Insertion-trigger residues differentially modulate endosomal escape by cytotoxic necrotizing factor toxins.

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Delivery Systems

Background:

• Bacterial protein toxins offer targeted cytosolic delivery of therapeutic cargo.
• Efficient endosomal escape is crucial for bacterial toxin-inspired drug delivery (BTIDD) vehicles to prevent lysosomal degradation.
• The cytotoxic necrotizing factor (CNF) family is a valuable model for studying cargo delivery due to its modular nature and homologous toxins.

Purpose of the Study:

• To investigate the factors influencing endosomal escape and cargo delivery efficiency in CNF toxins.
• To identify specific amino acid residues responsible for differential endosomal acidification sensitivity among CNF toxins.
• To explore the potential for fine-tuning BTIDD platforms by modifying toxin properties.

Main Methods:

• Comparative analysis of CNFy and CNF3 sensitivity to endosomal acidification inhibitors.
• Site-directed mutagenesis to swap key amino acid residues in the translocation domain of CNFy and CNF3.
• Assessment of cargo delivery efficiency under conditions that modulate endosomal acidification and trafficking (NH4Cl, EGA, ABMA).

Main Results:

• CNF3 exhibits lower sensitivity to endosomal acidification inhibitors compared to CNFy, CNF1, and CNF2.
• Two specific amino acid residues in the translocation domain differentiate the endosomal escape mechanisms of CNFy and CNF3.
• Modifying these residues altered toxin sensitivity and cargo delivery efficiency, mimicking the behavior of the other toxin.
• Cargo delivery by CNFy, but not CNF3, requires trafficking to the acidic late endosome.

Conclusions:

• Specific amino acid residues within the CNF translocation domain critically influence endosomal escape and cargo delivery.
• The differential requirement for late endosomal acidification can be modulated by altering these residues.
• These findings provide a basis for designing more efficient BTIDD platforms by fine-tuning endosomal escape mechanisms.