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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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UBP5 is involved in various stress responses of C. neoformans.A. Temperature sensitivity at 30°C, 37°C,39°C. B. Oxidative and nitrosative stress assay. C. High salts and osmotic stress assay. D. Cell-wall and cell-membrane stress assay. Strains were grown to saturation at 30°C in liquid YPD medium, 10-fold serially diluted (1–106 dilutions), and spotted 3 µl cells on YPD or YNB agar (containing different stress-inducing agents). Incubated them for five days and then photographed.
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pone-0038326-g002: UBP5 is involved in various stress responses of C. neoformans.A. Temperature sensitivity at 30°C, 37°C,39°C. B. Oxidative and nitrosative stress assay. C. High salts and osmotic stress assay. D. Cell-wall and cell-membrane stress assay. Strains were grown to saturation at 30°C in liquid YPD medium, 10-fold serially diluted (1–106 dilutions), and spotted 3 µl cells on YPD or YNB agar (containing different stress-inducing agents). Incubated them for five days and then photographed.

Mentions: Since the ubiquitin-proteasome system plays important roles in regulating stress responses in eukaryotes [33], we tested the sensitivity of each mutant to a variety of in vitro stresses. Unlike the WT and other DUB mutant strains, only the ubp5Δ mutant exhibited temperature sensitivity (ts), with a partial growth defect at 37°C and complete growth inhibition at 39°C (Figure 2A). The ts phenotype was not rescued by the addition of 1 M sorbitol (data not shown) as was observed for a C. neoformans tps1Δ mutant that is also thermosensitive [35]. Moreover, deletion of UBP5 resulted in slower growth compared to WT even in rich medium at 30°C (Figure 2A and Figure S1). To examine whether this poor growth phenotype was due to carbon source preference, we also compared its growth using different carbon sources (YP agar medium with 3% galactose, 3% lactate, 3% glycerol, and 2% raffinose). No growth improvement was observed when the ubp5Δ strain grew in the media with alternate carbon sources (data not shown).


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)

UBP5 is involved in various stress responses of C. neoformans.A. Temperature sensitivity at 30°C, 37°C,39°C. B. Oxidative and nitrosative stress assay. C. High salts and osmotic stress assay. D. Cell-wall and cell-membrane stress assay. Strains were grown to saturation at 30°C in liquid YPD medium, 10-fold serially diluted (1–106 dilutions), and spotted 3 µl cells on YPD or YNB agar (containing different stress-inducing agents). Incubated them for five days and then photographed.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375289&req=5

pone-0038326-g002: UBP5 is involved in various stress responses of C. neoformans.A. Temperature sensitivity at 30°C, 37°C,39°C. B. Oxidative and nitrosative stress assay. C. High salts and osmotic stress assay. D. Cell-wall and cell-membrane stress assay. Strains were grown to saturation at 30°C in liquid YPD medium, 10-fold serially diluted (1–106 dilutions), and spotted 3 µl cells on YPD or YNB agar (containing different stress-inducing agents). Incubated them for five days and then photographed.
Mentions: Since the ubiquitin-proteasome system plays important roles in regulating stress responses in eukaryotes [33], we tested the sensitivity of each mutant to a variety of in vitro stresses. Unlike the WT and other DUB mutant strains, only the ubp5Δ mutant exhibited temperature sensitivity (ts), with a partial growth defect at 37°C and complete growth inhibition at 39°C (Figure 2A). The ts phenotype was not rescued by the addition of 1 M sorbitol (data not shown) as was observed for a C. neoformans tps1Δ mutant that is also thermosensitive [35]. Moreover, deletion of UBP5 resulted in slower growth compared to WT even in rich medium at 30°C (Figure 2A and Figure S1). To examine whether this poor growth phenotype was due to carbon source preference, we also compared its growth using different carbon sources (YP agar medium with 3% galactose, 3% lactate, 3% glycerol, and 2% raffinose). No growth improvement was observed when the ubp5Δ strain grew in the media with alternate carbon sources (data not shown).

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