Changing the receptor specificity of anthrax toxin

Area of Science:

• Bacterial Toxinology
• Molecular Biology
• Cellular Biology

Background:

• Bacterial toxins utilize cell-surface receptors for entry and intracellular delivery of enzymatic components.
• Anthrax toxin's protective antigen (PA) binds to ANTXR1/ANTXR2, facilitating effector protein transport into host cells.
• Understanding and manipulating toxin-receptor interactions are crucial for developing targeted therapies.

Purpose of the Study:

• To engineer the anthrax toxin's protective antigen (PA) to alter its cell-surface receptor specificity.
• To investigate the potential of modified PA for targeted delivery of effector proteins into specific cell populations.
• To explore applications in biological research and medicine, including cell ablation.

Main Methods:

• A double mutation was introduced into domain 4 of PA to abolish native receptor binding.
• Epidermal growth factor (EGF) or diphtheria toxin receptor-binding domain was fused to the C terminus of the mutated PA.
• Functional assays were performed using cell lines expressing specific receptors (A431, CHO-K1) to assess effector protein transport.
• Competition assays with free EGF were used to confirm receptor-mediated uptake.

Main Results:

• The PA-EGF fusion protein successfully transported effector proteins into EGF receptor-expressing cells (A431) but not into cells lacking the receptor (CHO-K1).
• Free EGF inhibited effector protein transport mediated by the PA-EGF fusion protein in A431 cells.
• Fusion of the diphtheria toxin receptor-binding domain redirected effector protein transport via the diphtheria toxin receptor.
• These findings demonstrate the feasibility of altering PA's receptor specificity.

Conclusions:

• The receptor specificity of anthrax toxin's protective antigen (PA) can be readily modified through protein engineering.
• Engineered PA fusion proteins offer a versatile platform for targeted delivery of effector proteins.
• These modified toxins hold promise for research applications and potential therapeutic strategies, including selective cell ablation.