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Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Förstner P, Bayer F, Kalu N, Felsen S, Förtsch C, Aloufi A, Ng DY, Weil T, Nestorovich EM, Barth H - Biomacromolecules (2014)

Bottom Line: Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development.These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells.We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center , D-89081 Ulm, Germany.

ABSTRACT
Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

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PAMAM dendrimer G1 does not affect enzyme activity and cell bindingof C2 toxin. (A) PAMAM dendrimer G1 does not inhibit the ADP-ribosylationof actin by C2I in vitro. HeLa lysate (40 μg of protein in 25μL) was incubated for 30 min at 37 °C in the presence orabsence of G1. The lysate was treated with or without 10 ng/mL C2Iand incubated for 15 min at 37 °C with 10 μM biotin-labeledNAD+. The proteins were separated by SDS-PAGE, blotted onto nitrocelluloseand the ADP-ribosylated (i.e., biotin-labeled) actin was detectedby Western blotting (right panel). The intensity of bands was determinedby densitometry using the Adobe Photoshop 7.0 software (left panel).Values are given as mean ± SD (n = 3) and significancewas tested between C2I-treated samples with or without G1 by usingthe Student’s t test (ns = not significant).(B) PAMAM dendrimer G1 does not inhibit the receptor binding of C2toxin. HeLa cells were incubated for 30 min at 4 °C in serum-freemedium with C2 toxin (800 ng/mL C2IIa + 400 ng/mL C2I) in the presenceor absence of 10 μM G1. As a control, cells were incubated withfresh serum-free medium. Then the medium was removed and cells werewashed to remove any unbound toxin. After 25 μL of ADP-ribosylationbuffer was added, cells were scraped of and lysed. ADP-ribosylationof actin was detected by Western blotting, as described in A.
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fig6: PAMAM dendrimer G1 does not affect enzyme activity and cell bindingof C2 toxin. (A) PAMAM dendrimer G1 does not inhibit the ADP-ribosylationof actin by C2I in vitro. HeLa lysate (40 μg of protein in 25μL) was incubated for 30 min at 37 °C in the presence orabsence of G1. The lysate was treated with or without 10 ng/mL C2Iand incubated for 15 min at 37 °C with 10 μM biotin-labeledNAD+. The proteins were separated by SDS-PAGE, blotted onto nitrocelluloseand the ADP-ribosylated (i.e., biotin-labeled) actin was detectedby Western blotting (right panel). The intensity of bands was determinedby densitometry using the Adobe Photoshop 7.0 software (left panel).Values are given as mean ± SD (n = 3) and significancewas tested between C2I-treated samples with or without G1 by usingthe Student’s t test (ns = not significant).(B) PAMAM dendrimer G1 does not inhibit the receptor binding of C2toxin. HeLa cells were incubated for 30 min at 4 °C in serum-freemedium with C2 toxin (800 ng/mL C2IIa + 400 ng/mL C2I) in the presenceor absence of 10 μM G1. As a control, cells were incubated withfresh serum-free medium. Then the medium was removed and cells werewashed to remove any unbound toxin. After 25 μL of ADP-ribosylationbuffer was added, cells were scraped of and lysed. ADP-ribosylationof actin was detected by Western blotting, as described in A.

Mentions: Taken together, these results clearly demonstrate that PAMAMdendrimersG0 and G1 interfere with the mode of action of C2 toxin but give nohints on an underlying reason. However, the data indicate that 10μM of G1 did neither inhibit the ADP-ribosylation of actin byC2I in vitro (Figure 6A), nor the binding ofC2 toxin to its cell surface receptor (Figure 6B).


Cationic PAMAM dendrimers as pore-blocking binary toxin inhibitors.

Förstner P, Bayer F, Kalu N, Felsen S, Förtsch C, Aloufi A, Ng DY, Weil T, Nestorovich EM, Barth H - Biomacromolecules (2014)

PAMAM dendrimer G1 does not affect enzyme activity and cell bindingof C2 toxin. (A) PAMAM dendrimer G1 does not inhibit the ADP-ribosylationof actin by C2I in vitro. HeLa lysate (40 μg of protein in 25μL) was incubated for 30 min at 37 °C in the presence orabsence of G1. The lysate was treated with or without 10 ng/mL C2Iand incubated for 15 min at 37 °C with 10 μM biotin-labeledNAD+. The proteins were separated by SDS-PAGE, blotted onto nitrocelluloseand the ADP-ribosylated (i.e., biotin-labeled) actin was detectedby Western blotting (right panel). The intensity of bands was determinedby densitometry using the Adobe Photoshop 7.0 software (left panel).Values are given as mean ± SD (n = 3) and significancewas tested between C2I-treated samples with or without G1 by usingthe Student’s t test (ns = not significant).(B) PAMAM dendrimer G1 does not inhibit the receptor binding of C2toxin. HeLa cells were incubated for 30 min at 4 °C in serum-freemedium with C2 toxin (800 ng/mL C2IIa + 400 ng/mL C2I) in the presenceor absence of 10 μM G1. As a control, cells were incubated withfresh serum-free medium. Then the medium was removed and cells werewashed to remove any unbound toxin. After 25 μL of ADP-ribosylationbuffer was added, cells were scraped of and lysed. ADP-ribosylationof actin was detected by Western blotting, as described in A.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4215879&req=5

fig6: PAMAM dendrimer G1 does not affect enzyme activity and cell bindingof C2 toxin. (A) PAMAM dendrimer G1 does not inhibit the ADP-ribosylationof actin by C2I in vitro. HeLa lysate (40 μg of protein in 25μL) was incubated for 30 min at 37 °C in the presence orabsence of G1. The lysate was treated with or without 10 ng/mL C2Iand incubated for 15 min at 37 °C with 10 μM biotin-labeledNAD+. The proteins were separated by SDS-PAGE, blotted onto nitrocelluloseand the ADP-ribosylated (i.e., biotin-labeled) actin was detectedby Western blotting (right panel). The intensity of bands was determinedby densitometry using the Adobe Photoshop 7.0 software (left panel).Values are given as mean ± SD (n = 3) and significancewas tested between C2I-treated samples with or without G1 by usingthe Student’s t test (ns = not significant).(B) PAMAM dendrimer G1 does not inhibit the receptor binding of C2toxin. HeLa cells were incubated for 30 min at 4 °C in serum-freemedium with C2 toxin (800 ng/mL C2IIa + 400 ng/mL C2I) in the presenceor absence of 10 μM G1. As a control, cells were incubated withfresh serum-free medium. Then the medium was removed and cells werewashed to remove any unbound toxin. After 25 μL of ADP-ribosylationbuffer was added, cells were scraped of and lysed. ADP-ribosylationof actin was detected by Western blotting, as described in A.
Mentions: Taken together, these results clearly demonstrate that PAMAMdendrimersG0 and G1 interfere with the mode of action of C2 toxin but give nohints on an underlying reason. However, the data indicate that 10μM of G1 did neither inhibit the ADP-ribosylation of actin byC2I in vitro (Figure 6A), nor the binding ofC2 toxin to its cell surface receptor (Figure 6B).

Bottom Line: Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development.These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells.We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmacology and Toxicology, University of Ulm Medical Center , D-89081 Ulm, Germany.

ABSTRACT
Dendrimers are unique highly branched macromolecules with numerous groundbreaking biomedical applications under development. Here we identified poly(amido amine) (PAMAM) dendrimers as novel blockers for the pore-forming B components of the binary anthrax toxin (PA63) and Clostridium botulinum C2 toxin (C2IIa). These pores are essential for delivery of the enzymatic A components of the internalized toxins from endosomes into the cytosol of target cells. We demonstrate that at low μM concentrations cationic PAMAM dendrimers block PA63 and C2IIa to inhibit channel-mediated transport of the A components, thereby protecting HeLa and Vero cells from intoxication. By channel reconstitution and high-resolution current recording, we show that the PAMAM dendrimers obstruct transmembrane PA63 and C2IIa pores in planar lipid bilayers at nM concentrations. These findings suggest a new potential role for the PAMAM dendrimers as effective polyvalent channel-blocking inhibitors, which can protect human target cells from intoxication with binary toxins from pathogenic bacteria.

Show MeSH
Related in: MedlinePlus