Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis. This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung. Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S. schenckii and several extracellular matrix (ECM) proteins. The binding of two morphological phases of S. schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated. Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control. Cell adhesion was assessed by ELISA with a rabbit anti-S. schenckii antiserum. The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent). The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P<0.05) when compared to the adhesion obtained with BSA. No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen. Our results indicate that S. schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus.

Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients. Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases. Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn). Both yeast cells and conidia of S. schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays. Adhesion of yeast cells to Fn is dose dependent and saturable. S. schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments. While adhesion to Fn was increased by Ca 2؉ , inhibition assays demonstrated that it was not RGD dependent. A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells. The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties. These results provide new data concerning S. schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.Infections caused by the dimorphic mycopathogen Sporothrix schenckii have increased in recent years mainly in immunocompromised patients (7,17). S. schenckii is found as mycelium in its saprophytic form and as yeast cells in human lesions. S. schenckii can cause either limited cutaneous lesions or invasive, disseminated infections (17). Systemic sporotrichosis may be due to conidia inhalation (8) or bloodstream dissemination from a cutaneous lesion (4). Risk factors such as alcoholism, diabetes, and extensive use of immunosuppressive drugs may predispose to severe infections, including pulmonary and osteoarticular sporotrichosis (17).Adherence of pathogenic microorganisms to host tissues is regarded as a prerequisite for dissemination. Microbial adherence has been studied extensively in pathogenic bacteria (15) and fungi such as Candida albicans (37), Aspergillus fumigatus (3), and Blastomyces dermatitidis (19), but little is known about the adherence mechanisms in S. schenckii.Extracellular matrices (ECM) are covered by epithelial and endothelial cells. However, cell injury and exposure of subendothelial ECM may occur during infections (20). Fibronectin (Fn), a large (440 kDa) dimeric glycoprotein, is a multifunctional ECM protein with a central role in cell adhesion and spreading (29). Fn has been implicated in adherence of several pathogens such as Aspergillus fumigatus (31, 39), Candida albicans (21), Candida tropicalis (1, 5), and Pneumocystis carinii (33). Adherence to Fn is a virulence factor for Staphylococcus aureus, since an Fn binding protein is important for internalization of this bacterium by nonprofessional phagocytes (36). Different mechanisms seem to be involved in these adhesion processes. Either a protein-protein or a lectin-like interaction was described for several fungal pathogens (14,35,39). It was also reported that adherence of A. fumigatus to Fn varied in accordance with ...

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