Antibodies Against Glycolipids Enhance Antifungal Activity of Macrophages and Reduce Fungal Burden After Infection with Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a fungal disease endemic in Latin America. Polyclonal antibodies to acidic glycosphingolipids (GSLs) from Paracoccidioides brasiliensis opsonized yeast forms in vitro increasing phagocytosis and reduced the fungal burden of infected animals. Antibodies to GSL were active in both prophylactic and therapeutic protocols using a murine intratracheal infection model. Pathological examination of the lungs of animals treated with antibodies to GSL showed well-organized granulomas and minimally damaged parenchyma compared to the untreated control. Murine peritoneal macrophages activated by IFN-γ and incubated with antibodies against acidic GSLs more effectively phagocytosed and killed P. brasiliensis yeast cells as well as produced more nitric oxide compared to controls. The present work discloses a novel target of protective antibodies against P. brasiliensis adding to other well-studied mediators of the immune response to this fungus.

Peptides of the constant region of antibodies display fungicidal activity.

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Metallopeptidase inhibitors arrest vital biological processes in the fungal pathogen Scedosporium apiospermum.

Scedosporium apiospermum is an emerging agent of opportunistic mycoses in humans. Previously, we showed that mycelia of S. apiospermum secreted metallopeptidases which were directly linked to the destruction of key host proteins. In this study, we analysed the effect of metallopeptidase inhibitors on S. apiospermum development. As germination of inhaled conidia is a crucial event in the infectious process of S. apiospermum, we studied the morphological transformation induced by the incubation of conidia in Sabouraud-dextrose medium at 37 °C. After 6 h, some conidia presented a small projection resembling a germ-tube. A significant increase, around sixfold, in the germ-tube length was found after 12 h, and hyphae were exclusively observed after 24 h. Three distinct metallopeptidase inhibitors were able to arrest the transformation of conidia into hyphae in different ways; for instance, 1,10-phenanthroline (PHEN) completely blocked this process at 10 µmol l(-1), while ethylenediamine tetraacetic acid (EDTA) and ethylene glycol-bis (ß-aminoethyl ether; EGTA) only partially inhibited the differentiation at up to 10 mmol l(-1). EGTA did not promote any significant reduction in the conidial growth, while PHEN and EDTA, both at 10 mmol l(-1), inhibited the proliferation around 100% and 65%, respectively. The secretion of polypeptides into the extracellular environment and the metallopeptidase activity secreted by mycelia were completely inhibited by PHEN. These findings suggest that metallo-type enzymes could be potential targets for future therapeutic interventions against S. apiospermum.

Pseudallescheria boydii releases metallopeptidases capable of cleaving several proteinaceous compounds.

Pseudallescheria boydii is an opportunistic filamentous fungus that causes serious infections in humans. Virulence attributes expressed by P. boydii are unknown. Conversely, peptidases are incriminated as virulence factors in several pathogenic fungi. Here we investigated the extracellular peptidase profile in P. boydii. After growth on Sabouraud for 7 days, mycelia of P. boydii were incubated for 20 h in PBS-glucose. The cell-free PBS-glucose supernatant was submitted to SDS-PAGE and 12 secretory polypeptides were observed. Two of these polypeptides (28 and 35 kD) presented proteolytic activity when BSA was used as a copolymerized substrate. The extracellular peptidases were most active in acidic pH (5.5) and fully inhibited by 1,10-phenanthroline, a zinc-metallopeptidase inhibitor. Other metallo-, cysteine, serine and aspartic proteolytic inhibitors did not significantly alter these activities. To confirm that these enzymes belong to the metallo-type peptidases, the apoenzymes were obtained by dialysis against chelating agents, and supplementation with different cations, especially Cu(2+) and Zn(2+), restored their activities. Except for gelatin, both metallopeptidases hydrolyzed various co-polymerized substrates, including human serum albumin, casein, hemoglobin and IgG. Additionally, the metallopeptidases were able to cleave different soluble proteinaceous substrates such as extracellular matrix components and sialylated proteins. All these hydrolyses were inhibited by 1,10-phenanthroline. Interestingly, Scedosporium apiospermum (the anamorph of P. boydii) produced a distinct extracellular peptidase profile. Collectively, our results demonstrated for the first time the expression of acidic extracellular metallopeptidases in P. boydii capable of degrading several proteinaceous compounds that could help the fungus to escape from natural human barriers and defenses.

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells.

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.

Structure and biological functions of fungal cerebrosides

Ceramide monohexosides (CMHs, cerebrosides) are glycosphingolipids composed of a hydrophobic ceramide linked to one sugar unit. In fungal cells, CMHs are very conserved molecules consisting of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids, and a carbohydrate portion consisting of one residue of glucose or galactose. 9-Methyl 4,8-sphingadienine-containing ceramides are usually glycosylated to form fungal cerebrosides, but the recent description of a ceramide dihexoside (CDH) presenting phytosphingosine in Magnaporthe grisea suggests the existence of alternative pathways of ceramide glycosylation in fungal cells. Along with their unique structural characteristics, fungal CMHs have a peculiar subcellular distribution and striking biological properties. In Pseudallescheria boydii, Candida albicans, Cryptococcus neoformans, Aspergillus nidulans, A. fumigatus, and Schizophyllum commune, CMHs are apparently involved in morphological transitions and fungal growth. The elucidation of structural and functional aspects of fungal cerebrosides may therefore contribute to the design of new antifungal agents inhibiting growth and differentiation of pathogenic species.

Pesquisa de fungos em 20 pacientes com polipose nasossinusal / Fungal research in 20 patientes with nasal polyposis

Introduçäo: Polipose nasossinusal é uma doença freqüente na prática clínica otorrinolaringológica. Sua associaçäo com fungos, porém, tem recebido mais atençäo apenas nos últimos 20 anos, principalmente após o reconhecimento da sinusite fúngica alérgica. Objetivo: Identificar a presença de fungos nos seios paranasais de pacientes portadores de polipose nasossinusal e analisar a reatividade destes pacientes para aeroalérgenos. Forma de Estudo: Clínico prospectivo. Material e Método: Avaliamos de forma prospectiva 20 pacientes com diagnóstico de polipose nasossinusal, que se apresentaram ao ambulatório de Otorrinolaringologia do Hospital Universitário Clementino Fraga Filho. Durante o tratamento cirúrgico, a secreçäo removida dos seios paranasais foi colocada em Sabouraud com cloranfenicol e submetida à análise histopatológica. Foram dosados níveis séricos de IgE total, IgE específica para Aspergillus fumigatus e contagem de eosinófilos do sangue periférico. Testes cutâneos com antígenos de Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Blattella germânica, Blattella americana e Aspergillus fumigatus foram realizados .Resultados: Seis pacientes (30 por cento) apresentaram crescimento fúngico, sendo quatro Aspergillus sp, um Candida tropicalis e outro Cladophialophora carrionii. Treze (68,42 por cento) apresentaram teste de puntura positivo para os aeroalérgenos acima citados. Em dez pacientes (50 por cento) foi encontrada eosinofilia. Níveis séricos elevados de IgE total foram encontrados em oito (44,4 por cento). Nível sérico elevado de IgE específica para Aspergillus fumigatus foi encontrado em apenas um. Conclusäo: Salientamos a importância da pesquisa de fungos e de uma investigaçäo do sistema imune nos pacientes com polipose nasossinusal para ser feito um diagnóstico preciso e tratamento adequado

Partial characterization of polysaccharides from Fusarium solani

A crude polysaccharide obtained from mycelium of Fusarium solani by treatment with 2 per center KOH/2h/100§C and fractionated by gel filtration chromatography yielded three fractions denoted L1,L2 and L3. Chemical analysis of the crude polysaccharide showed the presence od 89,5 per center total carbohydrate, 4 per center protin 14 per center uronic acid, traces of phosphate and hexosamine. Mannose, galactose, glucose and unidentifid pentose, were present in a 27.5:34:34.5:4 molar ratio.