Studies On The Endocytic Trafficking And Furin Protease-induced Cleavage Of Several Recombinant Pseudomonas Exotoxin Proteins In Hela Cells

Pseudomonas exotoxin A (PEA), produced by the Gram-negative bacterium Pseudomonas aeruginosa, is a toxic protein capable of inactivating the eukaryotic elongation factor 2 (EF-2) by transferring the adenosine diphosphate (ADP)-ribose group from nicotinamide adenine dinucleotide (NAD+) to EF-2. ADP-ribosylated EF-2 is inactive in participating in the ribosome-mediated peptide chain elongation, thus arresting the protein synthesis process in the intoxicated cells. Cells that are unable to synthesize essential proteins eventually die, although PEA is also known to intoxicate cells through other parallel processes, such as inducing apoptosis. The PEA-mediated cell intoxication is a complex cellular and molecular process. Numerous efforts and endeavors have been dedicated to exploring the targeted receptors on cytoplasm, the trafficking routes and processes, and the molecular mechanism of intoxication of PEA in the cell cytosol.In this work, we designed four different recombinant toxins fused with fluorescent proteins and studied their endocytic and intracellular trafficking processes using laser confocal microscopy and immunocytochemistry. The dynamics of toxin trafficking were quantitated in real times. The intracellular organelles visited by PEA during trafficking were identified. The cleavage efficiency of PEA to express toxicity was estimated. The results showed that medium acidification is not an important factor in affecting binding of PEAs to cytoplasmic receptors, internalization to the early endosomes, late endosomes, and trans-Golgi network. Modifying the C terminus by the signal sequence KDEL promoted PEA trafficking efficiency to endoplasmic reticulum (ER), indicating that PEA trafficking is mediated by the KDEL receptors retrieval system in ER.The domain III of PEA is a 38 KDa fragment possessing ADP-ribosylating activity, releasable after cleavage by furin in the domain II to express full toxicity. We constructed a fusion toxin eBFP-PE44-eGFP (BPG) to estimate the cleavage efficiency by furin-like proteases using the fluorescent resonance energy transferring (FRET) method. The cleavage efficiency of BPG at 2 hrs following endocytosis was approximately 5%. The cleavage process was mediated by low pH, consistent with the view that medium acidification could promote toxicity of PEA. Immunocytochemical labeling indicated that most of the activated domain III fragments were still located within ER after 3 hrs, indicating that only a small fraction of the internalized PEAs translocated to the cytosol and mediated ADP-ribosylation of EF-2.