Structure-function analysis of Bacillus anthracis edema factor by using monoclonal antibodies

Area of Science:

• Molecular Biology
• Immunology
• Bacteriology

Background:

• Bacillus anthracis edema toxin comprises protective antigen (PA) and edema factor (EF).
• Edema factor (EF) is a calcium- and calmodulin-dependent adenylate cyclase.
• Understanding EF's structure is crucial for developing targeted interventions.

Purpose of the Study:

• To identify antigenic regions of Bacillus anthracis edema factor (EF).
• To map the binding sites of monoclonal antibodies (mAbs) against EF.
• To determine the EF region involved in interaction with protective antigen (PA).

Main Methods:

• Competitive-binding enzyme-linked immunosorbent assay (ELISA) using paired monoclonal antibodies (mAbs).
• Radioiodination of EF (125I-EF) to study binding to cell-bound PA.
• Acid hydrolysis and formic acid cleavage of EF to generate fragments.
• Immunoblotting to map mAb binding sites on EF fragments.

Main Results:

• Five distinct antigenic regions of EF were identified using mAbs.
• Two mAbs (9F5 and 7G10) inhibited EF binding to PA.
• Only mAb 9F5 inhibited the cellular elongation response induced by edema toxin.
• Cleavage of EF yielded N-terminal (18 kDa), central (53 kDa), and C-terminal (17 kDa) fragments.
• mAbs 9F5 and 7G10 mapped to the N-terminal 18 kDa fragment (amino acids 1-156).
• mAb 1E6 mapped to the C-terminal 17 kDa fragment.
• The remaining seven mAbs mapped to the central 53 kDa fragment.
• mAbs 7G10 and 9F5 identified an antigenic region within amino acids 1-156 of EF.

Conclusions:

• The N-terminal region of EF (amino acids 1-156) is critical for its interaction with PA.
• This interaction is essential for the formation of functional edema toxin.
• Monoclonal antibodies targeting this region hold potential for therapeutic development against anthrax edema toxin.