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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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Inhibition of cathepsin B enzymatic activity enhances the antifungal effects against C. neoformans.C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either 50 µg/ml cathepsin B, 10 µg/ml calpain inhibitor, 50 or 100 µM cathepsin B & L inhibitor, 20 µM CA-074, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the numbers of CFU in the wells were determined. A) Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference compared to the results for H99 in phosphate buffer alone (p < 0.05) and τ indicates a significant difference compared to the initial the inocula (white bar) (p < 0.05). B) The percent cathepsin B enzymatic activity following incubation with each different inhibitor was measured using the Cathepsin B Activity Assay Kit. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. Asterisks * show significant differences in activity compared to cathepsin B alone (p < 0.05).
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f4: Inhibition of cathepsin B enzymatic activity enhances the antifungal effects against C. neoformans.C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either 50 µg/ml cathepsin B, 10 µg/ml calpain inhibitor, 50 or 100 µM cathepsin B & L inhibitor, 20 µM CA-074, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the numbers of CFU in the wells were determined. A) Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference compared to the results for H99 in phosphate buffer alone (p < 0.05) and τ indicates a significant difference compared to the initial the inocula (white bar) (p < 0.05). B) The percent cathepsin B enzymatic activity following incubation with each different inhibitor was measured using the Cathepsin B Activity Assay Kit. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. Asterisks * show significant differences in activity compared to cathepsin B alone (p < 0.05).

Mentions: Given our observations that the 50 µg/ml concentration of cathepsin B significantly inhibited the growth of C. neoformans strain H99 when compared to the growth in the phosphate buffer alone, we determined whether the inhibition of growth could be reversed by inhibiting the enzymatic activity of cathepsin B. For this, C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either cathepsin B, an inhibitor, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the CFUs were determined. The inhibitors used were calpain inhibitor at 10 µg/ml, cathepsin B & L inhibitor at 50 or 100 µM, and CA-074 at 20 µM. Calpain inhibitor is only soluble in DMSO, which is toxic to C. neoformans at higher concentrations, so higher concentrations of the calpain inhibitor could not be used in our assay (data not shown). CA-074 is a specific inhibitor of only cathepsin B, cathepsin B&L inhibitor inhibits cathepsin B and L only, and calpain is a general inhibitor of cathepsin activity. However, because our assays use only purified cathepsin B and not the entire DC lysosomal extract, the specificity of the inhibitors is not a factor. Surprisingly, our results showed that the addition of any cathepsin B inhibitor, at the concentrations used, enhanced the antifungal activity of cathepsin B rather than abrogating its activity (Figure 4A). To determine whether this observation was a result of the failure of the inhibitors to prohibit enzymatic activity of cathepsin B, we measured the percent inhibition of the cathepsin B enzymatic activity with each inhibitor (Figure 4B). We found that each inhibitor did significantly inhibit the enzymatic activity of cathepsin B. The calpain inhibitor showed 47.34% inhibition (52.66% activity) at 10 µg/ml, the cathepsin B&L inhibitor showed 55.67% inhibition (44.33% activity) at 50 µM and 95.83% inhibition (4.13% activity) at 100 µM, and CA-074 showed 95.69% inhibition (4.31% activity) at 20 µM, suggesting that cathepsin B may have non-enzymatic activity against C. neoformans.


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

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

Inhibition of cathepsin B enzymatic activity enhances the antifungal effects against C. neoformans.C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either 50 µg/ml cathepsin B, 10 µg/ml calpain inhibitor, 50 or 100 µM cathepsin B & L inhibitor, 20 µM CA-074, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the numbers of CFU in the wells were determined. A) Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference compared to the results for H99 in phosphate buffer alone (p < 0.05) and τ indicates a significant difference compared to the initial the inocula (white bar) (p < 0.05). B) The percent cathepsin B enzymatic activity following incubation with each different inhibitor was measured using the Cathepsin B Activity Assay Kit. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. Asterisks * show significant differences in activity compared to cathepsin B alone (p < 0.05).
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Related In: Results  -  Collection

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Show All Figures
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f4: Inhibition of cathepsin B enzymatic activity enhances the antifungal effects against C. neoformans.C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either 50 µg/ml cathepsin B, 10 µg/ml calpain inhibitor, 50 or 100 µM cathepsin B & L inhibitor, 20 µM CA-074, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the numbers of CFU in the wells were determined. A) Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. An asterisk * indicates a significant difference compared to the results for H99 in phosphate buffer alone (p < 0.05) and τ indicates a significant difference compared to the initial the inocula (white bar) (p < 0.05). B) The percent cathepsin B enzymatic activity following incubation with each different inhibitor was measured using the Cathepsin B Activity Assay Kit. Data shown are means ± standard errors of the means (SEM) of the cumulative results of 3 independent experiments. Asterisks * show significant differences in activity compared to cathepsin B alone (p < 0.05).
Mentions: Given our observations that the 50 µg/ml concentration of cathepsin B significantly inhibited the growth of C. neoformans strain H99 when compared to the growth in the phosphate buffer alone, we determined whether the inhibition of growth could be reversed by inhibiting the enzymatic activity of cathepsin B. For this, C. neoformans strain H99 yeast cells were incubated in phosphate buffer alone, in phosphate buffer with either cathepsin B, an inhibitor, or cathepsin B plus an inhibitor for 24 h at 37°C, following which the CFUs were determined. The inhibitors used were calpain inhibitor at 10 µg/ml, cathepsin B & L inhibitor at 50 or 100 µM, and CA-074 at 20 µM. Calpain inhibitor is only soluble in DMSO, which is toxic to C. neoformans at higher concentrations, so higher concentrations of the calpain inhibitor could not be used in our assay (data not shown). CA-074 is a specific inhibitor of only cathepsin B, cathepsin B&L inhibitor inhibits cathepsin B and L only, and calpain is a general inhibitor of cathepsin activity. However, because our assays use only purified cathepsin B and not the entire DC lysosomal extract, the specificity of the inhibitors is not a factor. Surprisingly, our results showed that the addition of any cathepsin B inhibitor, at the concentrations used, enhanced the antifungal activity of cathepsin B rather than abrogating its activity (Figure 4A). To determine whether this observation was a result of the failure of the inhibitors to prohibit enzymatic activity of cathepsin B, we measured the percent inhibition of the cathepsin B enzymatic activity with each inhibitor (Figure 4B). We found that each inhibitor did significantly inhibit the enzymatic activity of cathepsin B. The calpain inhibitor showed 47.34% inhibition (52.66% activity) at 10 µg/ml, the cathepsin B&L inhibitor showed 55.67% inhibition (44.33% activity) at 50 µM and 95.83% inhibition (4.13% activity) at 100 µM, and CA-074 showed 95.69% inhibition (4.31% activity) at 20 µM, suggesting that cathepsin B may have non-enzymatic activity against C. neoformans.

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