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Changing the receptor specificity of anthrax toxin.

Mechaly A, McCluskey AJ, Collier RJ - MBio (2012)

Bottom Line: We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor.Bacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment.By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Unlabelled: The actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.

Importance: Bacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment. By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell. Our results show that the receptor specificity of the transport protein of anthrax toxin may be readily changed, raising the possibility that receptor-redirected forms of protective antigen (PA) and PA homologs may be useful for research and medical applications requiring modification or ablation of designated populations of cells.

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mPA-EGF transports LF and EF into EGFR-bearing cells. (A) A431 cells (1 × 106) were treated with 100 nM LF plus 10 nM PA or PA variant for 3 h. Cell lysates were prepared, fractionated by SDS-PAGE, and transferred to a PVDF membrane, and MEK1 cleavage was evaluated by Western blotting with anti-MEK1 antibody. For a control, GAPDH was monitored with anti-GAPDH antibodies. (B) A431 cells (3.5 × 104) were exposed to 50 nM EF plus 10 nM PA or PA variant for 1 h. A competition enzyme-linked immunoassay was performed to detect the intracellular concentration of cAMP, based on a standard curve, following the protocol of the manufacturer (Cell Signaling Technology). The column labeled “Control” corresponds to A431 cells treated with EF in the absence of PA. Each bar represents the average of experiments performed in quadruplicate.
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fig4: mPA-EGF transports LF and EF into EGFR-bearing cells. (A) A431 cells (1 × 106) were treated with 100 nM LF plus 10 nM PA or PA variant for 3 h. Cell lysates were prepared, fractionated by SDS-PAGE, and transferred to a PVDF membrane, and MEK1 cleavage was evaluated by Western blotting with anti-MEK1 antibody. For a control, GAPDH was monitored with anti-GAPDH antibodies. (B) A431 cells (3.5 × 104) were exposed to 50 nM EF plus 10 nM PA or PA variant for 1 h. A competition enzyme-linked immunoassay was performed to detect the intracellular concentration of cAMP, based on a standard curve, following the protocol of the manufacturer (Cell Signaling Technology). The column labeled “Control” corresponds to A431 cells treated with EF in the absence of PA. Each bar represents the average of experiments performed in quadruplicate.

Mentions: We tested the ability of mPA-EGF to translocate LF and EF, the native effector moieties of anthrax toxin, into A431 cells. LF inactivates mitogen-activated protein kinase kinases (MEKs) by cleaving near their N termini (3, 5), and we measured LF entry by Western blotting of cell lysates with an anti-MEK1 antibody after incubating cells with LF plus PA or a PA variant. MEK1 was cleaved completely with LF in combination with PA or mPA-EGF, but not in combination with the translocation-deficient mutant PAF427H (Fig. 4A). We measured entry of EF using an enzyme-linked competition assay to determine the intracellular level of cyclic AMP (cAMP) and observed a 400-fold elevation of cAMP when mPA-EGF was used as the transporter (Fig. 4B). This level was ~100 times higher than that observed when wild-type PA (WT PA) was used as the transporter. The level of cAMP when mPA or PAF427H was used as the transporter was identical to the background level. The strong elevation observed with mPA-EGF was likely due in part to the high level of EGF receptor (EGFR) on the A431 cells. The fact that cleavage of MEK1 was complete when LF was delivered via WT PA or mPA-EGF, whereas the amount of cAMP generated by EF delivered by mPA-EGF was vastly greater than when EF entered via WT PA may reflect differences in reaction kinetics and the levels of substrates of the two effectors within cells.


Changing the receptor specificity of anthrax toxin.

Mechaly A, McCluskey AJ, Collier RJ - MBio (2012)

mPA-EGF transports LF and EF into EGFR-bearing cells. (A) A431 cells (1 × 106) were treated with 100 nM LF plus 10 nM PA or PA variant for 3 h. Cell lysates were prepared, fractionated by SDS-PAGE, and transferred to a PVDF membrane, and MEK1 cleavage was evaluated by Western blotting with anti-MEK1 antibody. For a control, GAPDH was monitored with anti-GAPDH antibodies. (B) A431 cells (3.5 × 104) were exposed to 50 nM EF plus 10 nM PA or PA variant for 1 h. A competition enzyme-linked immunoassay was performed to detect the intracellular concentration of cAMP, based on a standard curve, following the protocol of the manufacturer (Cell Signaling Technology). The column labeled “Control” corresponds to A431 cells treated with EF in the absence of PA. Each bar represents the average of experiments performed in quadruplicate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3569862&req=5

fig4: mPA-EGF transports LF and EF into EGFR-bearing cells. (A) A431 cells (1 × 106) were treated with 100 nM LF plus 10 nM PA or PA variant for 3 h. Cell lysates were prepared, fractionated by SDS-PAGE, and transferred to a PVDF membrane, and MEK1 cleavage was evaluated by Western blotting with anti-MEK1 antibody. For a control, GAPDH was monitored with anti-GAPDH antibodies. (B) A431 cells (3.5 × 104) were exposed to 50 nM EF plus 10 nM PA or PA variant for 1 h. A competition enzyme-linked immunoassay was performed to detect the intracellular concentration of cAMP, based on a standard curve, following the protocol of the manufacturer (Cell Signaling Technology). The column labeled “Control” corresponds to A431 cells treated with EF in the absence of PA. Each bar represents the average of experiments performed in quadruplicate.
Mentions: We tested the ability of mPA-EGF to translocate LF and EF, the native effector moieties of anthrax toxin, into A431 cells. LF inactivates mitogen-activated protein kinase kinases (MEKs) by cleaving near their N termini (3, 5), and we measured LF entry by Western blotting of cell lysates with an anti-MEK1 antibody after incubating cells with LF plus PA or a PA variant. MEK1 was cleaved completely with LF in combination with PA or mPA-EGF, but not in combination with the translocation-deficient mutant PAF427H (Fig. 4A). We measured entry of EF using an enzyme-linked competition assay to determine the intracellular level of cyclic AMP (cAMP) and observed a 400-fold elevation of cAMP when mPA-EGF was used as the transporter (Fig. 4B). This level was ~100 times higher than that observed when wild-type PA (WT PA) was used as the transporter. The level of cAMP when mPA or PAF427H was used as the transporter was identical to the background level. The strong elevation observed with mPA-EGF was likely due in part to the high level of EGF receptor (EGFR) on the A431 cells. The fact that cleavage of MEK1 was complete when LF was delivered via WT PA or mPA-EGF, whereas the amount of cAMP generated by EF delivered by mPA-EGF was vastly greater than when EF entered via WT PA may reflect differences in reaction kinetics and the levels of substrates of the two effectors within cells.

Bottom Line: We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor.Bacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment.By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT

Unlabelled: The actions of many bacterial toxins depend on their ability to bind to one or more cell-surface receptors. Anthrax toxin acts by a sequence of events that begins when the protective-antigen (PA) moiety of the toxin binds to either one of two cell-surface proteins, ANTXR1 and ANTXR2, and is proteolytically activated. The activated PA self-associates to form oligomeric pore precursors, which, in turn, bind the enzymatic moieties of the toxin and transport them to the cytosol. We introduced a double mutation into domain 4 of PA to ablate its native receptor-binding function and fused epidermal growth factor (EGF) to the C terminus of the mutated protein. The resulting fusion protein transported enzymatic effector proteins into a cell line that expressed the EGF receptor (A431 cells), but not into a line lacking this receptor (CHO-K1 cells). Addition of excess free EGF blocked transport of effector proteins into A431 cells via the fusion protein, but not via native PA. We also showed that fusing the diphtheria toxin receptor-binding domain to the C terminus of the mutated PA channeled effector-protein transport through the diphtheria toxin receptor. PA fusion proteins with altered receptor specificity may be useful in biological research and could have practical applications, including ablation or perturbation of selected populations of cells in vivo.

Importance: Bacterial toxins that act within mammalian cells have receptor-dependent mechanisms to transport their enzymatic components to the cytoplasmic compartment. By inactivating or otherwise modifying their respective intracellular targets, these intracellular effectors disrupt metabolic pathways and in some cases cause death of the cell. Our results show that the receptor specificity of the transport protein of anthrax toxin may be readily changed, raising the possibility that receptor-redirected forms of protective antigen (PA) and PA homologs may be useful for research and medical applications requiring modification or ablation of designated populations of cells.

Show MeSH
Related in: MedlinePlus