Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Bordetella pertussis Toxin

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.

A binding motif for Hsp90 in the A chains of ADP‐ribosylating toxins that move from the endoplasmic reticulum to the cytosol

“…Another example of this difference is the influence of peptidyl prolyl cis ‐ trans isomerases on the intoxication process: These enzymes are required for the activity of endosome‐translocating ADPRTs (Barth, ; Ernst et al, ) but are dispensable for the cellular activity of Ctx (Burress et al, ). Interestingly, peptidyl prolyl cis ‐ trans isomerases do appear to be involved with Ptx intoxication (Ernst et al, ). These collective observations support a general role, with toxin‐to‐toxin differences, for Hsp90, Hsc70, and peptidyl prolyl cis ‐ trans isomerases in A chain translocation for the family of ADPRTs.…”

Immunomodulation as a Novel Strategy for Prevention and Treatment of Bordetella spp. Infections

“…The enzymatically active (A) subunit of PTX, PTS1, detaches from the rest of the toxin in the ER and unfolds due to its thermal instability. It is then transported into the cytosol with the help of cyclophilin (Cyps), an important protein folding helper enzyme that also is required to facilitate membrane translocation from early endosomes into the cytosol of various ADP-ribosylating toxins (311)(312)(313). Inhibiting Cyps activity has been shown to in turn inhibit membrane translocation and protect cells from intoxication with PTX and others (311).…”

“…It has been used as the primary agent in immunosuppressive regimens such as grafts and transplants since the 1980s. It is now suggested that CsA might interfere with the translocation of PTS1 from the ER into the cytosol; it may also play a role in reassembling the unfolded PTS1 subunit (311).…”

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo

“…Both CypA and Cyp40 directly bound to PTS1 in vitro and application of pharmacological Cyp inhibitors such as cyclosporine A (CsA) protected CHO-K1 cells from intoxication with PT. In the presence of Cyp inhibitor, less PTS1 reached the cytosol and there was less ADP-ribosylated Giα, implicating that Cyps are required for uptake of PTS1 into the cytosol 29,33 .…”

“…We recently demonstrated that specific members of the cyclophilin (Cyp) family, namely CypA and Cyp40 are involved in uptake of PTS1 into the cytosol of target cells 29 . Cyps are members of the peptidyl prolyl cis/trans isomerases (PPIases), which are protein folding helper enzymes that catalyze the rotation of peptide bonds in proteins and assist chaperones such as heat shock protein (Hsp) 90 in protein folding/refolding [30][31][32] .…”