Identification of Host Factors Required for Anthrax Lethal Toxin Intoxication Using Chemical Genetic and RNAi Approaches

Abstract

Bacterial toxins have co-opted host cell machinery in order to enter cells and exert their deleterious effects. Anthrax toxin is composed of the receptor binding protein protective antigen (PA), and the enzymatic subunits lethal factor (LF) and edema factor (EF), which form the binary toxin complexes lethal toxin, LeTx (PA + LF), and edema toxin, EdTx (PA + EF). PA binds to receptors on the surface of host cells and shuttles LF and EF into cells through the endocytic pathway. Upon endosome acidification, PA oligomers insert into the endosomal membrane and form functional pores that deliver LF and EF into the cytoplasm. Translocation of the N-terminal domain of LF, (LF_N), through PA pores formed in lipid bilayers in vitro does not require host machinery. However, translocation of the related fusion protein (LF_N)-DTA across the membrane of toxin-loaded endosomes in vitro requires the addition of cytosolic translocation factors that include the COPI coatamer complex. We performed high-throughput small molecule and RNAi screens to identify host factors required for LF translocation, using LeTx-induced cell death as a phenotype. We describe the characterization of small molecule inhibitors of LeTx-induced cell death that inhibit toxin entry. Further, we describe the role of the endosomal chaperone GRP78 and the cytoplasmic CCT chaperonin in toxin translocation. RNAi knockdown of GRP78 and CCT subunits inhibited LeTx and EdTx delivered through the endocytic pathway. CCT knockdown additionally inhibited translocation of LF through PA pores formed directly in the plasma membrane, while GRP78 had no effect. Furthermore, we show that the role of GRP78 in toxin translocation is specific to translocation from the early endosome. Together with biochemical data, we propose that GRP78 facilitates translocation by unfolding LF and EF at near-neutral pH. In addition, we show that in CCT-knockdown cells, lethal levels of toxin reach the endosome, suggesting that CCT has a role in translocation and/or refolding of LF and EF. These studies highlight previously unidentified strategies used by anthrax toxin to hijack host cellular machinery in order to gain access to the cytosol.