Abstract
The Bordetella pertussis toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.
Highlights
The Bordetella (B.) pertussis toxin (PT) is a multi-subunit protein toxin consisting of an enzymatically active (A) subunit, namely PTS1, which is non-covalently associated with a pentameric binding/transport (B) subunit [1,2]
Earlier we reported that Cyps are required to facilitate the membrane translocation from early endosomes into the cytosol of clostridial binary toxins, diphtheria toxin and Photorhabdus luminescens PTC3 toxin, which display ADP-ribosyltransferase activity [27,28,29,30,31]
Chinese hamster ovary (CHO)-K1 cells respond to treatment with PT by a characteristic clustering and a reduced cell number compared to untreated cells
Summary
The Bordetella (B.) pertussis toxin (PT) is a multi-subunit protein toxin consisting of an enzymatically active (A) subunit, namely PTS1, which is non-covalently associated with a pentameric binding/transport (B) subunit [1,2]. In the ER, PTS1 is detached from the B pen tamer after the binding of ATP to the central ‘pore’ of the B oligomer [12,13,14]. Due to its thermal instability, the detached PTS1 is in an unfolded conformation, which makes it a substrate for the ER-associated degradation (ERAD) pathway, which transports PTS1 from the ER into the cytosol [15,16,17]. The subsequent ubiquitin-dependent degradation by the proteasome is circumvented because PTS1 does not contain lysine residues, which are required for ubiquitination of proteins [18]
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