title: 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.
authors.affiliation: Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Louise H
authors.initials: LH
authors.lastname: Slater
authors.forename: Erik C
authors.initials: EC
authors.lastname: Hett
authors.forename: Anne E
authors.initials: AE
authors.lastname: Clatworthy
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Mark
authors.forename: Deborah T
authors.initials: DT
authors.lastname: Hung
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism* / Q000502:physiology; D001427:Bacterial Toxins / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D056404:Chaperonin Containing TCP-1 / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism* / Q000382:microbiology; D011992:Endosomes / Q000378:metabolism; D054884:Host-Pathogen Interactions; D008264:Macrophages / Q000166:cytology / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D021381:Protein Transport / Q000235:genetics; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C030325:anthrax toxin; D001618:beta-Lactamases; D056404:Chaperonin Containing TCP-1
keywords: TCP-1; TRiC