IngentaConnect Anthrax toxin fusion proteins for intracellular delivery of macro...

Abstract:

The dominant role played by the anthrax toxin in Bacillus anthracis pathogenesis shows that the toxin has evolved to be an efficient system for delivering its two catalytic protein components, oedema factor and lethal factor (LF), into the cytosol of host cells. This system involves binding of the protective antigen (PA) toxin component to a ubiquitous (and still unidentified) receptor, proteolytic activation at the cell surface, internalization by endocytosis and translocation through an early endosome membrane to the cytosol ( Leppla 1995). We and colleagues showed that the system can be exploited to deliver heterologous polypeptides to the cytosol ( Arora et al. 1992 ; Milne et al. 1995 ). This work used the catalytic domains of other toxins which are normally translocated across membranes ( Arora & Leppla 1994) .

Immunity to intracellular pathogens depends on the cytosolic processing of antigens to produce peptides that are presented on the cell surface bound to MHC Class I molecules. The anthrax toxin delivery system provides a way to mimic this process. We made a fusion protein containing the (non-catalytic) amino terminal domain of LF and the gp120 envelope glycoprotein of HIV-1. Administration of this recombinant protein along with PA to antigen-presenting cells sensitized them to cytolysis by cytotoxic T-cells specific to gp120 peptides ( Goletz et al. 1997 ).

Further exploitation of the anthrax toxin system as a cell-targeting reagent would be facilitated by achieving cell type specificity. The recent determination of the PA structure ( Petosa et al. 1997 ) allows rational engineering to modify or replace the receptor-binding domain with specific ligand structures. A model system was produced by fusing a c-Myc peptide to the carboxyl terminus of PA so as to target hybridoma cells expressing cell surface antibodies to this peptide. Killing of the hybridoma cells was shown to be specific by competition with the peptide and with non-toxic mutants of PA ( Varughese et al. 1998 ).

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Anthrax toxin fusion proteins for intracellular delivery of macromolecules