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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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Confirmation of the presence of Cathepsin B in BMDC lysosomal extracts.Lysosomal extracts prepared from BMDCs were examined for the presence of cathepsin B. For ELISA analysis, fractions were taken during each step of the lysosomal purification process to examine the amount of cathepsin B present. Recombinant mouse cathepsin B in the pro-form was used as the positive control for Western blot analysis and for the ELISA standard. Cathepsin B was detected by separating the lysosomal extracts through SDS-page then transferring to a PVDF membrane and probing with anti-human/mouse cathepsin B. Recombinant mouse cathepsin B in the pro-form is 39 kDa, and active cathepsin B is 25-26 kDa. A). Western blot analysis of cathepsin B shows the presence of cathepsin B in two different samples of BMDC lysosomal extract. The blot is representative of three separate experiments. B) ELISA shows that 4000 pg cathepsin B per mg protein is present in BMDC lysosomal extracts. Aliquots of the different stages in the lysosomal purification process showed that cathepsin B is found primarily in the lysosomal fraction. Data shown are means ± standard error of the mean (SEM) of three independent experiments.
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f3: Confirmation of the presence of Cathepsin B in BMDC lysosomal extracts.Lysosomal extracts prepared from BMDCs were examined for the presence of cathepsin B. For ELISA analysis, fractions were taken during each step of the lysosomal purification process to examine the amount of cathepsin B present. Recombinant mouse cathepsin B in the pro-form was used as the positive control for Western blot analysis and for the ELISA standard. Cathepsin B was detected by separating the lysosomal extracts through SDS-page then transferring to a PVDF membrane and probing with anti-human/mouse cathepsin B. Recombinant mouse cathepsin B in the pro-form is 39 kDa, and active cathepsin B is 25-26 kDa. A). Western blot analysis of cathepsin B shows the presence of cathepsin B in two different samples of BMDC lysosomal extract. The blot is representative of three separate experiments. B) ELISA shows that 4000 pg cathepsin B per mg protein is present in BMDC lysosomal extracts. Aliquots of the different stages in the lysosomal purification process showed that cathepsin B is found primarily in the lysosomal fraction. Data shown are means ± standard error of the mean (SEM) of three independent experiments.

Mentions: In order to definitively confirm that cathepsin B was present in our DC lysosomal extract, the extract was examined for the presence of cathepsin B by Western blot analysis. Proteins from two separate preparations of DC lysosomal extract were separated by SDS-PAGE and transferred to a PVDF membrane. Blots were probed with anti-human/mouse cathepsin B monoclonal antibody. Recombinant mouse cathepsin B in the pro-form (39 kDa), was used as a positive control. Active cathepsin B is approximately 25–26 kDa40. The Western blot showed the presence of cathepsin B in two separate preparations of BMDC lysosomal extract (Figure 3A). We next determined the concentration of cathepsin B in the BMDC lysosomal extracts by ELISA. Aliquots of each step during the lysosomal purification process were examined to quantify the amount of cathepsin B present. The ELISA showed that an average amount of 4000 pg/mg protein of cathepsin B is present in BMDC lysosomal extracts (Figure 3B).


Mechanisms of dendritic cell lysosomal killing of Cryptococcus.

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

Confirmation of the presence of Cathepsin B in BMDC lysosomal extracts.Lysosomal extracts prepared from BMDCs were examined for the presence of cathepsin B. For ELISA analysis, fractions were taken during each step of the lysosomal purification process to examine the amount of cathepsin B present. Recombinant mouse cathepsin B in the pro-form was used as the positive control for Western blot analysis and for the ELISA standard. Cathepsin B was detected by separating the lysosomal extracts through SDS-page then transferring to a PVDF membrane and probing with anti-human/mouse cathepsin B. Recombinant mouse cathepsin B in the pro-form is 39 kDa, and active cathepsin B is 25-26 kDa. A). Western blot analysis of cathepsin B shows the presence of cathepsin B in two different samples of BMDC lysosomal extract. The blot is representative of three separate experiments. B) ELISA shows that 4000 pg cathepsin B per mg protein is present in BMDC lysosomal extracts. Aliquots of the different stages in the lysosomal purification process showed that cathepsin B is found primarily in the lysosomal fraction. Data shown are means ± standard error of the mean (SEM) of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Confirmation of the presence of Cathepsin B in BMDC lysosomal extracts.Lysosomal extracts prepared from BMDCs were examined for the presence of cathepsin B. For ELISA analysis, fractions were taken during each step of the lysosomal purification process to examine the amount of cathepsin B present. Recombinant mouse cathepsin B in the pro-form was used as the positive control for Western blot analysis and for the ELISA standard. Cathepsin B was detected by separating the lysosomal extracts through SDS-page then transferring to a PVDF membrane and probing with anti-human/mouse cathepsin B. Recombinant mouse cathepsin B in the pro-form is 39 kDa, and active cathepsin B is 25-26 kDa. A). Western blot analysis of cathepsin B shows the presence of cathepsin B in two different samples of BMDC lysosomal extract. The blot is representative of three separate experiments. B) ELISA shows that 4000 pg cathepsin B per mg protein is present in BMDC lysosomal extracts. Aliquots of the different stages in the lysosomal purification process showed that cathepsin B is found primarily in the lysosomal fraction. Data shown are means ± standard error of the mean (SEM) of three independent experiments.
Mentions: In order to definitively confirm that cathepsin B was present in our DC lysosomal extract, the extract was examined for the presence of cathepsin B by Western blot analysis. Proteins from two separate preparations of DC lysosomal extract were separated by SDS-PAGE and transferred to a PVDF membrane. Blots were probed with anti-human/mouse cathepsin B monoclonal antibody. Recombinant mouse cathepsin B in the pro-form (39 kDa), was used as a positive control. Active cathepsin B is approximately 25–26 kDa40. The Western blot showed the presence of cathepsin B in two separate preparations of BMDC lysosomal extract (Figure 3A). We next determined the concentration of cathepsin B in the BMDC lysosomal extracts by ELISA. Aliquots of each step during the lysosomal purification process were examined to quantify the amount of cathepsin B present. The ELISA showed that an average amount of 4000 pg/mg protein of cathepsin B is present in BMDC lysosomal extracts (Figure 3B).

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