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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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DC-derived lysosomal extract kills Cryptococcus in vitro.C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells at 2.5 x 105 cells/ml were incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, and CFU in the wells determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer alone, and black bars indicate cryptococcal cells incubated with lysosomal extract. Data shown are means ± standard errors of the means (SEM) of the cumulative results of three independent experiments. An asterisk * indicates a significant difference compared to the results for the yeast incubated in phosphate buffer alone (P < 0.0001) and τ indicates a significant difference compared to the results for inocula (P < 0.0001).
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f1: DC-derived lysosomal extract kills Cryptococcus in vitro.C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells at 2.5 x 105 cells/ml were incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, and CFU in the wells determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer alone, and black bars indicate cryptococcal cells incubated with lysosomal extract. Data shown are means ± standard errors of the means (SEM) of the cumulative results of three independent experiments. An asterisk * indicates a significant difference compared to the results for the yeast incubated in phosphate buffer alone (P < 0.0001) and τ indicates a significant difference compared to the results for inocula (P < 0.0001).

Mentions: Previous studies have shown that DC-derived lysosomal extract can kill C. neoformans serotype A strain 145 in vitro20. To confirm DC lysosomal activity against other cryptococcal strains and serotypes, C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells were individually incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, following which the CFU in the wells were determined. We found that the DC-derived lysosomal extracts have significant antifungal activity against all cryptococcal strains tested, leading to complete killing of each strain of Cryptococcus (Figure 1). Because all strains and serotypes were equally affected, further studies were conducted using the highly pathogenic C. neoformans strain H99.


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

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

DC-derived lysosomal extract kills Cryptococcus in vitro.C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells at 2.5 x 105 cells/ml were incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, and CFU in the wells determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer alone, and black bars indicate cryptococcal cells incubated with lysosomal extract. Data shown are means ± standard errors of the means (SEM) of the cumulative results of three independent experiments. An asterisk * indicates a significant difference compared to the results for the yeast incubated in phosphate buffer alone (P < 0.0001) and τ indicates a significant difference compared to the results for inocula (P < 0.0001).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: DC-derived lysosomal extract kills Cryptococcus in vitro.C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells at 2.5 x 105 cells/ml were incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, and CFU in the wells determined as described in Materials and Methods. White bars indicate inoculum, gray bars indicate cryptococcal cells incubated in phosphate buffer alone, and black bars indicate cryptococcal cells incubated with lysosomal extract. Data shown are means ± standard errors of the means (SEM) of the cumulative results of three independent experiments. An asterisk * indicates a significant difference compared to the results for the yeast incubated in phosphate buffer alone (P < 0.0001) and τ indicates a significant difference compared to the results for inocula (P < 0.0001).
Mentions: Previous studies have shown that DC-derived lysosomal extract can kill C. neoformans serotype A strain 145 in vitro20. To confirm DC lysosomal activity against other cryptococcal strains and serotypes, C. neoformans serotype A strain H99, C. gattii serotype B strain R265, C. gattii serotype C strain WSA87, and C. neoformans serotype D strain R4249 yeast cells were individually incubated in phosphate buffer alone or in phosphate buffer with lysosomal extract for 24 h at 37°C, following which the CFU in the wells were determined. We found that the DC-derived lysosomal extracts have significant antifungal activity against all cryptococcal strains tested, leading to complete killing of each strain of Cryptococcus (Figure 1). Because all strains and serotypes were equally affected, further studies were conducted using the highly pathogenic C. neoformans strain H99.

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