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Pleiotropic effects of deubiquitinating enzyme Ubp5 on growth and pathogenesis of Cryptococcus neoformans.

Fang W, Price MS, Toffaletti DL, Tenor J, Betancourt-Quiroz M, Price JL, Pan WH, Liao WQ, Perfect JR - PLoS ONE (2012)

Bottom Line: Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents).Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans.Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Medical Mycology, PLA Key Laboratory of Mycosis, Institute of Dermatology and Mycosis of Changzheng Hospital, Second Military Medical University, Shanghai, China.

ABSTRACT
Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

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Structure analysis of deubiquitinating enzymes.A. Domain structure of cryptococcal deubiquitinating enzymes. 7 DUB enzymes in this study belong to three subfamilies (USP, OTU, and JAMM) on the basis of their catalytic domains. USP and OTU domain DUBs are cysteine proteases, JAMM domain DUB is metalloprotease. We retrieved domain architectures for each DUB using the Pfam databases (http://pfam.sanger.ac.uk/). B. Comparison of Ubp5 orthologs between C. neoformans and other species. Each Ubp5 ortholog diagram shows functional protein domains, which were identified by the Pfam database. C. Phylogenetic tree analysis of Ubp5 orthologs is depicted by Clustal W alignment from DNASTAR software (versions 6.13). Protein sequences of Ubp5 orthologs were retrieved from the following database: C. neoformans Ubp5 (CNAG_05650) from the C. neoformans var. grubii H99 database of the Broad Institute, and its orthologs among other eukaryotic species from the protein databank of NCBI website. Cn, Cryptococcus neoformans; Um, Ustilago maydis; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ca, Candidia albicans; Af, Aspergillus fumigatus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens.
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pone-0038326-g001: Structure analysis of deubiquitinating enzymes.A. Domain structure of cryptococcal deubiquitinating enzymes. 7 DUB enzymes in this study belong to three subfamilies (USP, OTU, and JAMM) on the basis of their catalytic domains. USP and OTU domain DUBs are cysteine proteases, JAMM domain DUB is metalloprotease. We retrieved domain architectures for each DUB using the Pfam databases (http://pfam.sanger.ac.uk/). B. Comparison of Ubp5 orthologs between C. neoformans and other species. Each Ubp5 ortholog diagram shows functional protein domains, which were identified by the Pfam database. C. Phylogenetic tree analysis of Ubp5 orthologs is depicted by Clustal W alignment from DNASTAR software (versions 6.13). Protein sequences of Ubp5 orthologs were retrieved from the following database: C. neoformans Ubp5 (CNAG_05650) from the C. neoformans var. grubii H99 database of the Broad Institute, and its orthologs among other eukaryotic species from the protein databank of NCBI website. Cn, Cryptococcus neoformans; Um, Ustilago maydis; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ca, Candidia albicans; Af, Aspergillus fumigatus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens.

Mentions: Bioinformatic analysis of the putative cryptococcal DUB proteins in this study showed that five of them belong to the USP subfamily (the largest one in C. neoformans includes 15 DUBs), while the other two belong to either the OTU or JAMM subfamilies. Many of these DUBs have amino-terminal extensions while some have carboxyl-terminal extensions (Figure 1A). Even within the same subfamily, their catalytic domains exhibit significant differences in length, suggesting that there must be different insertions in the catalytic domains of many isoforms. Previous studies have confirmed that DUBs do display specificity for both substrates and particular ubiquitin chain types, yet they all share the basic role of cleaving off ubiquitin from other adduct or fusion proteins [34], [20], [21]. These extensions and insertions may contribute to the functional diversity of the DUB enzymes. Interestingly, we found that deletion of UBP5 generated many intriguing phenotypic changes in C. neoformans that were linked to virulence. Therefore, we focused in detail on UBP5 for its relationship to pathogenesis.


Pleiotropic effects of deubiquitinating enzyme Ubp5 on growth and pathogenesis of Cryptococcus neoformans.

Fang W, Price MS, Toffaletti DL, Tenor J, Betancourt-Quiroz M, Price JL, Pan WH, Liao WQ, Perfect JR - PLoS ONE (2012)

Structure analysis of deubiquitinating enzymes.A. Domain structure of cryptococcal deubiquitinating enzymes. 7 DUB enzymes in this study belong to three subfamilies (USP, OTU, and JAMM) on the basis of their catalytic domains. USP and OTU domain DUBs are cysteine proteases, JAMM domain DUB is metalloprotease. We retrieved domain architectures for each DUB using the Pfam databases (http://pfam.sanger.ac.uk/). B. Comparison of Ubp5 orthologs between C. neoformans and other species. Each Ubp5 ortholog diagram shows functional protein domains, which were identified by the Pfam database. C. Phylogenetic tree analysis of Ubp5 orthologs is depicted by Clustal W alignment from DNASTAR software (versions 6.13). Protein sequences of Ubp5 orthologs were retrieved from the following database: C. neoformans Ubp5 (CNAG_05650) from the C. neoformans var. grubii H99 database of the Broad Institute, and its orthologs among other eukaryotic species from the protein databank of NCBI website. Cn, Cryptococcus neoformans; Um, Ustilago maydis; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ca, Candidia albicans; Af, Aspergillus fumigatus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens.
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pone-0038326-g001: Structure analysis of deubiquitinating enzymes.A. Domain structure of cryptococcal deubiquitinating enzymes. 7 DUB enzymes in this study belong to three subfamilies (USP, OTU, and JAMM) on the basis of their catalytic domains. USP and OTU domain DUBs are cysteine proteases, JAMM domain DUB is metalloprotease. We retrieved domain architectures for each DUB using the Pfam databases (http://pfam.sanger.ac.uk/). B. Comparison of Ubp5 orthologs between C. neoformans and other species. Each Ubp5 ortholog diagram shows functional protein domains, which were identified by the Pfam database. C. Phylogenetic tree analysis of Ubp5 orthologs is depicted by Clustal W alignment from DNASTAR software (versions 6.13). Protein sequences of Ubp5 orthologs were retrieved from the following database: C. neoformans Ubp5 (CNAG_05650) from the C. neoformans var. grubii H99 database of the Broad Institute, and its orthologs among other eukaryotic species from the protein databank of NCBI website. Cn, Cryptococcus neoformans; Um, Ustilago maydis; Sp, Schizosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ca, Candidia albicans; Af, Aspergillus fumigatus; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Hs, Homo sapiens.
Mentions: Bioinformatic analysis of the putative cryptococcal DUB proteins in this study showed that five of them belong to the USP subfamily (the largest one in C. neoformans includes 15 DUBs), while the other two belong to either the OTU or JAMM subfamilies. Many of these DUBs have amino-terminal extensions while some have carboxyl-terminal extensions (Figure 1A). Even within the same subfamily, their catalytic domains exhibit significant differences in length, suggesting that there must be different insertions in the catalytic domains of many isoforms. Previous studies have confirmed that DUBs do display specificity for both substrates and particular ubiquitin chain types, yet they all share the basic role of cleaving off ubiquitin from other adduct or fusion proteins [34], [20], [21]. These extensions and insertions may contribute to the functional diversity of the DUB enzymes. Interestingly, we found that deletion of UBP5 generated many intriguing phenotypic changes in C. neoformans that were linked to virulence. Therefore, we focused in detail on UBP5 for its relationship to pathogenesis.

Bottom Line: Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents).Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans.Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Key Laboratory of Molecular Medical Mycology, PLA Key Laboratory of Mycosis, Institute of Dermatology and Mycosis of Changzheng Hospital, Second Military Medical University, Shanghai, China.

ABSTRACT
Ubiquitination is a reversible protein modification that influences various cellular processes in eukaryotic cells. Deubiquitinating enzymes remove ubiquitin, maintain ubiquitin homeostasis and regulate protein degradation via the ubiquitination pathway. Cryptococcus neoformans is an important basidiomycete pathogen that causes life-threatening meningoencephalitis primarily in the immunocompromised population. In order to understand the possible influence deubiquitinases have on growth and virulence of the model pathogenic yeast Cryptococcus neoformans, we generated deletion mutants of seven putative deubiquitinase genes. Compared to other deubiquitinating enzyme mutants, a ubp5Δ mutant exhibited severely attenuated virulence and many distinct phenotypes, including decreased capsule formation, hypomelanization, defective sporulation, and elevated sensitivity to several external stressors (such as high temperature, oxidative and nitrosative stresses, high salts, and antifungal agents). Ubp5 is likely the major deubiquitinating enzyme for stress responses in C. neoformans, which further delineates the evolutionary divergence of Cryptococcus from the model yeast S. cerevisiae, and provides an important paradigm for understanding the potential role of deubiquitination in virulence by other pathogenic fungi. Other putative deubiquitinase mutants (doa4Δ and ubp13Δ) share some phenotypes with the ubp5Δ mutant, illustrating functional overlap among deubiquitinating enzymes in C. neoformans. Therefore, deubiquitinating enzymes (especially Ubp5) are essential for the virulence composite of C. neoformans and provide an additional yeast survival and propagation advantage in the host.

Show MeSH
Related in: MedlinePlus