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ABSTRACTScytalidium dimidiatumis a pigmented dematiaceous coelomycete that typically causes chronic superficial skin diseases and onychomycosis, as well as deeper infections, such as subcutaneous abscesses, mycetoma, and even fungemia in immunocompromised patients. A second species, Scytalidium hyalinum, has hyaline hyphae and arthroconidia and is considered by some authors to be an albino mutant of S. dimidiatum. This study aimed to confirm the presence of melanin or melanin-like compounds (which have been previously implicated in the virulence of other fungal pathogens) in S. dimidiatumfrom a patient with multiple subcutaneous nodules. Treatment of the hyphae and arthroconidia with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles, which were stable free radicals, consistent with their identification as melanins. Extracted melanin particles from S. dimidiatumcultures were labeled by melanin-binding monoclonal antibodies (MAbs) from Sporothrix schenckii, Aspergillus fumigatus, and Cryptococcus neoformans. Lesional skin from the patient infected with S. dimidiatumcontained fungal cells that were labeled by melanin-binding MAbs, and digestion of the tissue yielded dark particles that were also reactive. S. hyalinumwas also subjected to the melanin extraction protocol, but no dark particles were yielded.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

The cell-associated and extracellular enzymatic activities were examined in a total of 10 Penicillium marneffeiisolates. Both mycelia and yeast expressed alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, whereas a variety of other enzyme activities, including trypsin, chymotrypsin and alpha-fucosidase were absent. There was some inter-isolate variation in both mycelia and yeast in the activities of other enzymes such as esterases and galactosidases. Enzyme activities did not change significantly over the course of culturing in three representative isolates.

ABSTRACTAdhesion of Penicillium marneffeiconidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffeiconidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Immunofluorescence microscopy demonstrated that laminin bound to the surface of Penicillium marneffei conidia. Attachment of P. marneffei conidia in an adherence assay was inhibited by soluble laminin and anti-laminin antibody. N-Acetylneuraminic acid abolished adherence, indicating an interaction mediated by a sialic acid-specific lectin.

Forty-six isolates of Penicillium marneffei were differentiated into two DNA types on the basis of their restriction fragment length polymorphisms. Of the 22 human isolates of P. marneffei, 16 (72.7%) were type I and 6 (27.3%) were type II. Of the 23 bamboo rat isolates, 20 from Rhizomys sumatrensis were type I and 3 from Cannomys badius were type II. The soil isolate was type II. These data represent the first molecular epidemiological study of this important emerging fungal pathogen.

ABSTRACTImmunofluorescence microscopy demonstrated that laminin bound to the surface of Penicillium marneffeiconidia. Attachment ofP. marneffeiconidia in an adherence assay was inhibited by soluble laminin and anti-laminin antibody.N-Acetylneuraminic acid abolished adherence, indicating an interaction mediated by a sialic acid-specific lectin.

The cell-associated and extracellular enzymatic activities were examined in a total of 10 Penicillium marneffei isolates. Both mycelia and yeast expressed alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, whereas a variety of other enzyme activities, including trypsin, chymotrypsin and a-fucosidase were absent. There was some inter-isolate variation in both mycelia and yeast in the activities of other enzymes such as esterases and galactosidases. Enzyme activities did not change significantly over the course of culturing in three representative isolates.