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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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Purified lysosomal enzymes inhibit C. neoformans growth in vitro.C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (A), cathepsin D (B), or cathepsin L (C) for 24 h at 37°C, following which the CFU in the wells were determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer, hashed bars indicate cryptococcal cells incubated with 10 µg/ml cathepsin, and black bars indicate cryptococcal cells incubated with 50 µg/ml cathepsin. 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 incubated in phosphate buffer alone (p < 0.05).
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f2: Purified lysosomal enzymes inhibit C. neoformans growth in vitro.C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (A), cathepsin D (B), or cathepsin L (C) for 24 h at 37°C, following which the CFU in the wells were determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer, hashed bars indicate cryptococcal cells incubated with 10 µg/ml cathepsin, and black bars indicate cryptococcal cells incubated with 50 µg/ml cathepsin. 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 incubated in phosphate buffer alone (p < 0.05).

Mentions: Many lysosomal components of DCs can contribute to killing and degradation of phagocytosed organisms. Lysosomal enzymes, such as cathepsins, are known to be involved in intracellular degradation and processing for antigen presentation3637. Therefore, we chose to examine the role of cathepsins in the killing of C. neoformans strain H99. In order to examine the role of cathepsins in cryptococcal killing or inhibition, C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (Figure 2A), cathepsin D (Figure 2B), or cathepsin L (Figure 2C) for 24 h at 37°C, following which the CFUs were determined. These concentrations were chosen to represent physiological concentration found in cell lysates (10 µg)3839 and excess concentration (50 µg). Results showed that cathepsin B significantly inhibited the growth of C. neoformans at 10 µg/ml, and at 50 µg/ml showed even greater inhibition almost to inocula levels (Figure 2A). Incubation of C. neoformans with cathepsin D at 10 µg/ml and 50 µg/ml had no effect on the growth when compared to growth in the phosphate buffer alone (Figure 2B). Cathepsin L inhibited the growth of C. neoformans at both 10 µg/ml and 50 µg/ml compared to the growth in the phosphate buffer alone, but this was not a statistically significant difference (Figure 2C). Because the only significant inhibition of C. neoformans growth was observed following incubation with cathepsin B, further studies focused only on cathepsin B.


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

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

Purified lysosomal enzymes inhibit C. neoformans growth in vitro.C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (A), cathepsin D (B), or cathepsin L (C) for 24 h at 37°C, following which the CFU in the wells were determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer, hashed bars indicate cryptococcal cells incubated with 10 µg/ml cathepsin, and black bars indicate cryptococcal cells incubated with 50 µg/ml cathepsin. 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 incubated in phosphate buffer alone (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472389&req=5

f2: Purified lysosomal enzymes inhibit C. neoformans growth in vitro.C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (A), cathepsin D (B), or cathepsin L (C) for 24 h at 37°C, following which the CFU in the wells were determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer, hashed bars indicate cryptococcal cells incubated with 10 µg/ml cathepsin, and black bars indicate cryptococcal cells incubated with 50 µg/ml cathepsin. 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 incubated in phosphate buffer alone (p < 0.05).
Mentions: Many lysosomal components of DCs can contribute to killing and degradation of phagocytosed organisms. Lysosomal enzymes, such as cathepsins, are known to be involved in intracellular degradation and processing for antigen presentation3637. Therefore, we chose to examine the role of cathepsins in the killing of C. neoformans strain H99. In order to examine the role of cathepsins in cryptococcal killing or inhibition, C. neoformans strain H99 yeast cells were incubated in phosphate buffer with 10 µg/ml or 50 µg/ml of cathepsin B (Figure 2A), cathepsin D (Figure 2B), or cathepsin L (Figure 2C) for 24 h at 37°C, following which the CFUs were determined. These concentrations were chosen to represent physiological concentration found in cell lysates (10 µg)3839 and excess concentration (50 µg). Results showed that cathepsin B significantly inhibited the growth of C. neoformans at 10 µg/ml, and at 50 µg/ml showed even greater inhibition almost to inocula levels (Figure 2A). Incubation of C. neoformans with cathepsin D at 10 µg/ml and 50 µg/ml had no effect on the growth when compared to growth in the phosphate buffer alone (Figure 2B). Cathepsin L inhibited the growth of C. neoformans at both 10 µg/ml and 50 µg/ml compared to the growth in the phosphate buffer alone, but this was not a statistically significant difference (Figure 2C). Because the only significant inhibition of C. neoformans growth was observed following incubation with cathepsin B, further studies focused only on cathepsin B.

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