Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells

Katharina Ernst,Holger Barth,Anna Anastasia,Patricia Hauff,Meike Hohwieler,Thomas Jank,Klaus Aktories,Anna Katharina Ückert,Michel R Popoff,Matthias Beck,Johannes Schmid,Manfred Frick,Cordelia Schiene-Fischer,Marlen Hägele,Michael Fauler,Martin Müller,Alexander Kleger

doi:10.1038/s41598-017-02882-y

Abstract

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

Highlights

Clostridium (C.) perfringens iota toxin, C. botulinum C2 toxin and C. difficile toxin CDT belong to the group of binary actin ADP-ribosylating toxins causing severe enterotoxicity in humans and animals[1,2,3,4]
We demonstrate for the first time that the enzyme components of these toxins interact with Hsp[70] in the cytosol of living cells, indicating the importance of Hsp[70] for efficient uptake of clostridial binary toxins into the host cell cytosol
In line with previous results, the enzyme components do not bind to the C3bot protein of C. botulinum or FKBP12, a small FK506-binding protein (FKBP) isoform of the peptidyl-prolyl cis/trans isomerases (PPIases) family, demonstrating the specificity of this binding

Summary

Introduction

Clostridium (C.) perfringens iota toxin, C. botulinum C2 toxin and C. difficile toxin CDT belong to the group of binary actin ADP-ribosylating toxins causing severe enterotoxicity in humans and animals[1,2,3,4] They are secreted by bacteria as two non-linked proteins, which interact on the surface of target cells. Iota and CDT share the same cell surface receptor, the lipolysis-stimulated lipoprotein receptor (LSR)[23, 24] and exploit in addition to LSR CD44 for uptake[25] Despite these differences between C2 toxin and iota-like toxins, a common membrane translocation mechanism involving requirement of the host cell chaperone Hsp[90] and peptidyl-prolyl cis/trans isomerases (PPIases) of the cyclophilin (Cyp) and FK506-binding protein (FKBP) families is evident We demonstrate for the first time that the enzyme components of these toxins interact with Hsp[70] in the cytosol of living cells, indicating the importance of Hsp[70] for efficient uptake of clostridial binary toxins into the host cell cytosol

Methods

Results

Conclusion