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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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Disruption of UBP5 significantly down-regulates the capsule production.A. ubp5Δ mutants have a capsule defect. The WT strain H99 and all the DUB mutants were cultured on DME medium for capsule production at 37°C for 3 days. Capsule was assessed by staining with India ink and visualizing at 100 magnification (scale bar  =  10 µm). B. Relative capsule volume detection. Total (cell and capsule) and cell-only diameter were measured via Photoshop Software for 50 cells for each strain. Then calculated the relative ratio of capsule with the formula ([Total Volume-Packed Volume]/Total Volume). After statistical analysis, the capsule production of ubp5Δ strain (7.7±0.48%) significantly decreased compared with either WT (43.4±6.41%) or reconstituted strain (37.4±8.01%) (P<0.001). C. Cryptocrit for Capsule Assay. After overnight growth, each culture was inoculated into tissue culture flasks with 20 mL DMEM medium in capsule-inducing condition for three days. Cells were killed with 10% formalin (V/V) for 5 minutes, and then counted. The samples were standardized to 3.2×108 cells/mL. The difference between ubp5Δ (21.28±3.00) and complemented (42.8±0.84) or WT (35.8±1.48) strains was P<0.001. D. Capsule transfer assay. Capsule material secreted from wild-type or ubp5Δ cells into conditioned medium (CM) can be attached to acapsular acceptor cells (cap59Δ). cap59Δ cell cannot shed capsule polysaccharide, thus its CM was used as a negative control. Cells were labeled with monoclonal antibody mAb18B7 and Alexa Fluror anti-mouse IgG (secondary antibody) was used for immunofluorescence.
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pone-0038326-g003: Disruption of UBP5 significantly down-regulates the capsule production.A. ubp5Δ mutants have a capsule defect. The WT strain H99 and all the DUB mutants were cultured on DME medium for capsule production at 37°C for 3 days. Capsule was assessed by staining with India ink and visualizing at 100 magnification (scale bar  =  10 µm). B. Relative capsule volume detection. Total (cell and capsule) and cell-only diameter were measured via Photoshop Software for 50 cells for each strain. Then calculated the relative ratio of capsule with the formula ([Total Volume-Packed Volume]/Total Volume). After statistical analysis, the capsule production of ubp5Δ strain (7.7±0.48%) significantly decreased compared with either WT (43.4±6.41%) or reconstituted strain (37.4±8.01%) (P<0.001). C. Cryptocrit for Capsule Assay. After overnight growth, each culture was inoculated into tissue culture flasks with 20 mL DMEM medium in capsule-inducing condition for three days. Cells were killed with 10% formalin (V/V) for 5 minutes, and then counted. The samples were standardized to 3.2×108 cells/mL. The difference between ubp5Δ (21.28±3.00) and complemented (42.8±0.84) or WT (35.8±1.48) strains was P<0.001. D. Capsule transfer assay. Capsule material secreted from wild-type or ubp5Δ cells into conditioned medium (CM) can be attached to acapsular acceptor cells (cap59Δ). cap59Δ cell cannot shed capsule polysaccharide, thus its CM was used as a negative control. Cells were labeled with monoclonal antibody mAb18B7 and Alexa Fluror anti-mouse IgG (secondary antibody) was used for immunofluorescence.

Mentions: Several pathogenic factors are essential for C. neoformans to invade the host, such as capsule, melanin, and urease. When incubated under capsule-inducing conditions for 72 hrs, an obvious capsule defect was observed for the ubp5Δ mutant, whereas the reconstituted strain and other DUB mutants all showed WT capsule production (Figure 3A). In order to exclude the confounding effect of thermosensitivity, the ubp5Δ mutant was also examined in capsule-inducing medium at 30°C. Similarly, about 90% of ubp5Δ mutant cells displayed a significantly reduced capsule while 10% produced capsule similar to WT (data not shown). We measured the relative capsule volume (capsule diameter/total diameter) for the WT, ubp5Δ and ubp5Δ+UBP5 strains, and found that the volume of the ubp5Δ capsule was significantly smaller than WT (P<0.001) (Figure 3B). These data were confirmed using an independent measurement of capsule volume based on packed-cell volume (Figure 3C).


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)

Disruption of UBP5 significantly down-regulates the capsule production.A. ubp5Δ mutants have a capsule defect. The WT strain H99 and all the DUB mutants were cultured on DME medium for capsule production at 37°C for 3 days. Capsule was assessed by staining with India ink and visualizing at 100 magnification (scale bar  =  10 µm). B. Relative capsule volume detection. Total (cell and capsule) and cell-only diameter were measured via Photoshop Software for 50 cells for each strain. Then calculated the relative ratio of capsule with the formula ([Total Volume-Packed Volume]/Total Volume). After statistical analysis, the capsule production of ubp5Δ strain (7.7±0.48%) significantly decreased compared with either WT (43.4±6.41%) or reconstituted strain (37.4±8.01%) (P<0.001). C. Cryptocrit for Capsule Assay. After overnight growth, each culture was inoculated into tissue culture flasks with 20 mL DMEM medium in capsule-inducing condition for three days. Cells were killed with 10% formalin (V/V) for 5 minutes, and then counted. The samples were standardized to 3.2×108 cells/mL. The difference between ubp5Δ (21.28±3.00) and complemented (42.8±0.84) or WT (35.8±1.48) strains was P<0.001. D. Capsule transfer assay. Capsule material secreted from wild-type or ubp5Δ cells into conditioned medium (CM) can be attached to acapsular acceptor cells (cap59Δ). cap59Δ cell cannot shed capsule polysaccharide, thus its CM was used as a negative control. Cells were labeled with monoclonal antibody mAb18B7 and Alexa Fluror anti-mouse IgG (secondary antibody) was used for immunofluorescence.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375289&req=5

pone-0038326-g003: Disruption of UBP5 significantly down-regulates the capsule production.A. ubp5Δ mutants have a capsule defect. The WT strain H99 and all the DUB mutants were cultured on DME medium for capsule production at 37°C for 3 days. Capsule was assessed by staining with India ink and visualizing at 100 magnification (scale bar  =  10 µm). B. Relative capsule volume detection. Total (cell and capsule) and cell-only diameter were measured via Photoshop Software for 50 cells for each strain. Then calculated the relative ratio of capsule with the formula ([Total Volume-Packed Volume]/Total Volume). After statistical analysis, the capsule production of ubp5Δ strain (7.7±0.48%) significantly decreased compared with either WT (43.4±6.41%) or reconstituted strain (37.4±8.01%) (P<0.001). C. Cryptocrit for Capsule Assay. After overnight growth, each culture was inoculated into tissue culture flasks with 20 mL DMEM medium in capsule-inducing condition for three days. Cells were killed with 10% formalin (V/V) for 5 minutes, and then counted. The samples were standardized to 3.2×108 cells/mL. The difference between ubp5Δ (21.28±3.00) and complemented (42.8±0.84) or WT (35.8±1.48) strains was P<0.001. D. Capsule transfer assay. Capsule material secreted from wild-type or ubp5Δ cells into conditioned medium (CM) can be attached to acapsular acceptor cells (cap59Δ). cap59Δ cell cannot shed capsule polysaccharide, thus its CM was used as a negative control. Cells were labeled with monoclonal antibody mAb18B7 and Alexa Fluror anti-mouse IgG (secondary antibody) was used for immunofluorescence.
Mentions: Several pathogenic factors are essential for C. neoformans to invade the host, such as capsule, melanin, and urease. When incubated under capsule-inducing conditions for 72 hrs, an obvious capsule defect was observed for the ubp5Δ mutant, whereas the reconstituted strain and other DUB mutants all showed WT capsule production (Figure 3A). In order to exclude the confounding effect of thermosensitivity, the ubp5Δ mutant was also examined in capsule-inducing medium at 30°C. Similarly, about 90% of ubp5Δ mutant cells displayed a significantly reduced capsule while 10% produced capsule similar to WT (data not shown). We measured the relative capsule volume (capsule diameter/total diameter) for the WT, ubp5Δ and ubp5Δ+UBP5 strains, and found that the volume of the ubp5Δ capsule was significantly smaller than WT (P<0.001) (Figure 3B). These data were confirmed using an independent measurement of capsule volume based on packed-cell volume (Figure 3C).

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