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

Abstract

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.

Novel Strategies to Inhibit Pertussis Toxin

“…For example, it is not known how the unfolded PTS1 regains its active conformation in the cytosol. However, a crucial role of cellular protein folding helper enzymes for uptake of PTS1 into the host-cell cytosol has been demonstrated (see Section 3.1 ) [ 41 , 42 , 43 ].…”

“…Therefore, we hypothesized that intracellular chaperones and protein folding helper enzymes might play a role for uptake and mode of action of PT. We showed that inhibitors of cellular chaperones protect cells from intoxication with PT [ 41 , 42 , 43 ]. We characterized the underlying mode of inhibition and demonstrated that chaperones of the heat shock family (Hsp90, Hsp70) and of the peptidyl prolyl cis/trans isomerase (PPIase) family (cyclophilins, Cyps and FK506-binding proteins, FKBPs) are required for the transport of the enzyme subunit of PT, as well as other ADP-ribosylating toxins into the target cell cytosol [ 58 , 61 , 62 , 63 , 64 , 65 ].…”

“…To investigate activity and/or inhibition of PT in vitro, the cell line CHO (Chinese hamster ovary) is used because these cells show a specific clustering morphology after treatment with purified PT [ 71 , 72 ]. Pre-incubation of CHO cells with either radicicol, VER, HA9, CsA or FK506 resulted in a decreased PT-induced clustering morphology and reduced ADP-ribosylated Gαi by PTS1 in lysates from PT-treated cells [ 41 , 42 ]. CsA and FK506 also prevented PT-mediated effects on cAMP signaling [ 41 ].…”

Bordetella Adenylate Cyclase Toxin Elicits Airway Mucin Secretion through Activation of the cAMP Response Element Binding Protein

“…PT enters cells through receptor-mediated endocytosis within clathrin-coated pits and follows the retrograde transport pathway through the Golgi apparatus into the endoplasmic reticulum (ER) [36][37][38]. From there, the unfolded S1 subunit translocates into cell cytosol through the ER-associated protein degradation (ERAD) retrotranslocon [39,40] with the assistance of the cytosolic chaperone cyclophilin [41,42]. Once in the cytosol, the S1 subunit catalyzes the transfer of the adenosine diphosphate (ADP)-ribose moiety of nicotinamide adenine dinucleotide (NAD + ) onto the Gα i/o inhibitory subunits of the heterotrimeric G-proteins, thus locking the various isoforms of Gα i/o inhibitory subunits in the inactive GDP-loaded state [43].…”

Exotoxin-Targeted Drug Modalities as Antibiotic Alternatives

“…For instance, small molecules have been identified which affect the endosomal maturation, 9 retrograde trafficking, 10 intracellular activatory proteolytic processing, 11 and intracellular chaperone-assisted activatory folding of exotoxins. 12 …”