Fibronectin-binding molecules of Scedosporium apiospermum: focus on adhesive events.

Scedosporium apiospermum is a widespread, emerging, and multidrug-resistant filamentous fungus that can cause localized and disseminated infections. The initial step in the infection process involves the adhesion of the fungus to host cells and/or extracellular matrix components. However, the mechanisms of adhesion involving surface molecules in S. apiospermum are not well understood. Previous studies have suggested that the binding of fungal receptors to fibronectin enhances its ability to attach to and infect host cells. The present study investigated the effects of fibronectin on adhesion events of S. apiospermum. The results revealed that conidial cells were able to bind to both immobilized and soluble human fibronectin in a typically dose-dependent manner. Moreover, fibronectin binding was virtually abolished in trypsin-treated conidia, suggesting the proteinaceous nature of the binding site. Western blotting assay, using fibronectin and anti-fibronectin antibody, evidenced 7 polypeptides with molecular masses ranging from 55 to 17 kDa in both conidial and mycelial extracts. Fibronectin-binding molecules were localized by immunofluorescence and immunocytochemistry microscopies at the cell wall and in intracellular compartments of S. apiospermum cells. Furthermore, a possible function for the fibronectin-like molecules of S. apiospermum in the interaction with host lung cells was assessed. Conidia pre-treated with soluble fibronectin showed a significant reduction in adhesion to either epithelial or fibroblast lung cells in a classically dose-dependent manner. Similarly, the pre-treatment of the lung cells with anti-fibronectin antibodies considerably diminished the adhesion. Collectively, the results demonstrated the presence of fibronectin-binding molecules in S. apiospermum cells and their role in adhesive events.

Anti-cryptococcal activity of ethanol crude extract and hexane fraction from Ocimum basilicum var. Maria bonita: mechanisms of action and synergism with amphotericin B and Ocimum basilicum essential oil.

CONTEXT: Ocimum basilicum L. (Lamiaceae) has been used in folk medicine to treat headaches, kidney disorders, and intestinal worms. OBJECTIVE: This study evaluates the anti-cryptococcal activity of ethanol crude extract and hexane fraction obtained from O. basilicum var. Maria Bonita leaves. MATERIALS AND METHODS: The MIC values for Cryptococcus sp. were obtained according to Clinical and Laboratory Standards Institute in a range of 0.3-2500 µg/mL. The checkerboard assay evaluated the association of the substances tested (in a range of 0.099-2500 µg/mL) with amphotericin B and O. basilicum essential oil for 48 h. The ethanol extract, hexane fraction and associations in a range of 0.3-2500 µg/mL were tested for pigmentation inhibition after 7 days of treatment. The inhibition of ergosterol synthesis and reduction of capsule size were evaluated after the treatment with ethanol extract (312 µg/mL), hexane fraction (78 µg/mL) and the combinations of essential oil + ethanol extract (78 µg/mL + 19.5 µg/mL, respectively) and essential oil + hexane fraction (39.36 µg/mL + 10 µg/mL, respectively) for 24 and 48 h, respectively. RESULTS: The hexane fraction presented better results than the ethanol extract, with a low MIC (156 µg/mL against C. neoformans T444 and 312 µg/mL against C. neoformans H99 serotype A and C. gattii WM779 serotype C). The combination of the ethanol extract and hexane fraction with amphotericin B and essential oil enhanced their antifungal activity, reducing the concentration of each substance needed to kill 100% of the inoculum. The substances tested were able to reduce the pigmentation, capsule size and ergosterol synthesis, which suggest they have important mechanisms of action. CONCLUSIONS: These results provide further support for the use of ethanol extracts of O. basilicum as a potential source of antifungal agents.

Comparative genomics of the major fungal agents of human and animal Sporotrichosis: Sporothrix schenckii and Sporothrix brasiliensis.

BACKGROUND: The fungal genus Sporothrix includes at least four human pathogenic species. One of these species, S. brasiliensis, is the causal agent of a major ongoing zoonotic outbreak of sporotrichosis in Brazil. Elsewhere, sapronoses are caused by S. schenckii and S. globosa. The major aims on this comparative genomic study are: 1) to explore the presence of virulence factors in S. schenckii and S. brasiliensis; 2) to compare S. brasiliensis, which is cat-transmitted and infects both humans and cats with S. schenckii, mainly a human pathogen; 3) to compare these two species to other human pathogens (Onygenales) with similar thermo-dimorphic behavior and to other plant-associated Sordariomycetes. RESULTS: The genomes of S. schenckii and S. brasiliensis were pyrosequenced to 17x and 20x coverage comprising a total of 32.3 Mb and 33.2 Mb, respectively. Pair-wise genome alignments revealed that the two species are highly syntenic showing 97.5% average sequence identity. Phylogenomic analysis reveals that both species diverged about 3.8-4.9 MYA suggesting a recent event of speciation. Transposable elements comprise respectively 0.34% and 0.62% of the S. schenckii and S. brasiliensis genomes and expansions of Gypsy-like elements was observed reflecting the accumulation of repetitive elements in the S. brasiliensis genome. Mitochondrial genomic comparisons showed the presence of group-I intron encoding homing endonucleases (HE's) exclusively in S. brasiliensis. Analysis of protein family expansions and contractions in the Sporothrix lineage revealed expansion of LysM domain-containing proteins, small GTPases, PKS type1 and leucin-rich proteins. In contrast, a lack of polysaccharide lyase genes that are associated with decay of plants was observed when compared to other Sordariomycetes and dimorphic fungal pathogens, suggesting evolutionary adaptations from a plant pathogenic or saprobic to an animal pathogenic life style. CONCLUSIONS: Comparative genomic data suggest a unique ecological shift in the Sporothrix lineage from plant-association to mammalian parasitism, which contributes to the understanding of how environmental interactions may shape fungal virulence. . Moreover, the striking differences found in comparison with other dimorphic fungi revealed that dimorphism in these close relatives of plant-associated Sordariomycetes is a case of convergent evolution, stressing the importance of this morphogenetic change in fungal pathogenesis.

Melanin in Fonsecaea pedrosoi: a trap for oxidative radicals.

BACKGROUND: The pathogenic fungus Fonsecaea pedrosoi constitutively produces the pigment melanin, an important virulence factor in fungi. Melanin is incorporated in the cell wall structure and provides chemical and physical protection for the fungus.We evaluated the production of nitric oxide (NO) in macrophages, the oxidative burst and the inducible nitric oxide synthase (i-NOS) activity in interactions between activated murine macrophages and F. pedrosoi. Experiments were carried out with or without tricyclazole (TC) treatment, a selective inhibitor of the dihydroxynaphthalene (DHN)-melanin biosynthesis pathway in F. pedrosoi. The paramagnetisms of melanin and the TC-melanin were analysed by electron spin resonance. The fungal growth responses to H2O2 and to S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide donor, were also evaluated. RESULTS: Melanised F. pedrosoi cells were more resistant to both H2O2 and NO. Nitrite was not detected in the supernatant of macrophages incubated with melanised fungal cells. However, i-NOS expression was unaffected by the presence of either untreated control F. pedrosoi or TC-treated F. pedrosoi. In addition, the inhibition of the DHN-melanin pathway by TC improved the oxidative burst capability of the macrophages. CONCLUSION: The NO-trapping ability of F. pedrosoi melanin is an important mechanism to escape the oxidative burst of macrophages.

Identification, antifungal susceptibility and scanning electron microscopy of a keratinolytic strain of Rhodotorula mucilaginosa: a primary causative agent of onychomycosis.

Onychomycosis is a dermatological problem of high prevalence that mainly affects the hallux toenail. Onychomycosis caused by the yeast Rhodotorula mucilaginosa was identified using colony morphology, light microscopy, urease and carbohydrate metabolism in a 57-year-old immunocompetent patient from Rio de Janeiro, Brazil. High-resolution scanning electron microscopy of nail fragments, processed by a noncoating method, led to the observation with fine detail of the structures of both nail and fungus involved in the infection. Yeasts were mainly found inside grooves in the nail. Budding yeasts presented a spiral pattern of growth and blastoconidia were found in the nail groove region. Keratinase assays and keratin enzymography revealed that this isolate was highly capable of degrading keratin. Antifungal susceptibility tests showed that the fungus was susceptible to low concentrations of amphotericin B and 5-flucytosine and resistant to high concentrations of fluconazole, itraconazole, voriconazole and terbinafine. These findings showed data for the first time concerning the interaction of R. mucilaginosa in toenail infection and suggest that this emerging yeast should also be considered an opportunistic primary causative agent of onychomycosis.

Ultrastructural characterization of melanosomes of the human pathogenic fungus Fonsecaea pedrosoi.

Melanin is a complex polymer widely distributed in nature and has been described as an important virulence factor in pathogenic fungi. In the majority of fungi, the mechanism of melanin formation remains unclear. In Fonsecaea pedrosoi, the major etiologic agent of chromoblastomycosis, melanin is stored in intracellular vesicles, named melanosomes. This paper details the ultrastructural aspects of melanin formation, its storage and transportation to the cell wall in the human pathogenic fungus F. pedrosoi. In this fungus, melanin synthesis within melanosomes also begins with a fibrillar matrix formation, displaying morphological and structural features similar to melanosomes from amphibian and mammalian cells. Silver precipitation based on Fontana-Masson technique for melanin detection and immunocytochemistry showed that melanosome fuses with fungal cell membrane where the melanin is released and reaches the cell wall. Melanin deposition in the fungal cell wall occurs in concentric layers. Antibodies raised against F. pedrosoi melanin revealed the sites of melanin production and storage in the melanosomes. In addition, a preliminary description of the elemental composition of this organelle by X-ray microanalysis and elemental mapping revealed the presence of calcium, phosphorus and iron concentrated in its matrix, suggesting a new functional role for these organelles as iron storage compartments.

Effects of tricyclazole (5-methyl-1,2,4-triazol[3,4] benzothiazole), a specific DHN-melanin inhibitor, on the morphology of Fonsecaea pedrosoi conidia and sclerotic cells.

The influence of tricyclazole (5-methyl-1,2,4-triazol[3,4]benzothiazole), a specific DHN-melanin inhibitor, on the cell walls and intracellular structures of Fonsecaea pedrosoi conidia and sclerotic cells was analyzed by transmission electron microscopy (TEM), deep-etching, and field emission scanning electron microscopy. The treatment of the fungus with 16 microg mL(-1) of tricyclazole (TC) did not significantly affect fungal viability, but electron microscopy observations showed several important morphological differences between TC-treated and non-TC treated cells. Control sclerotic cells presented patched granules, with an average diameter of 47 nm, on the cell surface, which were absent in TC-treated cells. Also, TC-treated sclerotic cells showed an undulated relief. TC treatment leads to an accumulation of electron lucent vacuoles in the fungal cytoplasm of both conidia and sclerotic cells, and treated conidia observed by deep etching showed a relevant thickening of the fungal cell wall. Together, these observations support the previous data of our group that F. pedrosoi synthesizes melanin in intracellular organelles. In addition, we suggest that melanin is not only an extracellular constituent but could also be dispersing all over the cell walls and could have an effective role in cross-linking different cell wall compounds that help maintain the regular shape of the cell wall.

Inhibition of melanin synthesis pathway by tricyclazole increases susceptibility of Fonsecaea pedrosoi against mouse macrophages.

Fonsecaea pedrosoi produces melanin, a pigment related to virulence in pathogenic fungi. To understand the involvement of melanin in the protection of fungi, the authors used tricyclazole to inhibit the melanin pathway in F. pedrosoi. Experiments of pigmentation suggested that F. pedrosoi uniquely produces dihydroxynaphthalene-melanin. Pigments produced on cultures modified or not with tricyclazole were extracted by an alkali-acid method and submitted to infrared and ion exchange chromatography analysis; also cytochemistry analysis for cationized ferritin of whole cells was carried out. This group of experiments showed that the tricyclazole treatment on F. pedrosoi produced a melanin-like pigment, but less negatively charged and with less affinity for iron ions than that without the tricyclazole treatment, and this in turn lead to a less negatively charge cell wall surface. Scanning electron microscopy of such pigments showed that the melanin from control cultures maintained their hyphae-like structures, which have been described as "melanin-ghosts," whereas the tricyclazole pigment showed an amorphous surface. Interaction of conidia from cultures of F. pedrosoi, modified by tricyclazole or not, with peritoneal activated macrophages suggested that tricyclazole causes higher association of fungus with macrophages, weakens the fungus capacity to destroy the macrophages, and diminishes the resistance to dry fracture procedures on samples prepared for high resolution scanning electron microscopy.