identificación de las especies fue realizada por estudios de características morfológicas, cromogénicas y bioquímicas (CHROMagar , Candifast, API 20 y API 32). Los resultados revelan predominio de especies noalbicans (52.7 por ciento), obteniéndose las siguientes frecuencias de aislamientos: C.albicans (47,3 por ciento), C. parapsilosis: 15,8 por ciento, C. glabrata: 13,2 por ciento, C. krusei: 11,8 por ciento, C. tropicalis: 10,6 por ciento y C. dubliniensis: 1,3 por ciento. Algunas de ellas pueden presentar resistencia primaria o secundaria a algunos antifúngicos de uso habitual, por lo cual es necesario incluir estudios de sensibilidad a estos, para una mejor conducta terapéutica.

In order to find out species causing human candidosis in positive HIV patients or in individuals suffering from other secondary immunodeficiencies and the incidence of species bearing a resistance ability to antifungal agents, 76 Candida isolations obtained from 61 cases of superficial and deep candidosis in children and adults were studied. Samples were collected from skin, annexa, mucosities, abscesses, catheters and diverse secretions, among others. The identification of species was carried out through studies on morphological, chromogenic and biochemical characteristics (CHROMagar, Candifast, API 20 and API 32). Results reveal a predominance of non-albican species (52,7 percent), and the following frequencies of isolation: C.albicans (47.3 percent), C. parapsilosis: 15.8 percent, C.glabrata: 13.2 percent, C. krusei: 11.8 percent, C. tropicalis: 10.6 percent and C. dubliniensis: 1.3 percent. Some of them may exhibit some primary or secondary resistance to certain antifungal agents of common use, this is why it is necessary to include studies on sensitivity of them so as to attain a better therapeutical behaviour.

A new in-vitro kinetic model to study the pharmacodynamics of antifungal agents: inhibition of the fungicidal activity of amphotericin B against Candida albicans by voriconazole.

The aim of this study was to develop and validate a new in-vitro kinetic model for the combination of two drugs with different half-lives, and to use this model for the study of the pharmacodynamic effects of amphotericin B and voriconazole, alone or in combination, against a strain of Candida albicans. Bolus doses of voriconazole and amphotericin B were administered to a starting inoculum of C. albicans. Antifungal-containing medium was eliminated and replaced by fresh medium using a peristaltic pump, with the flow-rate adjusted to obtain the desired half-life of the drug with the shorter half-life. A computer-controlled dosing pump compensated for the agent with the longer half-life. Voriconazole and amphotericin B half-lives were set to 6 and 24 h, respectively. Pharmacokinetic parameters were close to target values when both single doses and sequential doses were simulated. Voriconazole and amphotericin B administered alone demonstrated fungistatic and fungicidal activity, respectively. Simultaneous administration resulted in fungicidal activity, whereas pre-exposure of C. albicans to voriconazole, followed by amphotericin at 8 and 32 h, resulted in fungistatic activity similar to that observed with voriconazole alone. Using this model, which allowed a combination of antifungal agents with different half-lives, it was possible to demonstrate an antagonistic effect of voriconazole on the fungicidal activity of amphotericin B. The characteristics and clinical relevance of this interaction require further investigation.

Triazole-polyene antagonism in experimental invasive pulmonary aspergillosis: in vitro and in vivo correlation.

Combination antifungal therapy is increasingly used in the treatment of invasive aspergillosis. Whether the interaction between amphotericin B and triazoles is antagonistic against invasive aspergillosis is a controversial issue that is not likely to be resolved through a randomized clinical trial. Here, we found both in vitro and in vivo antagonism between liposomal amphotericin B and ravuconazole in simultaneous treatment of experimental invasive pulmonary aspergillosis in persistently neutropenic rabbits. Bliss independence-based drug-interaction modeling showed significant antagonism in vitro and in vivo, with the observed drug effects being 20%-69% lower than would be expected if the drugs were acting independently. These in vitro and in vivo findings of antagonism were consistent with the findings from Loewe additivity-based drug-interaction modeling. No pharmacokinetic interaction was found. The combination of a triazole and polyene may be antagonistic in the treatment of invasive pulmonary aspergillosis.

Specific antibody to Cryptococcus neoformans glucurunoxylomannan antagonizes antifungal drug action against cryptococcal biofilms in vitro.

The fungus Cryptococcus neoformans possesses a polysaccharide capsule and can form biofilms on medical devices. We investigated the efficacy that the combination of a specific antibody to the capsular polysaccharide and antifungal therapy has against cryptococcal biofilms. The antibody enhanced the susceptibility of planktonic cells to antifungal agents, but an antagonistic effect was observed for combination therapy against C. neoformans biofilms. Our findings suggest that antibody therapies for infectious diseases that involve biofilms may antagonize certain antimicrobial therapies, and they also imply that products of the immune response may contribute to drug resistance of biofilms formed in vivo.

Interaction of local anaesthetics with other antifungal agents against pathogenic Aspergillus.

Aspergillus spp. are responsible for an increasing number of fungal infections in immunocompromised and transplant patients. Local anaesthetics (LAs) are growth inhibitors of bacteria and yeasts. Subinhibitory concentrations of the LAs lidocaine and bupivacaine blocked the germination of Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger whilst also showing a positive interaction in vitro with the antifungal activity of amphotericin B, itraconazole and caspofungin and a negative interaction with voriconazole. At higher concentrations, both LAs present fungicidal activity against resting conidia owing to cell membrane lesions. Verapamil, nifedipine and lanthanum produced a similar inhibitory effect on conidia germination. Calcium chloride reverted the inhibitory effect of verapamil and LAs. This study highlights that drug interactions may affect the clinical efficacy of antifungals, either promoting or limiting their action.

Purification and characterization of a chitinase-like antifungal protein from black turtle bean with stimulatory effect on nitric oxide production by macrophages.

An antifungal protein was isolated from black turtle bean, Phaseolus vulgaris cv. "Black Turtle". The purified protein displayed an N-terminal amino acid sequence with 60-80% homology to chitinases. The isolated protein specifically inhibited two species of fungi, namely Fusarium oxysporum and Mycosphaerella arachidicola , among several phytopathogenic fungi tested. Its antifungal activity was retained after incubation at 60 degrees C for 15 minutes, diminished after exposure to 80 degrees C, and was completely abrogated after treatment at 100 degrees C. The antifungal protein had higher thermostability than the antifungal protein mollisin from chestnut. It stimulated nitric oxide production by murine peritoneal macrophages, but exerted neither mitogenic nor cytotoxic activity on murine splenocytes, activities that may be related to allergic responses.

[Clinical use of the interaction between cyclosporine A and its inhibitors]. / Kliniczne zastosowanie interakcji pomiedzy cyklosporyna A i lekami hamujacymi jej metabolizm.

This article concerns cyclosporine A and cyclosporine--sparing agents. The drugs are evaluated due to their clinical usefulness in elevating cyclosporine blood level and their safety in long-term administration. We analyzed imidazole antifungal agents, calcium channel blockers and, additionally, pharmacological properties of cyclosporine A.