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The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex.

Godinho RM, Crestani J, Kmetzsch L, Araujo Gde S, Frases S, Staats CC, Schrank A, Vainstein MH, Rodrigues ML - Sci Rep (2014)

Bottom Line: Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans.Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation.Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic.

View Article: PubMed Central - PubMed

Affiliation: 1] Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil [2].

ABSTRACT
Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes.

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Surface architecture and GXM release are affected by SNF7 deletion.(A). Determination of GXM in C. neoformans and C. gattii culture supernatants. Asterisks denote statistically significant differences (**, p < 0.005; ***, p < 0.001) between the values obtained for the mutant strains and those obtained for wild type (WT) and complemented (snf7Δ::SNF7) cells. (B). Microscopic analysis of WT, snf7Δ and snf7Δ::SNF7 strains of C. neoformans and C. gattii. India ink counterstaining and immunostaining (upper and middle panels, respectively) revealed that SNF7 disruption profoundly affects capsule formation. GXM and chitin are stained in green and blue respectively (middle panels). Scale bars represent 5 μm. Scanning electron microscopy of the C. neoformans and C. gattii strains (Lower panels). Scale bar represents 1 μm. (C). Quantification of capsular dimensions based on in silico measurements of the results illustrated in B (***, p < 0.001).
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f5: Surface architecture and GXM release are affected by SNF7 deletion.(A). Determination of GXM in C. neoformans and C. gattii culture supernatants. Asterisks denote statistically significant differences (**, p < 0.005; ***, p < 0.001) between the values obtained for the mutant strains and those obtained for wild type (WT) and complemented (snf7Δ::SNF7) cells. (B). Microscopic analysis of WT, snf7Δ and snf7Δ::SNF7 strains of C. neoformans and C. gattii. India ink counterstaining and immunostaining (upper and middle panels, respectively) revealed that SNF7 disruption profoundly affects capsule formation. GXM and chitin are stained in green and blue respectively (middle panels). Scale bars represent 5 μm. Scanning electron microscopy of the C. neoformans and C. gattii strains (Lower panels). Scale bar represents 1 μm. (C). Quantification of capsular dimensions based on in silico measurements of the results illustrated in B (***, p < 0.001).

Mentions: Quantification of secreted GXM in all strains by ELISA revealed that deletion of SNF7 nearly extinguished polysaccharide export in both C. neoformans and C. gattii (Figure 5A). Morphological analysis of the capsule by India ink counterstaining, immunostaining of GXM and scanning electron microscopy revealed a clear reduction in capsular dimensions in the snf7Δ mutants (Figure 5B), implying defects in capsular assembly. Determination of the capsule/cell diameter ratio confirmed a significant reduction (p < 0.0001) in capsular dimensions in both CN-snf7Δ and CG-snf7Δ cells (Figure 5C).


The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex.

Godinho RM, Crestani J, Kmetzsch L, Araujo Gde S, Frases S, Staats CC, Schrank A, Vainstein MH, Rodrigues ML - Sci Rep (2014)

Surface architecture and GXM release are affected by SNF7 deletion.(A). Determination of GXM in C. neoformans and C. gattii culture supernatants. Asterisks denote statistically significant differences (**, p < 0.005; ***, p < 0.001) between the values obtained for the mutant strains and those obtained for wild type (WT) and complemented (snf7Δ::SNF7) cells. (B). Microscopic analysis of WT, snf7Δ and snf7Δ::SNF7 strains of C. neoformans and C. gattii. India ink counterstaining and immunostaining (upper and middle panels, respectively) revealed that SNF7 disruption profoundly affects capsule formation. GXM and chitin are stained in green and blue respectively (middle panels). Scale bars represent 5 μm. Scanning electron microscopy of the C. neoformans and C. gattii strains (Lower panels). Scale bar represents 1 μm. (C). Quantification of capsular dimensions based on in silico measurements of the results illustrated in B (***, p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4151102&req=5

f5: Surface architecture and GXM release are affected by SNF7 deletion.(A). Determination of GXM in C. neoformans and C. gattii culture supernatants. Asterisks denote statistically significant differences (**, p < 0.005; ***, p < 0.001) between the values obtained for the mutant strains and those obtained for wild type (WT) and complemented (snf7Δ::SNF7) cells. (B). Microscopic analysis of WT, snf7Δ and snf7Δ::SNF7 strains of C. neoformans and C. gattii. India ink counterstaining and immunostaining (upper and middle panels, respectively) revealed that SNF7 disruption profoundly affects capsule formation. GXM and chitin are stained in green and blue respectively (middle panels). Scale bars represent 5 μm. Scanning electron microscopy of the C. neoformans and C. gattii strains (Lower panels). Scale bar represents 1 μm. (C). Quantification of capsular dimensions based on in silico measurements of the results illustrated in B (***, p < 0.001).
Mentions: Quantification of secreted GXM in all strains by ELISA revealed that deletion of SNF7 nearly extinguished polysaccharide export in both C. neoformans and C. gattii (Figure 5A). Morphological analysis of the capsule by India ink counterstaining, immunostaining of GXM and scanning electron microscopy revealed a clear reduction in capsular dimensions in the snf7Δ mutants (Figure 5B), implying defects in capsular assembly. Determination of the capsule/cell diameter ratio confirmed a significant reduction (p < 0.0001) in capsular dimensions in both CN-snf7Δ and CG-snf7Δ cells (Figure 5C).

Bottom Line: Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans.Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation.Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic.

View Article: PubMed Central - PubMed

Affiliation: 1] Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-902, Rio de Janeiro, Brazil [2].

ABSTRACT
Fungal pathogenesis requires a number of extracellularly released virulence factors. Recent studies demonstrating that most fungal extracellular molecules lack secretory tags suggest that unconventional secretion mechanisms and fungal virulence are strictly connected. Proteins of the endosomal sorting complex required for transport (ESCRT) have been recently associated with polysaccharide export in the yeast-like human pathogen Cryptococcus neoformans. Snf7 is a key ESCRT operator required for unconventional secretion in Eukaryotes. In this study we generated snf7Δ mutant strains of C. neoformans and its sibling species C. gattii. Lack of Snf7 resulted in important alterations in polysaccharide secretion, capsular formation and pigmentation. This phenotype culminated with loss of virulence in an intranasal model of murine infection in both species. Our data support the notion that Snf7 expression regulates virulence in C. neoformans and C. gattii by ablating polysaccharide and melanin traffic. These results are in agreement with the observation that unconventional secretion is essential for cryptococcal pathogenesis and strongly suggest the occurrence of still obscure mechanisms of exportation of non-protein molecules in Eukaryotes.

Show MeSH
Related in: MedlinePlus