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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 intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 8 μg/mL His6-C2I + 0.8 μg/mL PA63 in the presence or absence of 20 or 30 μM G0. For control(con), cells were left untreated or treated with G0 alone. After theindicated time points, pictures were taken to monitor the changesin cell morphology. (A) The morphology of cells after 3.5 h of toxin-treatmentis shown. (B) The percentage of rounded cells was determined fromthe pictures. The values are given as mean ± SD (n = 3). Significance was determined by the Student’s t test for cells treated with the toxin in the presenceof G0 against cells treated with the toxin in the absence of G0 (***p < 0.0005; ** = p < 0.005, *p < 0.05).
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fig7: PAMAM dendrimer G0 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 8 μg/mL His6-C2I + 0.8 μg/mL PA63 in the presence or absence of 20 or 30 μM G0. For control(con), cells were left untreated or treated with G0 alone. After theindicated time points, pictures were taken to monitor the changesin cell morphology. (A) The morphology of cells after 3.5 h of toxin-treatmentis shown. (B) The percentage of rounded cells was determined fromthe pictures. The values are given as mean ± SD (n = 3). Significance was determined by the Student’s t test for cells treated with the toxin in the presenceof G0 against cells treated with the toxin in the absence of G0 (***p < 0.0005; ** = p < 0.005, *p < 0.05).

Mentions: Since G0 andG1 blocked the transmembrane pores formed by C2IIa and protectiveantigen (PA63) in vitro and protected cells from intoxicationwith C2 toxin, we investigated whether these dendrimers also protectcells from intoxication with His6-tagged C2I which is deliveredinto the cytosol by PA63. Recently, it was demonstratedthat His6-tagged C2I translocates through PA63-pores.89 Most likely, positively chargedHis-residues at the N-terminus of C2I mediate the interaction withthe pore and the translocation of C2I by mimicking positive chargesin the N-terminal region of the lethal factor.89 Therefore, this system might be the ideal model to comparethe pore blocking effect of the dendrimers in a cell-based model sincethe same cargo protein, His-C2I, is delivered by two different translocationpores, C2IIa and PA63, across endosomal membranes and allowsthe monitoring of the cytotoxic effects via C2I-mediated cell rounding.Having confirmed that G0 and G1 inhibit the intoxication of cellswith C2IIa and His-C2I (Figure 5, Supporting Information, Figure S9), cells werechallenged with PA63 + His-C2I in the absence and presenceof G0 or G1. The results shown in Figures 7 and 8 indicatethat both dendrimers delayed the intoxication of cells by PA63 + His-C2I as less cells rounded up in the presence of G0 or G1,suggesting that these dendrimers block the translocation pores formedby PA63 in the membranes of acidified endosomes in intactcells.


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 intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 8 μg/mL His6-C2I + 0.8 μg/mL PA63 in the presence or absence of 20 or 30 μM G0. For control(con), cells were left untreated or treated with G0 alone. After theindicated time points, pictures were taken to monitor the changesin cell morphology. (A) The morphology of cells after 3.5 h of toxin-treatmentis shown. (B) The percentage of rounded cells was determined fromthe pictures. The values are given as mean ± SD (n = 3). Significance was determined by the Student’s t test for cells treated with the toxin in the presenceof G0 against cells treated with the toxin in the absence of G0 (***p < 0.0005; ** = p < 0.005, *p < 0.05).
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fig7: PAMAM dendrimer G0 protects cells from intoxication with His6-C2I/PA63. HeLa cells were incubated at 37 °Cwith 8 μg/mL His6-C2I + 0.8 μg/mL PA63 in the presence or absence of 20 or 30 μM G0. For control(con), cells were left untreated or treated with G0 alone. After theindicated time points, pictures were taken to monitor the changesin cell morphology. (A) The morphology of cells after 3.5 h of toxin-treatmentis shown. (B) The percentage of rounded cells was determined fromthe pictures. The values are given as mean ± SD (n = 3). Significance was determined by the Student’s t test for cells treated with the toxin in the presenceof G0 against cells treated with the toxin in the absence of G0 (***p < 0.0005; ** = p < 0.005, *p < 0.05).
Mentions: Since G0 andG1 blocked the transmembrane pores formed by C2IIa and protectiveantigen (PA63) in vitro and protected cells from intoxicationwith C2 toxin, we investigated whether these dendrimers also protectcells from intoxication with His6-tagged C2I which is deliveredinto the cytosol by PA63. Recently, it was demonstratedthat His6-tagged C2I translocates through PA63-pores.89 Most likely, positively chargedHis-residues at the N-terminus of C2I mediate the interaction withthe pore and the translocation of C2I by mimicking positive chargesin the N-terminal region of the lethal factor.89 Therefore, this system might be the ideal model to comparethe pore blocking effect of the dendrimers in a cell-based model sincethe same cargo protein, His-C2I, is delivered by two different translocationpores, C2IIa and PA63, across endosomal membranes and allowsthe monitoring of the cytotoxic effects via C2I-mediated cell rounding.Having confirmed that G0 and G1 inhibit the intoxication of cellswith C2IIa and His-C2I (Figure 5, Supporting Information, Figure S9), cells werechallenged with PA63 + His-C2I in the absence and presenceof G0 or G1. The results shown in Figures 7 and 8 indicatethat both dendrimers delayed the intoxication of cells by PA63 + His-C2I as less cells rounded up in the presence of G0 or G1,suggesting that these dendrimers block the translocation pores formedby PA63 in the membranes of acidified endosomes in intactcells.

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