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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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SNF7 disruption affects the expression of RIM101.(A). Conceptual basis for the analysis of Rim101 and Rim20 expression in C. neoformans and C. gattii strains. Rim101 activation depends on the recruitment of ESCRTI, ESCRTII and ESCRTIII components (Vps20 and Snf7) to the cytoplasmic face of the endosomal membrane. The Snf7 and Rim20/Rim13 protein complex interacts with the Rim101 precursor, which is further cleaved to release the active form of Rim101. This process results in the regulation of cryptococcal genes playing key roles in a number of cellular events. Adapted from Selvig and Alspaugh87. Expression levels of RIM101 and RIM20 were assessed in wild-type (WT), mutant (snf7) and complemented (snf7::SNF7) strains of C.neoformans (B) and C.gattii (C). The expression levels were normalized to the wild-type using the 2−ΔΔCt88.The experiments were performed with two biological samples, and each cDNA sample was analyzed in triplicate with each primer pair. (*, p < 0.05 in comparison with WT and complemented cells).
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f3: SNF7 disruption affects the expression of RIM101.(A). Conceptual basis for the analysis of Rim101 and Rim20 expression in C. neoformans and C. gattii strains. Rim101 activation depends on the recruitment of ESCRTI, ESCRTII and ESCRTIII components (Vps20 and Snf7) to the cytoplasmic face of the endosomal membrane. The Snf7 and Rim20/Rim13 protein complex interacts with the Rim101 precursor, which is further cleaved to release the active form of Rim101. This process results in the regulation of cryptococcal genes playing key roles in a number of cellular events. Adapted from Selvig and Alspaugh87. Expression levels of RIM101 and RIM20 were assessed in wild-type (WT), mutant (snf7) and complemented (snf7::SNF7) strains of C.neoformans (B) and C.gattii (C). The expression levels were normalized to the wild-type using the 2−ΔΔCt88.The experiments were performed with two biological samples, and each cDNA sample was analyzed in triplicate with each primer pair. (*, p < 0.05 in comparison with WT and complemented cells).

Mentions: The ESCRT machinery, including Snf7, plays a central role in resistance to higher pHs and tolerance to ionic lithium303132. Therefore, we analyzed the ability of the snf7Δ mutants to survive under these conditions. Analysis of fungal growth in pHs ranging from 7 to 9 suggested lower growth rates of both CN-snf7Δ and CG-snf7Δ in pHs above 7.5, in comparison to parental and reconstituted cells (Figure 2C; p < 0.05 for all comparisons between cells expressing Snf7 and snf7Δ strains). A similar result was obtained when the C. neoformans and C. gattii strains were cultivated in the presence of 150 mM LiCl2 (Figure 2D). The phenotypes described above in C. neoformans and other fungal species are related to the interaction between Snf7 and molecules belonging to the RIM101 pathway (Figure 3A), which includes Rim101p and Rim20p30333435. Therefore, to support the notion that the phenotypes we observed were linked to defects in Rim101p activation via interaction with Snf7/Rim20p we assessed the expression of RIM101 and RIM20. We observed that although the mRNA levels of RIM20 remained the same for all strains, the expression of RIM101 was significantly lower in both snf7Δ mutants (Figure 3B and C). This observation is in agreement with a lower activation of RIM101p due to lack of Snf7.


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)

SNF7 disruption affects the expression of RIM101.(A). Conceptual basis for the analysis of Rim101 and Rim20 expression in C. neoformans and C. gattii strains. Rim101 activation depends on the recruitment of ESCRTI, ESCRTII and ESCRTIII components (Vps20 and Snf7) to the cytoplasmic face of the endosomal membrane. The Snf7 and Rim20/Rim13 protein complex interacts with the Rim101 precursor, which is further cleaved to release the active form of Rim101. This process results in the regulation of cryptococcal genes playing key roles in a number of cellular events. Adapted from Selvig and Alspaugh87. Expression levels of RIM101 and RIM20 were assessed in wild-type (WT), mutant (snf7) and complemented (snf7::SNF7) strains of C.neoformans (B) and C.gattii (C). The expression levels were normalized to the wild-type using the 2−ΔΔCt88.The experiments were performed with two biological samples, and each cDNA sample was analyzed in triplicate with each primer pair. (*, p < 0.05 in comparison with WT and complemented cells).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4151102&req=5

f3: SNF7 disruption affects the expression of RIM101.(A). Conceptual basis for the analysis of Rim101 and Rim20 expression in C. neoformans and C. gattii strains. Rim101 activation depends on the recruitment of ESCRTI, ESCRTII and ESCRTIII components (Vps20 and Snf7) to the cytoplasmic face of the endosomal membrane. The Snf7 and Rim20/Rim13 protein complex interacts with the Rim101 precursor, which is further cleaved to release the active form of Rim101. This process results in the regulation of cryptococcal genes playing key roles in a number of cellular events. Adapted from Selvig and Alspaugh87. Expression levels of RIM101 and RIM20 were assessed in wild-type (WT), mutant (snf7) and complemented (snf7::SNF7) strains of C.neoformans (B) and C.gattii (C). The expression levels were normalized to the wild-type using the 2−ΔΔCt88.The experiments were performed with two biological samples, and each cDNA sample was analyzed in triplicate with each primer pair. (*, p < 0.05 in comparison with WT and complemented cells).
Mentions: The ESCRT machinery, including Snf7, plays a central role in resistance to higher pHs and tolerance to ionic lithium303132. Therefore, we analyzed the ability of the snf7Δ mutants to survive under these conditions. Analysis of fungal growth in pHs ranging from 7 to 9 suggested lower growth rates of both CN-snf7Δ and CG-snf7Δ in pHs above 7.5, in comparison to parental and reconstituted cells (Figure 2C; p < 0.05 for all comparisons between cells expressing Snf7 and snf7Δ strains). A similar result was obtained when the C. neoformans and C. gattii strains were cultivated in the presence of 150 mM LiCl2 (Figure 2D). The phenotypes described above in C. neoformans and other fungal species are related to the interaction between Snf7 and molecules belonging to the RIM101 pathway (Figure 3A), which includes Rim101p and Rim20p30333435. Therefore, to support the notion that the phenotypes we observed were linked to defects in Rim101p activation via interaction with Snf7/Rim20p we assessed the expression of RIM101 and RIM20. We observed that although the mRNA levels of RIM20 remained the same for all strains, the expression of RIM101 was significantly lower in both snf7Δ mutants (Figure 3B and C). This observation is in agreement with a lower activation of RIM101p due to lack of Snf7.

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