Evaluating TcAs for Use in Biotechnology Applications.

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• ABC toxin complexes (Tcs) are tripartite nano-syringe-like systems with potential applications in pesticides and peptide delivery.
• Their ability to form chimeric holotoxins and translocate payloads across membranes makes them ideal for protein engineering.
• Current limitations include a lack of understanding of receptor binding domains (RBDs) and limited biodistribution data in mammals.

Purpose of the Study:

• To develop strategies for modifying the targeting capabilities of the A subunit (TcA) from *Xenorhabdus nematophilus* (Xn-XptA2).
• To identify structural differences in Xn-XptA2 compared to other TcAs.
• To demonstrate the modularity of substituting RBDs into the Xn-XptA2 scaffold and provide initial biodistribution data in mice.

Main Methods:

• Comparative structural analysis of Xn-XptA2 with other TcAs.
• Engineering chimeric TcA constructs by substituting RBDs.
• In vivo biodistribution studies of TcA in mice.

Main Results:

• Novel structural differences were identified in Xn-XptA2.
• The modularity of substituting RBDs into the Xn-XptA2 scaffold was demonstrated.
• The first biodistribution data for any TcA in mice were obtained.

Conclusions:

• Targeted protein engineering of ABC Tcs is feasible through modification of TcA subunits.
• Substitution of RBDs offers a modular approach to alter targeting specificity.
• This study provides foundational data for the future development of Tcs for therapeutic or pesticidal applications.