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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 ?-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.

SUBMITTER: Slater LH

PROVIDER: S-EPMC3683768 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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

Slater Louise H LH Hett Erik C EC Clatworthy Anne E AE Mark Kevin G KG Hung Deborah T DT

Proceedings of the National Academy of Sciences of the United States of America 20130528 24

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 thi ...[more]

PMID: 23716698

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Project description:UnlabelledAnthrax toxin proteins from Bacillus anthracis constitute a highly efficient system for delivering cytotoxic enzymes to the cytosol of tumor cells. However, exogenous proteins delivered to the cytosol of cells are subject to ubiquitination on lysines and proteasomal degradation, which limit their potency. We created fusion proteins containing modified ubiquitins with their C-terminal regions fused to the Pseudomonas exotoxin A catalytic domain (PEIII) in order to achieve delivery and release of PEIII to the cytosol. Fusion proteins in which all seven lysines of wild-type ubiquitin were retained while the site cleaved by cytosolic deubiquitinating enzymes (DUBs) was removed were nontoxic, apparently due to rapid ubiquitination and proteasomal degradation. Fusion proteins in which all lysines of wild-type ubiquitin were substituted by arginine had high potency, exceeding that of a simple fusion lacking ubiquitin. This variant was less toxic to nontumor tissues in mice than the fusion protein lacking ubiquitin and was very efficient for tumor treatment in mice. The potency of these proteins was highly dependent on the number of lysines retained in the ubiquitin domain and on retention of the C-terminal ubiquitin sequence cleaved by DUBs. It appears that rapid cytosolic release of a cytotoxic enzyme (e.g., PEIII) that is itself resistant to ubiquitination is an effective strategy for enhancing the potency of tumor-targeting toxins.ImportanceBacterial toxins typically have highly efficient mechanisms for cellular delivery of their enzymatic components. Cytosolic delivery of therapeutic enzymes and drugs is an important topic in molecular medicine. We describe anthrax toxin fusion proteins containing ubiquitin as a cytosolic cleavable linker that improves the delivery of an enzyme to mammalian cells. The ubiquitin linker allowed modulation of potency in cells and in mice. This effective strategy for enhancing the intracellular potency of an enzyme may be useful for the cytosolic delivery and release of internalized drugs.

Dataset Information

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

Publications

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

Similar Datasets

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