In Vitro and In Vivo Effect of Peptides Derived from 14-3-3 Paracoccidioides spp. Protein.

BACKGROUND: Paracoccidioidomycosis (PCM) is a chronic disease that causes sequelae and requires prolonged treatment; therefore, new therapeutic approaches are necessary. In view of this, three peptides from Paracoccidioides brasiliensis 14-3-3 protein were selected based on its immunogenicity and therapeutic potential. METHODS: The in vitro antifungal activity and cytotoxicity of the 14-3-3 peptides were evaluated. The influence of the peptides in immunological and survival aspects was evaluated in vivo, using Galleria mellonella and the expression of antimicrobial peptide genes in Caenorhabditis elegans. RESULTS: None of the peptides were toxic to HaCaT (skin keratinocyte), MRC-5 (lung fibroblast), and A549 (pneumocyte) cell lines, and only P1 exhibited antifungal activity against Paracoccidioides spp. The peptides could induce an immune response in G. mellonella. Moreover, the peptides caused a delay in the death of Paracoccidioides spp. infected larvae. Regarding C. elegans, the three peptides were able to increase the expression of the antimicrobial peptides. These peptides had essential effects on different aspects of Paracoccidioides spp. infection showing potential for a therapeutic vaccine. Future studies using mammalian methods are necessary to validate our findings.

Candida auris: Epidemiology, risk factors, virulence, resistance, and therapeutic options.

Candida auris emerged as a pathogen resistant to multiple antifungal and has been associated with nosocomial outbreaks with high transmission capacity between hospitalized individuals. C. auris was first described in 2009, after being isolated from the external ear canal discharge of a patient in Japan. The difficulty in identification, incorrect use of antifungal drugs, and treatment failure are causes of high mortality. Since then, C. auris has been increasingly reported from East Asia to North America, with substantial fatalities and misidentification. This review aims at describing the epidemiology, virulence, risk factors, resistance, and therapeutic options in C. auris infections.

Antifungal activity of fluconazole-loaded natural rubber latex against Candida albicans.

AIM: This work aimed to produce a membrane based on fluconazole-loaded natural rubber latex (NRL), and study their interaction, drug release and antifungal susceptibility against Candida albicans. MATERIALS & METHODS: Fluconazole-loaded NRL membrane was obtained by casting method. RESULTS: The Fourier Transform Infrared Spectroscopy showed no modifications either in NRL or fluconazole after the incorporation. Mechanical test presented low Young's modulus and high strain, indicating the membranes have sufficient elasticity for biomedical application. The bio-membrane was able to release the drug and inhibit the growth of C. albicans as demonstrated by disk diffusion and macrodilution assays. CONCLUSION: The biomembrane was able to release fluconazole and inhibit the growth of C. albicans, representing a promising biomaterial for skin application.

Activity of 3'-hydroxychalcone against Cryptococcus gattii and toxicity, and efficacy in alternative animal models.

AIM: This work aimed to evaluate the activity of 3'-hydroxychalcone against Cryptococcus gattii in planktonic and biofilm forms and their toxicity using alternative animal models. MATERIALS & METHODS: Minimum inhibitory concentration and minimum fungicide concentration were determined. Biofilm formation and the susceptibility tests were performed by the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-5-[carbonyl(phenylamino)]-2H-tetrazolium hydroxide assay. Toxicity and efficacy were checked in Danio rerio and Galleria mellonella models. RESULTS: The compound 3'-hydroxychalcone showed fungicidal activity against C. gattii in both planktonic and biofilm forms. The toxicity in zebrafish embryos revealed a low lethal concentration. In G. mellonella, the compound did not show antifungal activity and larvae toxicity. CONCLUSION: Because of the activity of 3'-hydroxychalcone against C. gattii in vitro, molecular modifications should be made to improve efficacy and to reduce toxicity in vivo. [Formula: see text].

Synergistic effect of pedalitin and amphotericin B against Cryptococcus neoformans by in vitro and in vivo evaluation.

Cryptococcosis is an opportunistic fungal infection responsible for high morbidity and mortality in immunocompromised patients. Combination of antifungal substances is a promising way to increase the percentage of successful treatment. Pedalitin (PED) is a natural substance obtained from Pterogyne nitens. The aim of this study was to verify the efficacy of PED alone and in combination with amphotericin B (AmB) in vitro and in vivo against Cryptococcus spp. In the in vitro assay, minimum inhibitory concentrations (MICs) of 0.125 mg/L for AmB and 3.9 mg/L for PED were found when the substances were tested alone, whilst in the combination treatment the active concentration of both decreased, with MICs of 0.03 mg/L for AmB and 1 mg/L for PED. In the survival assay, fungal burden study and histopathological assays it was possible to study the efficacy of the substances alone and in combination. The efficacy of combination therapy was considered better than monotherapy as evaluated in a Galleria mellonella model and a murine model. Thus, the combination of PED and AmB is an interesting alternative for anticryptococcal fungal treatment. Moreover, a correlation was observed between the invertebrate and murine models for this antifungal treatment combination.

Metabolic profiles of planktonic and biofilm cells of Candida orthopsilosis.

AIM: This study aims to understand which Candida orthopsilosis protein aids fungus adaptation upon its switching from planktonic growth to biofilm. MATERIALS & METHODS: Ion mobility separation within mass spectrometry analysis combination were used. RESULTS: Proteins mapped for different biosynthetic pathways showed that selective ribosome autophagy might occur in biofilms. Glucose, used as a carbon source in the glycolytic flux, changed to glycogen and trehalose. CONCLUSION: Candida orthopsilosis expresses proteins that combine a variety of mechanisms to provide yeasts with the means to adjust the catalytic properties of enzymes. Adjustment of the enzymes helps modulate the biosynthesis/degradation rates of the available nutrients, in order to control and coordinate the metabolic pathways that enable cells to express an adequate response to nutrient availability.

A flow cytometry method for testing the susceptibility of Cryptococcus spp. to amphotericin B / / Un método de citometría de flujo para determinar la sensibilidad de Cryptococcus spp. a la anfotericina B

Background. Human fungal infections have increased at an alarming rate in recent years, particularly in immunocompromised individuals. Cryptococcosis is the second most prevalent systemic fungal infection worldwide, and the most prevalent systemic infection in immunocompromised individuals, representing more than 70% of cases. The incidence of cryptococcosis is high in people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. Aims. The aim of this research was to develop a rapid flow cytometric antifungal susceptibility test and to compare the results with the standard methods. Methods. A reference strain and clinical isolates of Cryptococcus neoformans and Cryptococcus gattii were tested for susceptibility to amphotericin B by flow cytometry using propidium iodide as indicator of viability. Flow cytometry (FC) results were compared with the minimum inhibitory concentration (MIC) values determined by microdilution. Results. The antifungal activity of amphotericin B ranged from MICs of 0.06 to 2 μg/ml for the 11 isolates studied. The same results were found by FC. Conclusions. The FC method allows same-day results, assisting in the selection of appropriate antifungal therapies. These results demonstrate an excellent correlation between FC and the classic methods of testing for susceptibility to antifungal agents. This rapid diagnosis method makes it possible to quickly administer effective therapeutic interventions, often saving lives (AU)

Antecedentes. En los últimos años, las infecciones por hongos en el ser humano, particularmente en individuos inmunodeprimidos, han aumentado a un ritmo alarmante. La criptococosis es la segunda infección fúngica sistémica más frecuente en todo el mundo, y también la infección sistémica más frecuente en personas inmunodeprimidas, lo que representa más del 70% de los casos. La incidencia de la criptococosis es alta en las personas con VIH/síndrome de inmunodeficiencia adquirida. Estudios recientes indican que hay un millón de casos de meningitis criptocócica en pacientes con síndrome de inmunodeficiencia adquirida por año en todo el mundo. Objetivos. El objetivo de esta investigación fue desarrollar una prueba rápida de sensibilidad antifúngica por citometría de flujo (CF) y comparar los resultados con los métodos estándar. Métodos. Se estudió la sensibilidad a la anfotericina B por CF, con yoduro de propidio como indicador de la viabilidad, de una cepa de referencia y varios aislamientos clínicos de Cryptococcus neoformans y Cryptococcus gattii. Resultados. La actividad antifúngica de la anfotericina B varió de una concentración mínima inhibitoria de 0,06 a 2 μg/ml para los 11 aislamientos estudiados. Los mismos resultados fueron encontrados por CF. Conclusiones. El método de la CF permite resultados el mismo día, lo que hace posible una rápida selección de los tratamientos antifúngicos adecuados. Estos resultados demuestran una excelente correlación entre el método de la CF y los procedimientos clásicos de pruebas de sensibilidad a los agentes antifúngicos. Este método de rastreo ágil hace que sea posible realizar rápidamente intervenciones terapéuticas eficaces y, a menudo, salvar vidas (AU)

Highlights in pathogenic fungal biofilms / Aspectos sobresalientes en la formación de biopelículas por hongos patógenos

A wide variety of fungi have demonstrated the ability to colonize surfaces and form biofilms. Most studies on fungal biofilms have focused on Candida albicans and more recently, several authors have reported the involvement of other genera of yeasts and Candida species, as well as of filamentous fungi in the formation of biofilms, including: Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula species, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pneumocystis species, Coccidioides immitis, Fusarium species, Saccharomyces cerevisiae, Trichosporon asahii, Mucorales and Blastoschizomyces. There is a current interest in describing the particular characteristics of the biofilm formation by of these fungi. A major concern is the control of biofilms, requiring knowledge of the biofilm mechanisms. However, our knowledge of these microbial communities is limited, due to the complexity of these systems and metabolic interactions that remain unknown. This mini-review aims to highlight recently discovered fungal biofilms and to compare them with the current knowledge on biofilms. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

Una amplia variedad de hongos poseen la capacidad para colonizar superficies y formar biopelículas (biofilms). La mayoría de los estudios efectuados sobre biopelículas de hongos han prestado atención a Candida albicans y, más recientemente, varios autores han descrito la implicación de otros géneros de levaduras y especies de Candida, al igual que de hongos filamentosos, en la formación de biopelículas, incluidos Cryptococcus neoformans, Cryptococcus gattii, especies de Rhodotorula, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, especies de Pneumocystis, Coccidioides immitis, especies de Fusarium, Saccharomyces cerevisiae, Trichosporon asahii, mucorales y Blastoschizomyces. En la actualidad suscita interés la descripción de las características particulares de la formación de biopelículas de estos hongos. Una preocupación importante es el control de las biopelículas, que requiere una comprensión de los mecanismos de su formación. Sin embargo, nuestros conocimientos sobre estas comunidades microbianas son limitados debido a la complejidad de estos sistemas y a las interacciones metabólicas que aún no conocemos. Esta revisión tiene como objetivo poner de relieve las biopelículas fúngicas descubiertas recientemente y compararlas con los conocimientos actuales disponibles sobre ellas.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Effectiveness of disinfectants used in hemodialysis against both Candida orthopsilosis and C. parapsilosis sensu stricto biofilms.

Biofilms have been observed in the fluid pathways of hemodialysis machines. The impacts of four biocides used for the disinfection of hemodialysis systems were tested against Candida parapsilosis sensu stricto and Candida orthopsilosis biofilms generated by isolates obtained from a hydraulic circuit that were collected in a hemodialysis unit. Acetic acid was shown to be the most effective agent against Candida biofilms. Strategies for effective disinfection procedures used for hemodialysis systems should also seek to kill and inhibit biofilms.

Characterization of Paracoccidioides brasiliensis PbDfg5p, a cell-wall protein implicated in filamentous growth.

The dimorphic fungus Paracoccidioides brasiliensis is the causative agent of the most frequent systemic mycosis in Latin America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and differentiate into the yeast parasitic phase. Here we describe the characterization of a Dfg5p (defective for filamentous growth) homologue of P. brasiliensis, a predictable cell wall protein, first identified in Saccharomyces cerevisiae. The protein, the cDNA and genomic sequences were analysed. The cloned cDNA was expressed in Escherichia coli and the purified rPbDfg5p was used to obtain polyclonal antibodies. Immunoelectron microscopy and biochemical studies demonstrated the presence of PbDfg5p in the fungal cell wall. Enzymatic treatments identified PbDfg5p as a beta-glucan linked protein that undergoes N-glycosylation. The rPbDfg5p bound to extracellular matrix components, indicating that those interactions could be important for initial steps leading to P. brasiliensis attachment and colonization of host tissues.

Differential gene expression by Paracoccidioides brasiliensis in host interaction conditions: representational difference analysis identifies candidate genes associated with fungal pathogenesis.

Paracoccidioides brasiliensis causes infection by the host inhalation of airborne propagules of the mycelia phase of the fungus. These particles reach the lungs, and disseminate to virtually all organs. Here we describe the identification of differentially expressed genes in studies of host-fungus interaction. We analyzed two cDNA populations of P. brasiliensis, one obtained from infected animals and the other an admixture of fungus and human blood thus mimicking the hematologic events of the fungal dissemination. Our analysis identified transcripts differentially expressed. Genes related to iron acquisition, melanin synthesis and cell defense were specially upregulated in the mouse model of infection. The upregulated transcripts of yeast cells during incubation with human blood were those predominantly related to cell wall remodeling/synthesis. The expression pattern of genes was independently confirmed in host conditions, revealing their potential role in the infection process. This work can facilitate functional studies of novel regulated genes that may be important for the survival and growth strategies of P. brasiliensis in humans.