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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 G0 protects cells from intoxicationwith C2 toxin.HeLa cells were incubated at 37 °C with 100 ng/mL His6-C2I + 200 ng/mL C2IIa in the presence or absence of 10 μMand 20 μM G0. For control (con), cells were left untreated ortreated with 20 μM G0 alone. Pictures were taken after 5 and24 h. (A) The morphology of cells after 5 h of C2 toxin-treatmentin the absence and presence of 20 μM G0 is shown. (B) The percentageof rounded cells was determined from the pictures. The values aregiven as mean ± SD (n = 3). Significance wasdetermined by the Student’s t test for cellstreated with C2 toxin in the presence of G0 against cells treatedwith C2 toxin in the absence of G0 (**p < 0.005,*p < 0.05).
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fig5: PAMAM dendrimer G0 protects cells from intoxicationwith C2 toxin.HeLa cells were incubated at 37 °C with 100 ng/mL His6-C2I + 200 ng/mL C2IIa in the presence or absence of 10 μMand 20 μM G0. For control (con), cells were left untreated ortreated with 20 μM G0 alone. Pictures were taken after 5 and24 h. (A) The morphology of cells after 5 h of C2 toxin-treatmentin the absence and presence of 20 μM G0 is shown. (B) The percentageof rounded cells was determined from the pictures. The values aregiven as mean ± SD (n = 3). Significance wasdetermined by the Student’s t test for cellstreated with C2 toxin in the presence of G0 against cells treatedwith C2 toxin in the absence of G0 (**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 G0 protects cells from intoxicationwith C2 toxin.HeLa cells were incubated at 37 °C with 100 ng/mL His6-C2I + 200 ng/mL C2IIa in the presence or absence of 10 μMand 20 μM G0. For control (con), cells were left untreated ortreated with 20 μM G0 alone. Pictures were taken after 5 and24 h. (A) The morphology of cells after 5 h of C2 toxin-treatmentin the absence and presence of 20 μM G0 is shown. (B) The percentageof rounded cells was determined from the pictures. The values aregiven as mean ± SD (n = 3). Significance wasdetermined by the Student’s t test for cellstreated with C2 toxin in the presence of G0 against cells treatedwith C2 toxin in the absence of G0 (**p < 0.005,*p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: PAMAM dendrimer G0 protects cells from intoxicationwith C2 toxin.HeLa cells were incubated at 37 °C with 100 ng/mL His6-C2I + 200 ng/mL C2IIa in the presence or absence of 10 μMand 20 μM G0. For control (con), cells were left untreated ortreated with 20 μM G0 alone. Pictures were taken after 5 and24 h. (A) The morphology of cells after 5 h of C2 toxin-treatmentin the absence and presence of 20 μM G0 is shown. (B) The percentageof rounded cells was determined from the pictures. The values aregiven as mean ± SD (n = 3). Significance wasdetermined by the Student’s t test for cellstreated with C2 toxin in the presence of G0 against cells treatedwith C2 toxin in the absence of G0 (**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