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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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Microscopy of C. neoformans treated with cathepsin B and cathepsin B + inhibitors show cryptococcal structural changes.C. neoformans was grown in the presence of cathepsin B, calpain inhibitor, CA-074, or cathepsin B with inhibitors for 12 h, fixed with 4% formaldehyde/1% glutaraldehyde, prepared for electron microscopy, and examined by SEM or TEM. A) SEM of C. neoformans strain H99 shows structural changes (arrows) in the organism following incubation with cathepsin B and cathepsin B + inhibitors, but not in untreated conditions. B) TEM of C. neoformans shows structural changes in cells treated with cathepsin B and cathepsin B + an inhibitor, but not in untreated cells or in those treated with only calpain inhibitor or CA-074. In addition, cells showing structural changes (arrows) also show ruptured cell walls but intact cell membranes. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM images and 25,000 X for TEM images. C) Purified BMDCs were incubated with C. neoformans yeast cells at a 2:1 ratio with 1 µg/ml of Oregon green-labeled 3C2 opsonizing antibody and 50 nM LysoTracker red. Samples were incubated at 37°C for 1 hour prior to imaging. A representative confocal image of C. neoformans organisms (green) shown in a LysoTracker red-positive compartment (red) with a similar crescent-shaped morphology as seen through electron microscopy. Confocal image is representative of 3 experiments performed, 30 fields imaged per experiment.
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f5: Microscopy of C. neoformans treated with cathepsin B and cathepsin B + inhibitors show cryptococcal structural changes.C. neoformans was grown in the presence of cathepsin B, calpain inhibitor, CA-074, or cathepsin B with inhibitors for 12 h, fixed with 4% formaldehyde/1% glutaraldehyde, prepared for electron microscopy, and examined by SEM or TEM. A) SEM of C. neoformans strain H99 shows structural changes (arrows) in the organism following incubation with cathepsin B and cathepsin B + inhibitors, but not in untreated conditions. B) TEM of C. neoformans shows structural changes in cells treated with cathepsin B and cathepsin B + an inhibitor, but not in untreated cells or in those treated with only calpain inhibitor or CA-074. In addition, cells showing structural changes (arrows) also show ruptured cell walls but intact cell membranes. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM images and 25,000 X for TEM images. C) Purified BMDCs were incubated with C. neoformans yeast cells at a 2:1 ratio with 1 µg/ml of Oregon green-labeled 3C2 opsonizing antibody and 50 nM LysoTracker red. Samples were incubated at 37°C for 1 hour prior to imaging. A representative confocal image of C. neoformans organisms (green) shown in a LysoTracker red-positive compartment (red) with a similar crescent-shaped morphology as seen through electron microscopy. Confocal image is representative of 3 experiments performed, 30 fields imaged per experiment.

Mentions: Due to the killing observed following incubation with cathepsin B and its inhibitors, we were interested in determining their effects on the fungus during in vitro incubation. To assess this, scanning electron microscopy (SEM) (Figure 5A) or transmission electron microscopy (TEM) (Figure 5B) was performed on C. neoformans after growth in the presence of phosphate buffer, cathepsin B, calpain inhibitor, CA-074, or cathepsin B + an inhibitor for 12 h. We chose to examine the cells after 12 hours of incubation in order to observe the effects of cathepsin B and either calpain inhibitor or CA-074 on live cells, since 24 h of incubation results in complete killing of C. neoformans. SEM of C. neoformans strain H99 showed structural changes in the organism following incubation with cathepsin B and cathepsin B + calpain inhibitor or CA-074 compared to untreated conditions (Figure 5A). TEM of C. neoformans strain H99 showed a crescent morphology indicative of structural changes and possible cell death41 due to cathepsin B and cathepsin B + calpain inhibitor or CA-074 treatment compared to untreated cells or cells treated with calpain inhibitor or CA-074 alone (Figure 5B). In addition, cells showing structural changes also show ruptured cell walls but intact cell membranes (Figure 5B). Quantification of EM data showed altered cryptococcal cell morphology (crescent-shaped) in 10% of untreated cells and in 17% and 15% of calpain or CA-074 treated cells, respectively. Further, cells treated with cathepsin B, calpain inhibitor + cathepsin B, or CA-074 + cathepsin B had statistically higher percentages of altered cell morphology (27%, 27%, and 25%, respectively) compared to cells treated with either untreated cells or compared to cells treated with inhibitors alone (P<0.0001). In support of these data, confocal microscopy using live BMDCs showed that C. neoformans (green) trafficking into DC lysosomal compartments (red) also adopts a similar crescent-shaped morphology (Figure 5C)


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

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

Microscopy of C. neoformans treated with cathepsin B and cathepsin B + inhibitors show cryptococcal structural changes.C. neoformans was grown in the presence of cathepsin B, calpain inhibitor, CA-074, or cathepsin B with inhibitors for 12 h, fixed with 4% formaldehyde/1% glutaraldehyde, prepared for electron microscopy, and examined by SEM or TEM. A) SEM of C. neoformans strain H99 shows structural changes (arrows) in the organism following incubation with cathepsin B and cathepsin B + inhibitors, but not in untreated conditions. B) TEM of C. neoformans shows structural changes in cells treated with cathepsin B and cathepsin B + an inhibitor, but not in untreated cells or in those treated with only calpain inhibitor or CA-074. In addition, cells showing structural changes (arrows) also show ruptured cell walls but intact cell membranes. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM images and 25,000 X for TEM images. C) Purified BMDCs were incubated with C. neoformans yeast cells at a 2:1 ratio with 1 µg/ml of Oregon green-labeled 3C2 opsonizing antibody and 50 nM LysoTracker red. Samples were incubated at 37°C for 1 hour prior to imaging. A representative confocal image of C. neoformans organisms (green) shown in a LysoTracker red-positive compartment (red) with a similar crescent-shaped morphology as seen through electron microscopy. Confocal image is representative of 3 experiments performed, 30 fields imaged per experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Microscopy of C. neoformans treated with cathepsin B and cathepsin B + inhibitors show cryptococcal structural changes.C. neoformans was grown in the presence of cathepsin B, calpain inhibitor, CA-074, or cathepsin B with inhibitors for 12 h, fixed with 4% formaldehyde/1% glutaraldehyde, prepared for electron microscopy, and examined by SEM or TEM. A) SEM of C. neoformans strain H99 shows structural changes (arrows) in the organism following incubation with cathepsin B and cathepsin B + inhibitors, but not in untreated conditions. B) TEM of C. neoformans shows structural changes in cells treated with cathepsin B and cathepsin B + an inhibitor, but not in untreated cells or in those treated with only calpain inhibitor or CA-074. In addition, cells showing structural changes (arrows) also show ruptured cell walls but intact cell membranes. Data shown are representative of 30 fields per condition imaged. Magnification is 5,000 X for SEM images and 25,000 X for TEM images. C) Purified BMDCs were incubated with C. neoformans yeast cells at a 2:1 ratio with 1 µg/ml of Oregon green-labeled 3C2 opsonizing antibody and 50 nM LysoTracker red. Samples were incubated at 37°C for 1 hour prior to imaging. A representative confocal image of C. neoformans organisms (green) shown in a LysoTracker red-positive compartment (red) with a similar crescent-shaped morphology as seen through electron microscopy. Confocal image is representative of 3 experiments performed, 30 fields imaged per experiment.
Mentions: Due to the killing observed following incubation with cathepsin B and its inhibitors, we were interested in determining their effects on the fungus during in vitro incubation. To assess this, scanning electron microscopy (SEM) (Figure 5A) or transmission electron microscopy (TEM) (Figure 5B) was performed on C. neoformans after growth in the presence of phosphate buffer, cathepsin B, calpain inhibitor, CA-074, or cathepsin B + an inhibitor for 12 h. We chose to examine the cells after 12 hours of incubation in order to observe the effects of cathepsin B and either calpain inhibitor or CA-074 on live cells, since 24 h of incubation results in complete killing of C. neoformans. SEM of C. neoformans strain H99 showed structural changes in the organism following incubation with cathepsin B and cathepsin B + calpain inhibitor or CA-074 compared to untreated conditions (Figure 5A). TEM of C. neoformans strain H99 showed a crescent morphology indicative of structural changes and possible cell death41 due to cathepsin B and cathepsin B + calpain inhibitor or CA-074 treatment compared to untreated cells or cells treated with calpain inhibitor or CA-074 alone (Figure 5B). In addition, cells showing structural changes also show ruptured cell walls but intact cell membranes (Figure 5B). Quantification of EM data showed altered cryptococcal cell morphology (crescent-shaped) in 10% of untreated cells and in 17% and 15% of calpain or CA-074 treated cells, respectively. Further, cells treated with cathepsin B, calpain inhibitor + cathepsin B, or CA-074 + cathepsin B had statistically higher percentages of altered cell morphology (27%, 27%, and 25%, respectively) compared to cells treated with either untreated cells or compared to cells treated with inhibitors alone (P<0.0001). In support of these data, confocal microscopy using live BMDCs showed that C. neoformans (green) trafficking into DC lysosomal compartments (red) also adopts a similar crescent-shaped morphology (Figure 5C)

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