Repositioning Lopinavir, an HIV Protease Inhibitor, as a Promising Antifungal Drug: Lessons Learned from Candida albicans-In Silico, In Vitro and In Vivo Approaches.

The repurposing strategy was applied herein to evaluate the effects of lopinavir, an aspartic protease inhibitor currently used in the treatment of HIV-infected individuals, on the globally widespread opportunistic human fungal pathogen Candida albicans by using in silico, in vitro and in vivo approaches in order to decipher its targets on fungal cells and its antifungal mechanisms of action. Secreted aspartic proteases (Saps) are the obviously main target of lopinavir. To confirm this hypothesis, molecular docking assays revealed that lopinavir bound to the Sap2 catalytic site of C. albicans as well as inhibited the Sap hydrolytic activity in a typically dose-dependent manner. The inhibition of Saps culminated in the inability of C. albicans yeasts to assimilate the unique nitrogen source (albumin) available in the culture medium, culminating with fungal growth inhibition (IC50 = 39.8 µM). The antifungal action of lopinavir was corroborated by distinct microscopy analyses, which evidenced drastic and irreversible changes in the morphology that justified the fungal death. Furthermore, our results revealed that lopinavir was able to (i) arrest the yeasts-into-hyphae transformation, (ii) disturb the synthesis of neutral lipids, including ergosterol, (iii) modulate the surface-located molecules, such as Saps and mannose-, sialic acid- and N-acetylglucosamine-containing glycoconjugates, (iv) diminish the secretion of hydrolytic enzymes, such as Saps and esterase, (v) negatively influence the biofilm formation on polystyrene surface, (vi) block the in vitro adhesion to epithelial cells, (vii) contain the in vivo infection in both immunocompetent and immunosuppressed mice and (viii) reduce the Sap production by yeasts recovered from kidneys of infected animals. Conclusively, the exposed results highlight that lopinavir may be used as a promising repurposing drug against C. albicans infection as well as may be used as a lead compound for the development of novel antifungal drugs.

Histoplasma capsulatum-induced extracellular DNA trap release in human neutrophils.

Neutrophils are leukocytes that are capable of eliminating both intra- and extracellular pathogens by mechanisms such as phagocytosis, degranulation, and release of neutrophil extracellular traps (NETs). Histoplasma capsulatum var. capsulatum (H. capsulatum) is a dimorphic fungus with a global distribution that causes histoplasmosis, a disease that is endemic in different geographic areas and is spreading worldwide. The release of NETs has been described as an important host defense mechanism against different fungi; however, there are no reports demonstrating that this process is implicated in neutrophil response to H. capsulatum infection. Therefore, the aim of this work is to investigate whether isolated human neutrophils release NETs in response to H. capsulatum and the potential mechanisms involved, as well as delineate the NETs antifungal activity. Using both confocal fluorescence and scanning electron microscopy techniques, we determined that NETs are released in vitro in response to H. capsulatum via an oxidative mechanism that is downstream of activation of the Syk and Src kinase pathways and is also dependent on CD18. NETs released in response to H. capsulatum yeasts involve the loss of neutrophil viability and are associated with elastase and citrullinated histones, however also can occur in a PAD4 histone citrullination independent pathway. This NETs also presented fungicidal activity against H. capsulatum yeasts. Our findings may contribute to the understanding of how neutrophils recognize and respond as immune effector cells to H. capsulatum, which may lead to better knowledge of histoplasmosis pathophysiology and treatment.

Domestic feline contribution in the transmission of Sporothrix in Rio de Janeiro State, Brazil: a comparison between infected and non-infected populations.

BACKGROUND: Sporotrichosis is a neglected zoonosis caused by pathogenic fungi belonging to the Sporothrix schenckii complex. In Rio de Janeiro state, this disease reached an epidemic status with over 4700 domestic felines and around 4000 humans affected since the mid-90s. The present study evaluated clinical and epidemiological aspects and also the frequency of colonization and infection by these fungi in healthy cats and among those with suspicious cutaneous lesions, inhabiting four Rio de Janeiro state distinct areas. RESULTS: Three hundred and seventy-one cats were included in two groups: 175 healthy cats [CRG] and 196 cats showing lesions suggesting sporotrichosis [SSG]. Mycological diagnosis allowed SSG animals to be divided in positive [104 cats; +SG] and negative [92 cats; -SG] groups. Nails, oral mucosa and lesions swabs were submitted to culture and potential colonies were subculture for micromorphologycal analysis, dimorphism and molecular tests. In the CRG, only one cat was colonized in the oral cavity [0.57%]; in the -SG group, four animals showed colonization of the nail and/or oral cavity [4.3%]; while the highest frequency of colonization [39.4%] was observed in the +SG. All molecularly typed isolates were identified as S. brasiliensis. CONCLUSION: The results obtained here indicate that healthy cats have a minor role in sporotrichosis transmission within the state of Rio de Janeiro. Conversely, a higher participation of diseased feline in sporotrichosis transmission was evidenced, especially by the colonization of their oral cavity. Sporothrix brasiliensis equally affects and colonizes animals from distinct Rio de Janeiro state areas. Thus, we hypothesize that sporotrichosis is a uniform endemic throughout the state, whose transmission depends mainly on the contact with cats with sporotrichosis. Since Rio de Janeiro displays a world unique epidemic model of the disease, not fully understood, data on the infected and non-infected animals can be of major importance for future strategies of sporotrichosis prevention and control. Finally, considering the importance of the current concept of "one health", the experience here observed can be helpful for distinct epizootias and/or zoonosis.

Peptidorhamnomannan negatively modulates the immune response in a scedosporiosis murine model.

In this study, we analyzed the impact of immunization with the peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium (Lomentospora) prolificans in a murine model of invasive scedosporiosis. Immunization with PRM decreased the survival of mice infected with S. prolificans. Immunization of mice with PRM led to decreased secretion of pro-inflammatory cytokines and chemokines but did not affect the secretion of IL-10. Mice immunized with PRM showed an increase in IgG1 secretion, which is an immunoglobulin linked to a nonprotective response. Splenocytes isolated from mice infected with S. prolificans and immunized with PRM showed no differences in the percentages of Th17 cells and no increase in the frequency of the CD4(+)CD62L(Low) T cell population. PRM-immunized mice showed a significant increase in the percentage of Treg cells. In summary, our results indicated that immunization with PRM did not assist or improve the immunological response against S. prolificans infection. PRM exacerbated the infection process by reducing the inflammatory response, thereby facilitating colonization, virulence and dissemination by the fungus.

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.

Candida haemulonii complex: species identification and antifungal susceptibility profiles of clinical isolates from Brazil.

OBJECTIVES: The emerging fungal pathogens comprising the Candida haemulonii complex (Candida haemulonii, Candida haemulonii var. vulnera and Candida duobushaemulonii) are notable for their antifungal resistance. Twelve isolates with phenotypic similarity to C. haemulonii were recovered from patients in Brazilian hospitals. Here we aimed to identify these isolates by a molecular approach, using the current classification of this fungal complex, and to evaluate their antifungal susceptibility profiles. METHODS: The fungal isolates were rechecked to certify their authentication by mycology methodologies and then characterized by ITS1-5.8S-ITS2 gene sequencing. A susceptibility assay was performed using the broth microdilution method published by CLSI (M27-A3/M27-S3). RESULTS: Based on biochemical tests, all Brazilian isolates were identified as C. haemulonii. After employing ITS sequencing, five isolates were identified as C. haemulonii, four as C. duobushaemulonii and three as C. haemulonii var. vulnera. All 12 clinical isolates were resistant to amphotericin B (MICs ranged from 2 to >16 mg/L) and fluconazole (MICs ≥ 64 mg/L). One isolate of C. haemulonii var. vulnera and two isolates of C. duobushaemulonii were susceptible-dose dependent to itraconazole, while the remaining isolates (75%) were resistant to this antifungal. Eight out of 12 isolates (66.7%) were resistant to voriconazole (MICs ≥ 16 mg/L), while all isolates were susceptible to caspofungin (MICs ≤ 0.5 mg/L). CONCLUSIONS: Our results reinforce the importance of molecular identification in differentiating species of the C. haemulonii complex. Moreover, the antifungal multiresistant profile of clinical isolates of the C. haemulonii complex represents a challenge to the treatment of such infections.

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.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii.

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

Mycelial forms of Pseudallescheria boydii present ectophosphatase activities.

Phosphatase activities were characterized in intact mycelial forms of Pseudallescheria boydii, which are able to hydrolyze the artificial substrate p-nitrophenylphosphate (p-NPP) to p-nitrophenol (p-NP) at a rate of 41.41+/-2.33 nmol p-NP per h per mg dry weight, linearly with increasing time and with increasing cell density. MgCl2, MnCl2 and ZnCl2 were able to increase the (p-NPP) hydrolysis while CdCl2 and CuCl2 inhibited it. The (p-NPP) hydrolysis was enhanced by increasing pH values (2.5-8.5) over an approximately 5-fold range. High sensitivity to specific inhibitors of alkaline and acid phosphatases suggests the presence of both acid and alkaline phosphatase activities on P. boydii mycelia surface. Cytochemical localization of the acid and alkaline phosphatase showed electron-dense cerium phosphate deposits on the cell wall, as visualized by electron microscopy. The product of p-NPP hydrolysis, inorganic phosphate (Pi), and different inhibitors for phosphatase activities inhibited p-NPP hydrolysis in a dose-dependent manner, but only the inhibition promoted by sodium orthovanadate and ammonium molybdate is irreversible. Intact mycelial forms of P. boydii are also able to hydrolyze phosphoaminoacids with different specificity.

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.

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii.

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.

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.

Glucosylceramides in Colletotrichum gloeosporioides are involved in the differentiation of conidia into mycelial cells.

Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.

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

Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation.

Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.

A peptidorhamnomannan from the mycelium of Pseudallescheria boydii is a potential diagnostic antigen of this emerging human pathogen.

The ascomycete Pseudallescheria boydii is an emerging human pathogen frequently found in soil and polluted water. A peptidopolysaccharide antigen has been isolated from mycelial forms of P. boydii, and characterized using chemical and immunological methods. Monosaccharide composition, methylation analysis, and (1)H- and (13)C-NMR spectra indicated the presence of a rhamnomannan with a structure distinct from those of similar components isolated from other fungi, containing Rhap(1-->3)Rhap epitopes on side chains which may be linked (1-->3) to (1-->6)-linked mannose. The peptidorhamnomannan from P. boydii reacted poorly with an antiserum raised against whole cells of Sporothrix schenckii and strongly with one against P. boydii hyphae. These characteristics and immunological differences suggest that this major rhamnose-containing antigen of P. boydii may be useful for the specific diagnosis of infections attributable to this fungus.

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.