Resumo

Cryptococcus neoformans (CN) is a pathogenic fungus responsible for a high mortality in immunosuppressed patients, including those infected with the human immunodeficiency virus. The most important virulence determinant of CN is the presence of a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is synthesized in the cytoplasm and exported to the outer side of the cell inside the lumen of small vesicles with morphological characteristics that are similar to those found in multivesicular bodies (MVBs). MVBs participate in vesicular mechanisms of unconventional secretion of cytoplasmic molecules. Formation of MVB-associated vesicles is dependent on the biological activity of various proteins of the endosomal sorting complexes required for transport (ESCRT). The vacuolar protein Snf7 is a central component of the ESCRT-III complex, which is involved in addressing several proteins to the MVB formation route. In this work, SNF7 gene deletion mutants were constructed by biolistic transformation of C. neoformans, resulting in strains snf7Δ and snf7Δ::SNF7 (reconstituted). The ability of these strains to secrete GXM was compared by enzyme linked immunosorbent assay (ELISA) with a monoclonal antibody raised against the polysaccharide. The snf7Δ strain manifested defects in GXM secretion. Dimensional determination of GXM-containing vesicles by dynamic light scattering revealed that the mutant cells exported the polysaccharide in vesicles with reduced diameter. The defective GXM secretion was translated into a reduced ability of the mutant to induce capsular growth, as demonstrated by counterstaining with India ink, immunofluorescence using an anti-GXM monoclonal antibody (18B7) and scanning electron microscopy. The snf7Δ mutant also lost its ability to produce melanin when cultured at 30 or 37oC and under copper deprivation. The SNF7-derived defects in polysaccharide secretion, capsule formation and pigmentation resulted in an avirulent phenotype of C. neoformans in a murine intranasal infection model. Taken together, these results demonstrate that the SNF7 gene governs virulence in the C. neoformans model by interfering with unconventional mechanisms of secretion and, consequently, with the metabolism of polysaccharides and pigments.