Molecular cloning and biochemical characterization of a Cu,Zn-superoxide dismutase from Scedosporium apiospermum.

A Cu,Zn-superoxide dismutase has been characterized from Scedosporium apiospermum, a fungus which often colonizes the respiratory tract of patients with cystic fibrosis. Enzyme production was stimulated by iron starvation. Purification was achieved from mycelial extract from 7-day-old cultures on Amberlite XAD-16. The purified enzyme presented a relative molecular mass of 16.4 kDa under reducing conditions and was inhibited by potassium cyanide and diethyldithiocarbamate, which are two known inhibitors of Cu,Zn-SODs. Its optimum pH was 7.0 and the enzyme retained full activity after pretreatment at temperatures up to 50 degrees C. Moreover, a 450-bp fragment of the gene encoding the enzyme was amplified by PCR using degenerate primers designed from sequence alignment of four fungal Cu,Zn-SODs. Sequence data from this fragment allowed us to design primers which were used to amplify by walking-PCR the flanking regions of the known fragment. SaSODC gene (890 bp) corresponded to a 154 amino acid polypeptide with a predicted molecular mass of 15.9 kDa. A database search for sequence homology revealed for the deduced amino acid sequence 72 and 83% identity rate with Cu,Zn-SODs from Aspergillus fumigatus and Neurospora crassa, respectively. To our knowledge, this enzyme is the first putative virulence factor of S. apiospermum to be characterized.

Immunofluorescence and flow cytometry analysis of fibronectin and laminin binding to Sporothrix schenckii yeast cells and conidia.

The adherence of Sporothrix schenckii yeast cells to several extracellular matrix (ECM) components has already been demonstrated, but the mechanisms of these interactions remained to be defined. In indirect immunofluorescence assays with polyclonal antibodies directed towards the ECM proteins, both hyphae and yeast cells of S. schenckii exhibited the ability to bind laminin and fibronectin. Flow cytometry confirmed the binding of these proteins, and revealed a significant greater binding capability for the yeast cells than for the conidia. Fibronectin and laminin binding was dose-dependent and specific. In addition, competition experiments with synthetic peptides mimicking the adhesive sequences of these proteins, or with cell wall fractions and carbohydrates constitutive of their sugar chains, were performed in order to specify the peptide or carbohydrate motifs involved in the recognition process. A 50% reduction was noticed in fibronectin binding in the presence of the synthetic peptide RGD, and a 38% reduction in laminin binding with the peptide YIGSR. Some carbohydrate-containing fractions of the yeast cell wall also inhibited the binding of fibronectin, but had no significant effect on laminin binding. Together, these results suggest the presence at the yeast surface of distinct receptors for laminin and fibronectin.

Evaluation of the in vitro and in vivo dimorphism of Sporothrix schenckii, Blastomyces dermatitidis, and Paracoccidioides brasiliensis isolates after preservation in mineral oil.

Morphological differentiation has commanded attention for its putative impact on the pathogenesis of invasive fungal infections. We evaluated in vitro and in vivo the dimorphism from mycelial to yeast-phase of Sporothrix schenckii, Blastomyces dermatitidis and Paracoccidioides brasiliensis isolates, two strains for each species, preserved in mineral oil. S. schenckii strains showed typical micromorphology at 25 degrees C but one strain was unable to complete the dimorphic process in vitro. After in vivo passage through mice the strains had the ability to turn into yeast-like cells and to form colonies on brain-heart infusion medium at 36 degrees C. B. dermatitidis strains grew as dirty white to brownish membranous colonies at 25 degrees C and their micromorphology showed thin filaments with single hyaline conidia. At 36 degrees C the colonies did not differ from those grown at 25 degrees C, but produced a transitional micromorphology. P. brasiliensis strains grew as cream-colored cerebriform colonies at 25 degrees C showing a transitional morphology. B. dermatitidis and P. brasiliensis strains did not turn into yeast-like cells in vivo. The present results demonstrate that B. dermatitidis and P. brasiliensis strains were unable to complete the dimorphic process even after in vivo passage, in contrast to the S. schenckii strain.

The in vitro interaction of Sporothrix schenckii with human endothelial cells is modulated by cytokines and involves endothelial surface molecules.

Sporothrix schenckii is the etiological agent of sporotrichosis, a subcutaneous mycosis that can evolve to systemic complications in immunocompromised patients. Interactions with endothelium are thought to be essential for systemic infections. In the present work, we studied the interaction between S. schenckii and human umbilical vein endothelial cells (HUVECs). S. schenckii interacts with HUVECs in a time-dependent manner. Morphological analysis showed that yeasts locate to interendothelial junctions. Ultrastructural studies showed that internalized yeasts were found inside endocytic vacuoles as early as 2 h, without causing any detectable damage to HUVECs after 24 h of infection. The viability of infected HUVECs was confirmed by the MTT assay. When HUVECs were treated with different concentrations of Interleukin-1beta or transforming growth factor-beta, a significant dose-dependent increase in cell-associated yeasts was observed. The preliminary analysis of the endothelial surface ligands for S. schenckii cells revealed two major molecules, with Mr of approximately 90 and 135 kDa. The interaction of endothelial cell surface molecules with S. schenckii yeast cells was modulated by divalent cations. This is the first demonstration that S. schenckii is able to adhere and invade endothelial cells without significantly affect cellular integrity. Our results suggest the contribution of cytokine-modulated calcium-dependent molecules to this process.

Relationships between respiration and susceptibility to azole antifungals in Candida glabrata.

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.

Ligação de células de levedura e conídios do Sporothrix schenckii à fibronectina e laminina / Binding of yeast cells and conídios of the fungus Sporothrix schenckii to the fibronectin and laminin

Sporothrix ashenckii é o agente etiológico da esporotricose uma micose cutânea e subcutânea. Geralmente as infeções têm seu início com a aderência dos patógenos à membrana de células do hospedeiro ou à matriz extracelular. Em nossos estudos, células do S. schenckii demonstraram a capacidade de ligar-se à laminina e à fibronectina purificadas nas formas solúvel e imobilizada. Além disso, a ligação a fibronectina imobilizada foi aumentada na presença de cálcio sugerido a presença de adesinas dependentes de íons divalentes. Mais ainda, alguns monossacarídeos não exibiram efeito na ligação à fibronectina. Entretanto, um extrato rico em glicídios obtidos a partir do fracionamento de um glicopeptideo da parede celular em coluna MonoQ inibiu em até 30 por cento a ligação de células de levedura a fibronectina. Esses dados sugerem a participação de açúcares na ligação à proteína imobilizada. A citometria do fluxo confirmou a ligação de fibronectina e lamina solúveis ao fungo, mas o fibrinogênio não demostrou capacidade adesiva para ambas as formas morfológicas de S. schenkii. Ligação da lamina e fibronectina solúveis foi específica e dose-dependente. Experimentos de competição com peptídeos que mimetizam sequências adesivas presentes nas proteínas da MEC ou com carboidratos presentes nas cadeias glicídicas de fibronectina e laminina foram realizados para identificar os peptídeos ou carbodratos envolvidos no reconhecimento fungo-matriz. Uma redução de até 85 por cento na ligação a fibronectina foi observada na presença de peptídeos do tipo RGD e para laminina a redução foi de aproximadamente 70 por cento com o peptídeo YIGSR. Além disso, em testes com fibronectina e laminina solúveis, a ligação a células de levedura foi aumentada de forma marcante na presença de ácido siálico. As frações da parede obtidas por troca aniônica também foram capazes de inibir a ligação da fibronectina solúvel ao S. schenckii mas teve efeito na interação com laminina. Estes dados sugerem, portanto, a presença na superfície das células de levedura de receptores distintos, modulados por ácido siálico, capazes de interagir com laminina e fibronectina. Os eventos de ligação aqui descritos podem ter papel no estabelecimento da infecção.