Adaptation of Clostridium difficile toxin A for use as a protein translocation system.
A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.
Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI 48202, USA.
Stephanie M
SM
Kern
Andrew L
AL
Feig
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics / Q000378:metabolism*; D050939:Gene Fusion; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases; D018942:Macrolides / Q000494:pharmacology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells
D001427:Bacterial Toxins; D004768:Enterotoxins; D018942:Macrolides; D011993:Recombinant Fusion Proteins; C041348:tcdA protein, Clostridium difficile; C057620:bafilomycin A; D049452:Green Fluorescent Proteins; D008156:Luciferases