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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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Effects of SNF7 disruption on fungal growth under different conditions.(A). Wild type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii were grown in YPD broth with shaking at 30°C or 37°C. Growth measurements (OD600) were performed at 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. (B). Ten-fold serial dilutions of all strains were plated on LCM + Cu and LCM agar plates. The plates were incubated for 2 days at 30°C or 37°C. Fungal growth in LCM + Cu represented the control condition. (C). Growth rates of WT, mutant and complemented strains at pHs 7, 7.5, 8, 8.5 and 9. (D). Growth of WT, snf7Δ and snf7Δ::SNF7 strains on YPD agar plates supplemented with 150 mM LiCl2. Experiments were performed in triplicate and representative examples are shown.
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f2: Effects of SNF7 disruption on fungal growth under different conditions.(A). Wild type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii were grown in YPD broth with shaking at 30°C or 37°C. Growth measurements (OD600) were performed at 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. (B). Ten-fold serial dilutions of all strains were plated on LCM + Cu and LCM agar plates. The plates were incubated for 2 days at 30°C or 37°C. Fungal growth in LCM + Cu represented the control condition. (C). Growth rates of WT, mutant and complemented strains at pHs 7, 7.5, 8, 8.5 and 9. (D). Growth of WT, snf7Δ and snf7Δ::SNF7 strains on YPD agar plates supplemented with 150 mM LiCl2. Experiments were performed in triplicate and representative examples are shown.

Mentions: We first evaluated whether the growth rates of the snf7Δ strains varied under different conditions. C. neoformans and C. gattii mutant strains showed normal growth at both 30 and 37°C, in comparison to parental and reconstituted strains (Figure 2A). Since copper acquisition is important for C. neoformans melanization and capsule formation2829 we also evaluated the ability of the snf7Δ mutants to grow under copper deprivation conditions. Once again, the mutants showed normal growth rates, in comparison to WT and complemented cells (Figure 2B).


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)

Effects of SNF7 disruption on fungal growth under different conditions.(A). Wild type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii were grown in YPD broth with shaking at 30°C or 37°C. Growth measurements (OD600) were performed at 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. (B). Ten-fold serial dilutions of all strains were plated on LCM + Cu and LCM agar plates. The plates were incubated for 2 days at 30°C or 37°C. Fungal growth in LCM + Cu represented the control condition. (C). Growth rates of WT, mutant and complemented strains at pHs 7, 7.5, 8, 8.5 and 9. (D). Growth of WT, snf7Δ and snf7Δ::SNF7 strains on YPD agar plates supplemented with 150 mM LiCl2. Experiments were performed in triplicate and representative examples are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effects of SNF7 disruption on fungal growth under different conditions.(A). Wild type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii were grown in YPD broth with shaking at 30°C or 37°C. Growth measurements (OD600) were performed at 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. (B). Ten-fold serial dilutions of all strains were plated on LCM + Cu and LCM agar plates. The plates were incubated for 2 days at 30°C or 37°C. Fungal growth in LCM + Cu represented the control condition. (C). Growth rates of WT, mutant and complemented strains at pHs 7, 7.5, 8, 8.5 and 9. (D). Growth of WT, snf7Δ and snf7Δ::SNF7 strains on YPD agar plates supplemented with 150 mM LiCl2. Experiments were performed in triplicate and representative examples are shown.
Mentions: We first evaluated whether the growth rates of the snf7Δ strains varied under different conditions. C. neoformans and C. gattii mutant strains showed normal growth at both 30 and 37°C, in comparison to parental and reconstituted strains (Figure 2A). Since copper acquisition is important for C. neoformans melanization and capsule formation2829 we also evaluated the ability of the snf7Δ mutants to grow under copper deprivation conditions. Once again, the mutants showed normal growth rates, in comparison to WT and complemented cells (Figure 2B).

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