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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 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 10 μg/mL His6-C2I + 1 μg/mL PA63 in the presence or absence of 5 μM G1. For control (con),cells were left untreated or treated with G1 alone. After the indicatedtime points, pictures were taken to monitor the changes in cell morphology.(A) The morphology of cells after 4.5 h of toxin-treatment is shown.(B) The percentage of rounded cells was determined from the pictures.The values are given as mean ± SD (n = 6). Significancewas determined by the Student’s t test forcells treated with the toxin in the presence of G1 against cells treatedwith the toxin in the absence of G1 (***p < 0.0005;**p < 0.005; *p < 0.05).
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fig8: PAMAM dendrimer G1 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 10 μg/mL His6-C2I + 1 μg/mL PA63 in the presence or absence of 5 μM G1. For control (con),cells were left untreated or treated with G1 alone. After the indicatedtime points, pictures were taken to monitor the changes in cell morphology.(A) The morphology of cells after 4.5 h of toxin-treatment is shown.(B) The percentage of rounded cells was determined from the pictures.The values are given as mean ± SD (n = 6). Significancewas determined by the Student’s t test forcells treated with the toxin in the presence of G1 against cells treatedwith the toxin in the absence of G1 (***p < 0.0005;**p < 0.005; *p < 0.05).

Mentions: First, we tested theeffects of the PAMAM-dendrimers on HeLa cells. When cells were incubatedwith C2 toxin in the presence of G1 in the culture medium, less cellsrounded up compared to treatment of cells with the C2 toxin alone,as shown in Figure 4A for a 5.7 h incubationperiod. Cell rounding is the consequence of the toxin-induced depolymerizationof F-actin and therefore a specific parameter to monitor the cytotoxicmode of action of the actin-ADP-ribosylating toxins in the cytosolof cultured cells. A more detailed analysis with different concentrationsof C2 toxin revealed a time-dependent delay of intoxication by 10μM of G1 (Figure 4B). G1 alone had norelevant effects on cell morphology under these conditions (Figure 4B,C, Figure 8, and Supporting Information, Figure S3). Figure 4C shows the time- and concentration-dependent inhibitoryeffect of G1 on the intoxication of HeLa cells with C2 toxin overa 48 h incubation period. When PAMAM dendrimer G0 was used insteadof G1, widely comparable results were obtained (Figure 5) however, the protective effect against C2 toxin was strongerin the case of G1.


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 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 10 μg/mL His6-C2I + 1 μg/mL PA63 in the presence or absence of 5 μM G1. For control (con),cells were left untreated or treated with G1 alone. After the indicatedtime points, pictures were taken to monitor the changes in cell morphology.(A) The morphology of cells after 4.5 h of toxin-treatment is shown.(B) The percentage of rounded cells was determined from the pictures.The values are given as mean ± SD (n = 6). Significancewas determined by the Student’s t test forcells treated with the toxin in the presence of G1 against cells treatedwith the toxin in the absence of G1 (***p < 0.0005;**p < 0.005; *p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4215879&req=5

fig8: PAMAM dendrimer G1 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 10 μg/mL His6-C2I + 1 μg/mL PA63 in the presence or absence of 5 μM G1. For control (con),cells were left untreated or treated with G1 alone. After the indicatedtime points, pictures were taken to monitor the changes in cell morphology.(A) The morphology of cells after 4.5 h of toxin-treatment is shown.(B) The percentage of rounded cells was determined from the pictures.The values are given as mean ± SD (n = 6). Significancewas determined by the Student’s t test forcells treated with the toxin in the presence of G1 against cells treatedwith the toxin in the absence of G1 (***p < 0.0005;**p < 0.005; *p < 0.05).
Mentions: First, we tested theeffects of the PAMAM-dendrimers on HeLa cells. When cells were incubatedwith C2 toxin in the presence of G1 in the culture medium, less cellsrounded up compared to treatment of cells with the C2 toxin alone,as shown in Figure 4A for a 5.7 h incubationperiod. Cell rounding is the consequence of the toxin-induced depolymerizationof F-actin and therefore a specific parameter to monitor the cytotoxicmode of action of the actin-ADP-ribosylating toxins in the cytosolof cultured cells. A more detailed analysis with different concentrationsof C2 toxin revealed a time-dependent delay of intoxication by 10μM of G1 (Figure 4B). G1 alone had norelevant effects on cell morphology under these conditions (Figure 4B,C, Figure 8, and Supporting Information, Figure S3). Figure 4C shows the time- and concentration-dependent inhibitoryeffect of G1 on the intoxication of HeLa cells with C2 toxin overa 48 h incubation period. When PAMAM dendrimer G0 was used insteadof G1, widely comparable results were obtained (Figure 5) however, the protective effect against C2 toxin was strongerin the case of G1.

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