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Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.

Bewley MA, Naughton M, Preston J, Mitchell A, Holmes A, Marriott HM, Read RC, Mitchell TJ, Whyte MK, Dockrell DH - MBio (2014)

Bottom Line: LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β).The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP.We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom.

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Related in: MedlinePlus

Macrophages exposed to S. pneumoniae develop a progressive increase in lysosomal membrane permeabilization. (A to C) Monocyte-derived macrophages (MDM) were preloaded with 5 mg/ml of FITC-dextran molecules of the designated molecular mass and challenged with S. pneumoniae (D39) or pneumolysin-deficient (Stop) S. pneumoniae or with D39 in the presence of cytochalasin D (D39+CD). At the designated time postchallenge, cells were fixed and analyzed by fluorescence microscopy and the percentages of cells showing mixed punctate/diffuse or diffuse staining were determined. (A) Representative images of cells showing completely punctate FITC-dextran localization, mixed punctate/diffuse staining, or completely diffuse staining, taken from a strain D39-exposed population at 10 h postchallenge. (B and C) Localization of 10-kDa (B) and 40- or 250-kDa (C) FITC-dextran (n = 4). * = P < 0.05 for 10-kDa D39 versus 10-kDa D39+CD; ^ ^ ^ = P < 0.001 for 10-kDa D39 versus 10-kDa Stop; ++ = P < 0.01 for 40-kDa D39 versus 40-kDa Stop (two-way ANOVA). Data are expressed as means ± SEM.
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fig4: Macrophages exposed to S. pneumoniae develop a progressive increase in lysosomal membrane permeabilization. (A to C) Monocyte-derived macrophages (MDM) were preloaded with 5 mg/ml of FITC-dextran molecules of the designated molecular mass and challenged with S. pneumoniae (D39) or pneumolysin-deficient (Stop) S. pneumoniae or with D39 in the presence of cytochalasin D (D39+CD). At the designated time postchallenge, cells were fixed and analyzed by fluorescence microscopy and the percentages of cells showing mixed punctate/diffuse or diffuse staining were determined. (A) Representative images of cells showing completely punctate FITC-dextran localization, mixed punctate/diffuse staining, or completely diffuse staining, taken from a strain D39-exposed population at 10 h postchallenge. (B and C) Localization of 10-kDa (B) and 40- or 250-kDa (C) FITC-dextran (n = 4). * = P < 0.05 for 10-kDa D39 versus 10-kDa D39+CD; ^ ^ ^ = P < 0.001 for 10-kDa D39 versus 10-kDa Stop; ++ = P < 0.01 for 40-kDa D39 versus 40-kDa Stop (two-way ANOVA). Data are expressed as means ± SEM.

Mentions: The requirement for intracellular bacteria for the execution phase of macrophage apoptosis implicates intracellular microbial factors, but we know little concerning the extent of LMP and its relationship to the cellular localization of PLY after the initial trafficking of bacteria into the phagolysosome (40). Macrophages that have internalized pneumococci exhibit significant LLA from 10 h postinfection and show evidence of LMP as determined by translocation of lysosomal proteases into the cytoplasm (20). However, lysosomal integrity could theoretically be compromised earlier to a more modest extent, allowing efflux of small peptides before LLA and cathepsin release can be detected. To determine more accurately the kinetics and extent of lysosomal and phagolysosomal permeabilization, the translocation of fluorescein isothiocyanate (FITC)-dextran molecules of various sizes, represented by the replacement of the normal punctate staining pattern by a mixed punctate/diffuse or diffuse pattern (Fig. 4A), was measured. There was progressive translocation of 10-kDa FITC-dextran molecules into the cytosol, with the translocation visible 6 h after bacterial challenge (Fig. 4B). The redistribution of the 40-kDa FITC-dextran molecules occurred by the 10-h time point (Fig. 4C). In the cases of both the 10-kDa and the 40-kDa molecules, translocation predominantly resulted in a mixture of both diffuse staining and punctate staining, indicative of partial lysosomal/phagolysosomal efflux. In contrast, the 250-kDa FITC-dextran remained in the lysosomal/phagolysosomal compartment at all time points (Fig. 4C).


Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.

Bewley MA, Naughton M, Preston J, Mitchell A, Holmes A, Marriott HM, Read RC, Mitchell TJ, Whyte MK, Dockrell DH - MBio (2014)

Macrophages exposed to S. pneumoniae develop a progressive increase in lysosomal membrane permeabilization. (A to C) Monocyte-derived macrophages (MDM) were preloaded with 5 mg/ml of FITC-dextran molecules of the designated molecular mass and challenged with S. pneumoniae (D39) or pneumolysin-deficient (Stop) S. pneumoniae or with D39 in the presence of cytochalasin D (D39+CD). At the designated time postchallenge, cells were fixed and analyzed by fluorescence microscopy and the percentages of cells showing mixed punctate/diffuse or diffuse staining were determined. (A) Representative images of cells showing completely punctate FITC-dextran localization, mixed punctate/diffuse staining, or completely diffuse staining, taken from a strain D39-exposed population at 10 h postchallenge. (B and C) Localization of 10-kDa (B) and 40- or 250-kDa (C) FITC-dextran (n = 4). * = P < 0.05 for 10-kDa D39 versus 10-kDa D39+CD; ^ ^ ^ = P < 0.001 for 10-kDa D39 versus 10-kDa Stop; ++ = P < 0.01 for 40-kDa D39 versus 40-kDa Stop (two-way ANOVA). Data are expressed as means ± SEM.
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fig4: Macrophages exposed to S. pneumoniae develop a progressive increase in lysosomal membrane permeabilization. (A to C) Monocyte-derived macrophages (MDM) were preloaded with 5 mg/ml of FITC-dextran molecules of the designated molecular mass and challenged with S. pneumoniae (D39) or pneumolysin-deficient (Stop) S. pneumoniae or with D39 in the presence of cytochalasin D (D39+CD). At the designated time postchallenge, cells were fixed and analyzed by fluorescence microscopy and the percentages of cells showing mixed punctate/diffuse or diffuse staining were determined. (A) Representative images of cells showing completely punctate FITC-dextran localization, mixed punctate/diffuse staining, or completely diffuse staining, taken from a strain D39-exposed population at 10 h postchallenge. (B and C) Localization of 10-kDa (B) and 40- or 250-kDa (C) FITC-dextran (n = 4). * = P < 0.05 for 10-kDa D39 versus 10-kDa D39+CD; ^ ^ ^ = P < 0.001 for 10-kDa D39 versus 10-kDa Stop; ++ = P < 0.01 for 40-kDa D39 versus 40-kDa Stop (two-way ANOVA). Data are expressed as means ± SEM.
Mentions: The requirement for intracellular bacteria for the execution phase of macrophage apoptosis implicates intracellular microbial factors, but we know little concerning the extent of LMP and its relationship to the cellular localization of PLY after the initial trafficking of bacteria into the phagolysosome (40). Macrophages that have internalized pneumococci exhibit significant LLA from 10 h postinfection and show evidence of LMP as determined by translocation of lysosomal proteases into the cytoplasm (20). However, lysosomal integrity could theoretically be compromised earlier to a more modest extent, allowing efflux of small peptides before LLA and cathepsin release can be detected. To determine more accurately the kinetics and extent of lysosomal and phagolysosomal permeabilization, the translocation of fluorescein isothiocyanate (FITC)-dextran molecules of various sizes, represented by the replacement of the normal punctate staining pattern by a mixed punctate/diffuse or diffuse pattern (Fig. 4A), was measured. There was progressive translocation of 10-kDa FITC-dextran molecules into the cytosol, with the translocation visible 6 h after bacterial challenge (Fig. 4B). The redistribution of the 40-kDa FITC-dextran molecules occurred by the 10-h time point (Fig. 4C). In the cases of both the 10-kDa and the 40-kDa molecules, translocation predominantly resulted in a mixture of both diffuse staining and punctate staining, indicative of partial lysosomal/phagolysosomal efflux. In contrast, the 250-kDa FITC-dextran remained in the lysosomal/phagolysosomal compartment at all time points (Fig. 4C).

Bottom Line: LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β).The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP.We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection and Immunity, University of Sheffield Medical School, Sheffield, United Kingdom.

Show MeSH
Related in: MedlinePlus