Novel Use of a Cell‐Penetrating Peptide‐Adaptor System to Investigate Activity of Type III Secretion Effector Proteins in Mammalian Cells
Authors: Young Shaquanna M. , Dickson Robert L. , McMurry Jonathan L. 
Published: June 2021

Pathogenic bacteria utilize Type III Secretion Systems (T3SSs) to deliver virulent effector proteins for the subversion of host cell defenses. These effectors specifically target innate immune signaling pathways of host cells to ensure pathogen survival within the intestinal microbiota. This study investigated the activity of effector proteins AopP & AvrA derived from the human pathogens Aeromonas salmonicida and Salmonella enterica. AopP targets the nuclear factor‐kappa B (NF‐kB) pathway to promote cellular apoptosis while AvrA targets both the c‐Jun N‐terminal kinase (JNK) and NF‐kB pathways to hinder host immune response without apoptosis induction. Previous studies investigated effector activity via transfection or pathogen infection methods, but these methods have shortcomings, such as the cytotoxicity or immunogenicity of the viruses used for transfection or infection. An alternative strategy is to deliver proteins via a cell‐penetrating peptide (CPP)‐adaptor mechanism. CPPs fused with adaptors enable translocation of macromolecules across plasma membranes. The goal of this work was to develop a platform for efficient delivery of bacterial effectors directly into living cancer cells to study their biological activity in JNK and NF‐kB pathways and therapeutic potential. Our novel CPP‐adaptor (TAT‐CaM) system was used to deliver effectors into mammalian cells. Biolayer interferometry was used to assay the fast‐on, slow‐off, calcium‐dependent binding of TAT‐CaM to cargo (effectors). Cellular uptake of TAT‐CaM:Cargo complexes by mammalian cells was verified by confocal microscopy. Immunostaining was used to study activity of the effector proteins in JNK and NF‐kB pathways. TAT‐CaM bound to cargos with high affinity in a calcium rich solution and proceeded to disassociate in a solution containing EDTA, a chelating agent. Cargo proteins were successfully delivered into mammalian cells in the presence of TAT‐CaM but not in its absence, confirming the utility of our approach.. We found that AopP treatment readily induced apoptosis in mammalian cells. Dose‐response and time course experiments are currently being developed to refine parameters for alteration of cellular signaling pathways. Our CPP‐adaptor system allows for direct and efficient delivery of bioactive protein into living cells. This methodology allows, for the first time, the study of bacterial effectors independent of infection or DNA manipulation. Currently, we are using our CPP‐adaptor system to directly investigate the biological roles of these effectors in mammalian cells with a primary focus on NF‐kB and JNK signaling pathways. Developing the ability to alter pro‐apoptotic and immune signaling pathways using CPP‐mediated delivery could lead to new therapeutics for cancer that allow alteration of signaling pathways in a non‐toxic and rapid manner.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.