Sporotrichosis is a subcutaneous mycosis caused by pathogenic species of the Sporothrix genus. A new emerging species, Sporothrix brasiliensis, is related to cat-transmitted sporotrichosis and has severe clinical manifestations. The cell wall of pathogenic fungi is a unique structure and impacts directly on the host immune response. We reveal and compare the cell wall structures of Sporothrix schenckii and S. brasiliensis using high-pressure freezing electron microscopy to study the cell wall organization of both species. To analyze the components of the cell wall, we also used infrared and 13C and 1H NMR spectroscopy and the sugar composition was determined by quantitative high-performance anion-exchange chromatography. Our ultrastructural data revealed a bi-layered cell wall structure for both species, including an external microfibrillar layer and an inner electron-dense layer. The inner and outer layers of the S. brasiliensis cell wall were thicker than those of S. schenckii, correlating with an increase in the chitin and rhamnose contents. Moreover, the outer microfibrillar layer of the S. brasiliensis cell wall had longer microfibrils interconnecting yeast cells. Distinct from those of other dimorphic fungi, the cell wall of Sporothrix spp. lacked α-glucan component. Interestingly, glycogen α-particles were identified in the cytoplasm close to the cell wall and the plasma membrane. The cell wall structure as well as the presence of glycogen α-particles varied over time during cell culture. The structural differences observed in the cell wall of these Sporothrix species seemed to impact its uptake by monocyte-derived human macrophages. The data presented here show a unique cell wall structure of S. brasiliensis and S. schenckii during the yeast parasitic phase. A new cell wall model for Sporothrix spp. is therefore proposed that suggests that these fungi molt sheets of intact cell wall layers. This observation may have significant effects on localized and disseminated immunopathology.

No abstract

In this report we identified orthologues of fungal AGS1, RHO1, RHO2, RAC1 and CDC42 genes in the dimorphic fungus Paracoccidioides brasiliensis. Based on its homology to known fungal sequences, P. brasiliensis Ags1 was identified as an alpha-1,3-glucan synthase, while Rho1, Rho2, Rac1 and Cdc42 proteins were classified into the Rho1, Rho2, Rac1 and Cdc42 subgroups of fungal Rho GTPases, respectively. Of them, Rho1 is one of two subunits of a putative beta-1,3-glucan synthase complex, the other being the synthase itself (Fks1), while Rho2 has been associated to the alpha-1,3-glucan synthase (Ags1). Expression studies showed that mRNAs levels of RHO2 and AGS1 kept a direct relationship but the levels of RHO1 and FKS1 did not. P. brasiliensis RHO1 successfully restored growth of Saccharomyces cerevisiae rho1 mutant under restrictive temperature conditions. Chemical analyses of P. brasiliensis alpha-1,3-glucan, synthesized by Ags1p, indicated that it is essentially a linear polysaccharide, with <3% of alpha-1,4-linked glucose branches, occasionally attached as single units to the alpha-1,3-backbone.

From a 0.72-kb fragment universally generated in Paracoccidioides brasiliensis strains, primers were designed and tested on genomic DNA of this and other pathogenic fungi. They were specific and highly sensitive for P. brasiliensis DNA. Positive results were obtained when these were tested in clinical samples. Paracoccidioidomycosis (PCM), caused byParacoccidioides brasiliensis, is a chronic granulomatous systemic mycosis prevalent in rural areas of Latin America (16).

Sporothrix schenckii, Sporothrix brasiliensis, and Sporothrix globosa are the main causative agents of sporotrichosis, a human subcutaneous mycosis. Differences in virulence patterns are associated with each species but remain largely uncharacterized. The S. schenckii and S. brasiliensis cell wall composition and virulence are influenced by the culturing media, with little or no influence on S. globosa. By keeping constant the culturing media, we compared the cell wall composition of three S. schenckii and two S. brasiliensis strains, previously described as presenting different virulence levels on a murine model of infection. The cell wall composition of the five Sporothrix spp. strains correlated with the biochemical composition of the cell wall previously reported for the species. However, the rhamnose-to-β-glucan ratio exhibits differences among strains, with an increase in cell wall rhamnose-to-β-glucan ratio as their virulence increased. This relationship can be expressed mathematically, which could be an important tool for the determination of virulence in Sporothrix spp. Also, structural differences in rhamnomannan were found, with longer side chains present in strains with lower virulence reported for both species here studied, adding insight to the importance of this polysaccharide in the pathogenic process of these fungi.