CCT chaperonin complex is required for efficient delivery of anthrax toxin into the cytosol of host cells

Abstract

Bacterial toxins have evolved successful strategies for coopting host proteins to access the cytosol of host cells. Anthrax lethal factor (LF) enters the cytosol through pores in the endosomal membrane formed by anthrax protective antigen. Although in vitro models using planar lipid bilayers have shown that translocation can occur in the absence of cellular factors, recent studies using intact endosomes indicate that host factors are required for translocation in the cellular environment. In this study, we describe a high-throughput shRNA screen to identify host factors required for anthrax lethal toxin-induced cell death. The cytosolic chaperonin complex chaperonin containing t-complex protein 1 (CCT) was identified, and subsequent studies showed that CCT is required for efficient delivery of LF and related fusion proteins into the cytosol. We further show that knockdown of CCT inhibits the acid-induced delivery of LF and the fusion protein LFN-Bla (N terminal domain of LF fused to β-lactamase) across the plasma membrane of intact cells. Together, these results suggest that CCT is required for efficient delivery of enzymatically active toxin to the cytosol and are consistent with a direct role for CCT in translocation of LF through the protective antigen pore. TCP-1 | TRiCA nthrax toxin, a bacterial protein toxin produced by Bacillus anthracis, is an essential virulence factor that directly causes the pathology associated with anthrax disease. Anthrax toxin is a canonical AB toxin composed of the B subunit protective antigen (PA) and two enzymatic A subunits, lethal factor (LF) and edema factor (EF) (1). LF is a zinc metalloprotease that cleaves MAP kinase kinases (MEKs) and NLRP1 (2, 3), and EF is an adenylate cyclase that converts ATP to cAMP. Individually, the toxin components are not toxic, but a combination of PA and LF (lethal toxin; LT) causes death of mice and rapid caspase-1-dependent cell death of susceptible murine macrophages (4, 5), and a combination of PA and EF (edema toxin; ET) causes edema at the site of injection in experimental animals.Bacterial toxins have coopted numerous host factors to gain access to the cell and alter host physiology. Cytosolic delivery of anthrax toxin requires binding of PA protein to one of two anthrax toxin receptors (ANTXR1/2) on the surface of host cells, cleavage by a host furin protease, and oligomerization of receptor-associated PA 63 into heptamers or octamers (1, 6). After binding of LF and/or EF, the toxin-receptor complexes undergo endocytosis and trafficking to endosomes. Upon a decrease in endosomal pH, PA undergoes a conformational change and inserts into the membrane, forming a functional pore through which LF and EF translocate (1).Unfolding and translocation of the N-terminal domain of LF (LF N ) in vitro occurs in the absence of accessory factors (7). However, we have previously shown that the endosomal chaperone GRP78 (78 kDa glucose regulated protein) facilitates LF N unfolding in the mildly acidic early endosome (pH 6.5), but not the late endosome ...

Hijacking Multivesicular Bodies Enables Long-Term and Exosome-Mediated Long-Distance Action of Anthrax Toxin

“…LF can be artificially introduced into the cytosol, bypassing the endocytic pathway by briefly pulsing LT-pretreated cells with acidic pH (Slater et al, 2013). We added the vacuolar ATPase inhibitor Bafilomycin A during LT pre-treatment to ensure that none of the toxin complexes enter through endocytosis.…”

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

“…, ; Kashuba, Pokrovskaja, Klein, & Szekely, ; Lingappa et al . , ), and the human TRiC is also involved in translocation of the anthrax toxin into host cells (Slater, Hett, Clatworthy, Mark, & Hung, ). Although it is difficult to modulate TRiC expression in RBCs, the recent advances in using hematopoietic stem cell based screening to study malaria (Egan et al .…”

“…The human TRiC machinery is co-opted by other microbes. TRiC subunits are recruited by viral proteins where they may play a role in viral replication (Hong et al, 2001;Inoue et al, 2011;Kashuba, Pokrovskaja, Klein, & Szekely, 1999;Lingappa et al, 1994), and the human TRiC is also involved in translocation of the anthrax toxin into host cells (Slater, Hett, Clatworthy, Mark, & Hung, 2013). Although it is difficult to modulate TRiC expression in RBCs, the recent advances in using hematopoietic stem cell based screening to study malaria (Egan et al, 2015) may prove useful in clarifying the role of RBC TRiC in malaria protein export.…”

The chaperonin TRiC/CCT is essential for the action of bacterial glycosylating protein toxins like Clostridium difficile toxins A and B

“…The chaperonin inhibitor HSF1A has no effect on the protease activity of TcdB in vitro; however, we cannot exclude the possibility that TRiC/CCT5 plays a role in the refolding of the autoprotease domain of the clostridial glucosylating toxins. It was recently reported that TRiC/CCT is involved in the action of anthrax toxin (56). These studies focused on the lethal toxin (LFN) of the tripartite anthrax toxin, which is a zinc-dependent metalloprotease.…”