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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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Analysis of cryptococcal SNF7.(A). Phylogenetic analysis was performed by applying the neighbor-joining method with Snf7 amino acid sequences from distinct organisms as follows: S. cerevisiae, C. albicans, A. nidulans, P. brasiliensis, S. pombe, N. crassa, M. oryzae, A. thaliana, O. sativa, C. neoformans and C. gattii. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. Bootstrap values obtained with 1,000 re-samplings are displayed at the nodes. (B). Analysis of SNF7 disruption in C.neoformans and C. gattii by Southern blot. Genomic DNA (10 μg) from WT, snf7Δ and snf7Δ::SNF7 strains were digested with the Xbal restriction enzyme. The 3′ gene flank was used as probe in Southern hybridization. (C). For RT-PCR, cDNA from C. neoformans and C. gattii (WT, snf7Δ and snf7Δ::SNF7 strains) were used as template and genomic DNA of both fungi (WT cells) was used as positive control. Actin gene (ACT1) was used as reference gene for normalization of RT-PCR.
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f1: Analysis of cryptococcal SNF7.(A). Phylogenetic analysis was performed by applying the neighbor-joining method with Snf7 amino acid sequences from distinct organisms as follows: S. cerevisiae, C. albicans, A. nidulans, P. brasiliensis, S. pombe, N. crassa, M. oryzae, A. thaliana, O. sativa, C. neoformans and C. gattii. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. Bootstrap values obtained with 1,000 re-samplings are displayed at the nodes. (B). Analysis of SNF7 disruption in C.neoformans and C. gattii by Southern blot. Genomic DNA (10 μg) from WT, snf7Δ and snf7Δ::SNF7 strains were digested with the Xbal restriction enzyme. The 3′ gene flank was used as probe in Southern hybridization. (C). For RT-PCR, cDNA from C. neoformans and C. gattii (WT, snf7Δ and snf7Δ::SNF7 strains) were used as template and genomic DNA of both fungi (WT cells) was used as positive control. Actin gene (ACT1) was used as reference gene for normalization of RT-PCR.

Mentions: A database search of the C. gattii genome (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans_b/MultiHome.html- August 5, 2009, strain R265) revealed that the SNF7 (accession number CNBG_3856) coding region comprises 1,087 bp, includes five introns, and encodes a protein of 221 amino acids. A similar search in the C. neoformans genome (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) identified the putative SNF7 sequence (accession number CNAG_01583.2) based on the similarity to the corresponding C. gattii sequence (accession number CNBG_3856). The C. neoformansSNF7 ortholog is 1,413 bp long, contains five introns, and also encodes a putative 221 amino-acid protein. Phylogenetic analysis of Snf7 was performed with orthologues from distinct eukaryotic organisms (Figure 1A). The putative sequence of the C. gattii protein was highly similar to its C. neoformans counterpart (98% identity) and both display similarities to plant proteins (A. thaliana ~35% identity; O. sativa ~24% identity). In comparison to other fungi, the Snf7 sequences of both species studied here showed 45% identity with the S. cerevisiae protein and 43% identity with a C. albicans homologue.


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)

Analysis of cryptococcal SNF7.(A). Phylogenetic analysis was performed by applying the neighbor-joining method with Snf7 amino acid sequences from distinct organisms as follows: S. cerevisiae, C. albicans, A. nidulans, P. brasiliensis, S. pombe, N. crassa, M. oryzae, A. thaliana, O. sativa, C. neoformans and C. gattii. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. Bootstrap values obtained with 1,000 re-samplings are displayed at the nodes. (B). Analysis of SNF7 disruption in C.neoformans and C. gattii by Southern blot. Genomic DNA (10 μg) from WT, snf7Δ and snf7Δ::SNF7 strains were digested with the Xbal restriction enzyme. The 3′ gene flank was used as probe in Southern hybridization. (C). For RT-PCR, cDNA from C. neoformans and C. gattii (WT, snf7Δ and snf7Δ::SNF7 strains) were used as template and genomic DNA of both fungi (WT cells) was used as positive control. Actin gene (ACT1) was used as reference gene for normalization of RT-PCR.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Analysis of cryptococcal SNF7.(A). Phylogenetic analysis was performed by applying the neighbor-joining method with Snf7 amino acid sequences from distinct organisms as follows: S. cerevisiae, C. albicans, A. nidulans, P. brasiliensis, S. pombe, N. crassa, M. oryzae, A. thaliana, O. sativa, C. neoformans and C. gattii. The bar marker indicates the genetic distance, which is proportional to the number of amino acid substitutions. Bootstrap values obtained with 1,000 re-samplings are displayed at the nodes. (B). Analysis of SNF7 disruption in C.neoformans and C. gattii by Southern blot. Genomic DNA (10 μg) from WT, snf7Δ and snf7Δ::SNF7 strains were digested with the Xbal restriction enzyme. The 3′ gene flank was used as probe in Southern hybridization. (C). For RT-PCR, cDNA from C. neoformans and C. gattii (WT, snf7Δ and snf7Δ::SNF7 strains) were used as template and genomic DNA of both fungi (WT cells) was used as positive control. Actin gene (ACT1) was used as reference gene for normalization of RT-PCR.
Mentions: A database search of the C. gattii genome (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans_b/MultiHome.html- August 5, 2009, strain R265) revealed that the SNF7 (accession number CNBG_3856) coding region comprises 1,087 bp, includes five introns, and encodes a protein of 221 amino acids. A similar search in the C. neoformans genome (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) identified the putative SNF7 sequence (accession number CNAG_01583.2) based on the similarity to the corresponding C. gattii sequence (accession number CNBG_3856). The C. neoformansSNF7 ortholog is 1,413 bp long, contains five introns, and also encodes a putative 221 amino-acid protein. Phylogenetic analysis of Snf7 was performed with orthologues from distinct eukaryotic organisms (Figure 1A). The putative sequence of the C. gattii protein was highly similar to its C. neoformans counterpart (98% identity) and both display similarities to plant proteins (A. thaliana ~35% identity; O. sativa ~24% identity). In comparison to other fungi, the Snf7 sequences of both species studied here showed 45% identity with the S. cerevisiae protein and 43% identity with a C. albicans homologue.

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