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Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

Hole CR, Bui H, Wormley FL, Wozniak KL - Sci Rep (2012)

Bottom Line: Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes.Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment.Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, USA.

ABSTRACT
Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

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Cathepsin B and inhibitor treatment result in osmotic lysis of Cryptococcus neoformans.A) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFUs were determined by diluting and plating on YPD agar or osmotically supportive YPD media containing 1.4 M sorbitol. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference of the cathepsin B and inhibitor treated groups compared to the results for inocula compared (p < 0.05). A τ indicates a significant difference of the cathepsin B and inhibitor treated group grown on sorbitol YPD compared to the results grown on YPD (P < 0.0001). B) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 20 µM CA-074 for 24 h at 37°C, following which the cells were transferred to YPD broth or osmotically supportive YPD broth containing 1.4 M sorbitol for 8 h at 30°C. Following incubation, cells were fixed with 4% formaldehyde/1% glutaraldehyde and processed for electron microscopy. Quantified data (B) are shown from quantification of 30 fields per condition. C) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) show structural changes in the organism following incubation with CA-074 + cathepsin B (arrows) and abrogation of these changes following incubation in YPD+sorbitol. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM and 25,000 X for TEM images.
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f6: Cathepsin B and inhibitor treatment result in osmotic lysis of Cryptococcus neoformans.A) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFUs were determined by diluting and plating on YPD agar or osmotically supportive YPD media containing 1.4 M sorbitol. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference of the cathepsin B and inhibitor treated groups compared to the results for inocula compared (p < 0.05). A τ indicates a significant difference of the cathepsin B and inhibitor treated group grown on sorbitol YPD compared to the results grown on YPD (P < 0.0001). B) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 20 µM CA-074 for 24 h at 37°C, following which the cells were transferred to YPD broth or osmotically supportive YPD broth containing 1.4 M sorbitol for 8 h at 30°C. Following incubation, cells were fixed with 4% formaldehyde/1% glutaraldehyde and processed for electron microscopy. Quantified data (B) are shown from quantification of 30 fields per condition. C) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) show structural changes in the organism following incubation with CA-074 + cathepsin B (arrows) and abrogation of these changes following incubation in YPD+sorbitol. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM and 25,000 X for TEM images.

Mentions: Our results in Figure 5 showed that incubation with cathepsin B and calpain inhibitor lead to a compromised cell wall of C. neoformans. We hypothesized that the cell wall ruptures induced by cathepsin B and inhibitor treatment enhanced osmotic lysis of the cryptococcal cells, resulting in death. To examine this, C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFU in the wells were determined by diluting and plating on YPD agar or osmotically supportive YPD agar containing 1.4 M sorbitol. By plating on the osmotically supportive YPD agar, we almost completely abrogated the killing of C. neoformans (Figure 6). As an alternative approach, electron microscopy (EM) was performed following incubation in YPD + 1.4 M sorbitol broth using cathepsin B with the specific inhibitor CA-074. Both TEM and SEM confirmed that the addition of sorbitol reversed the crescent-shaped appearance of the organism (Figure 6B and 6C). Therefore, we conclude that the cathepsin B plus inhibitor treatment leads to osmotic lysis of the fungus.


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

Hole CR, Bui H, Wormley FL, Wozniak KL - Sci Rep (2012)

Cathepsin B and inhibitor treatment result in osmotic lysis of Cryptococcus neoformans.A) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFUs were determined by diluting and plating on YPD agar or osmotically supportive YPD media containing 1.4 M sorbitol. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference of the cathepsin B and inhibitor treated groups compared to the results for inocula compared (p < 0.05). A τ indicates a significant difference of the cathepsin B and inhibitor treated group grown on sorbitol YPD compared to the results grown on YPD (P < 0.0001). B) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 20 µM CA-074 for 24 h at 37°C, following which the cells were transferred to YPD broth or osmotically supportive YPD broth containing 1.4 M sorbitol for 8 h at 30°C. Following incubation, cells were fixed with 4% formaldehyde/1% glutaraldehyde and processed for electron microscopy. Quantified data (B) are shown from quantification of 30 fields per condition. C) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) show structural changes in the organism following incubation with CA-074 + cathepsin B (arrows) and abrogation of these changes following incubation in YPD+sorbitol. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM and 25,000 X for TEM images.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f6: Cathepsin B and inhibitor treatment result in osmotic lysis of Cryptococcus neoformans.A) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFUs were determined by diluting and plating on YPD agar or osmotically supportive YPD media containing 1.4 M sorbitol. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference of the cathepsin B and inhibitor treated groups compared to the results for inocula compared (p < 0.05). A τ indicates a significant difference of the cathepsin B and inhibitor treated group grown on sorbitol YPD compared to the results grown on YPD (P < 0.0001). B) C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 20 µM CA-074 for 24 h at 37°C, following which the cells were transferred to YPD broth or osmotically supportive YPD broth containing 1.4 M sorbitol for 8 h at 30°C. Following incubation, cells were fixed with 4% formaldehyde/1% glutaraldehyde and processed for electron microscopy. Quantified data (B) are shown from quantification of 30 fields per condition. C) Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) show structural changes in the organism following incubation with CA-074 + cathepsin B (arrows) and abrogation of these changes following incubation in YPD+sorbitol. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM and 25,000 X for TEM images.
Mentions: Our results in Figure 5 showed that incubation with cathepsin B and calpain inhibitor lead to a compromised cell wall of C. neoformans. We hypothesized that the cell wall ruptures induced by cathepsin B and inhibitor treatment enhanced osmotic lysis of the cryptococcal cells, resulting in death. To examine this, C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone or in phosphate buffer with 50 µg/ml cathepsin B and 10 µg/ml calpain inhibitor for 24 h at 37°C, following which the CFU in the wells were determined by diluting and plating on YPD agar or osmotically supportive YPD agar containing 1.4 M sorbitol. By plating on the osmotically supportive YPD agar, we almost completely abrogated the killing of C. neoformans (Figure 6). As an alternative approach, electron microscopy (EM) was performed following incubation in YPD + 1.4 M sorbitol broth using cathepsin B with the specific inhibitor CA-074. Both TEM and SEM confirmed that the addition of sorbitol reversed the crescent-shaped appearance of the organism (Figure 6B and 6C). Therefore, we conclude that the cathepsin B plus inhibitor treatment leads to osmotic lysis of the fungus.

Bottom Line: Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes.Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment.Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and The South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, USA.

ABSTRACT
Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

Show MeSH
Related in: MedlinePlus