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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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Disruption of SNF7 affects pigmentation but not extracellular enzyme activity.(A). Fungal cells were grown on Niger seed agar and L-DOPA plates to evaluate melanin production by wild-type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii. The cultures were incubated at 30°C and 37°C and monitored for 2 (Niger), 3 (L-DOPA 30°C) or 4 (L-DOPA 37°C) days of cultivation. (B). Phospholipase activity in egg yolk agar. All strains were inoculated on plates containing egg yolk agar and incubated at 30°C for 4 days, and Pz values were then determined. (C). Urease activity levels of WT, snf7Δ, snf7Δ::SNF7 cells. Fungal cells were incubated in urea broth with shaking (200 rpm) at 37°C for 24 hours for colorimetric determination. Statistical differences were not observed when parental, mutant and complemented strains were compared.
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f4: Disruption of SNF7 affects pigmentation but not extracellular enzyme activity.(A). Fungal cells were grown on Niger seed agar and L-DOPA plates to evaluate melanin production by wild-type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii. The cultures were incubated at 30°C and 37°C and monitored for 2 (Niger), 3 (L-DOPA 30°C) or 4 (L-DOPA 37°C) days of cultivation. (B). Phospholipase activity in egg yolk agar. All strains were inoculated on plates containing egg yolk agar and incubated at 30°C for 4 days, and Pz values were then determined. (C). Urease activity levels of WT, snf7Δ, snf7Δ::SNF7 cells. Fungal cells were incubated in urea broth with shaking (200 rpm) at 37°C for 24 hours for colorimetric determination. Statistical differences were not observed when parental, mutant and complemented strains were compared.

Mentions: Extracellularly released enzymes and exported pigments are fundamental for cryptococcal pathogenesis51736373839. Therefore, we analyzed whether SNF7 disruption would impact melanization and the extracellular activities of urease and phospholipase. Pigmentation was evaluated visually after growth of C. neoformans and C. gattii on both Niger seed and L-DOPA-containing solid media (Figure 4A). Under both conditions, the CN-snf7Δ mutant strain was similar to parental (WT) and complemented (reconstituted) strains in its ability to melanize at 30°C. However, when cultivated at 37°C, the mutant CN-snf7Δ showed reduced pigmentation. Differently, the CG-snf7Δ mutant showed subtle melanization defects when cultivated on L-DOPA agar at 30°C. At 37°C, the pigmentation defects were more evident. On Niger seed agar, SNF7 deletion in C. gattii affected pigmentation at 30°C, but not at 37°C. These results revealed an unexpected inter-species diversity in the relationship between Snf7 and pigmentation.


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)

Disruption of SNF7 affects pigmentation but not extracellular enzyme activity.(A). Fungal cells were grown on Niger seed agar and L-DOPA plates to evaluate melanin production by wild-type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii. The cultures were incubated at 30°C and 37°C and monitored for 2 (Niger), 3 (L-DOPA 30°C) or 4 (L-DOPA 37°C) days of cultivation. (B). Phospholipase activity in egg yolk agar. All strains were inoculated on plates containing egg yolk agar and incubated at 30°C for 4 days, and Pz values were then determined. (C). Urease activity levels of WT, snf7Δ, snf7Δ::SNF7 cells. Fungal cells were incubated in urea broth with shaking (200 rpm) at 37°C for 24 hours for colorimetric determination. Statistical differences were not observed when parental, mutant and complemented strains were compared.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Disruption of SNF7 affects pigmentation but not extracellular enzyme activity.(A). Fungal cells were grown on Niger seed agar and L-DOPA plates to evaluate melanin production by wild-type (WT), mutant (snf7Δ) and complemented (snf7Δ::SNF7) strains of C. neoformans and C. gattii. The cultures were incubated at 30°C and 37°C and monitored for 2 (Niger), 3 (L-DOPA 30°C) or 4 (L-DOPA 37°C) days of cultivation. (B). Phospholipase activity in egg yolk agar. All strains were inoculated on plates containing egg yolk agar and incubated at 30°C for 4 days, and Pz values were then determined. (C). Urease activity levels of WT, snf7Δ, snf7Δ::SNF7 cells. Fungal cells were incubated in urea broth with shaking (200 rpm) at 37°C for 24 hours for colorimetric determination. Statistical differences were not observed when parental, mutant and complemented strains were compared.
Mentions: Extracellularly released enzymes and exported pigments are fundamental for cryptococcal pathogenesis51736373839. Therefore, we analyzed whether SNF7 disruption would impact melanization and the extracellular activities of urease and phospholipase. Pigmentation was evaluated visually after growth of C. neoformans and C. gattii on both Niger seed and L-DOPA-containing solid media (Figure 4A). Under both conditions, the CN-snf7Δ mutant strain was similar to parental (WT) and complemented (reconstituted) strains in its ability to melanize at 30°C. However, when cultivated at 37°C, the mutant CN-snf7Δ showed reduced pigmentation. Differently, the CG-snf7Δ mutant showed subtle melanization defects when cultivated on L-DOPA agar at 30°C. At 37°C, the pigmentation defects were more evident. On Niger seed agar, SNF7 deletion in C. gattii affected pigmentation at 30°C, but not at 37°C. These results revealed an unexpected inter-species diversity in the relationship between Snf7 and pigmentation.

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