Auranofin Inhibits the Enzyme Activity of Pasteurella multocida Toxin PMT in Human Cells and Protects Cells from Intoxication

Abstract

The AB-type protein toxin from Pasteurella multocida (PMT) contains a functionally important disulfide bond within its catalytic domain, which must be cleaved in the host cell cytosol to render the catalytic domain of PMT into its active conformation. Here, we found that the reductive potential of the cytosol of target cells, and more specifically, the activity of the thioredoxin reductase (TrxR) is crucial for this process. This was demonstrated by the strong inhibitory effect of the pharmacological TrxR inhibitor auranofin, which inhibited the intoxication of target cells with PMT, as determined by analyzing the PMT-catalyzed deamidation of GTP-binding proteins (G-proteins) in the cytosol of cells. The amount of endogenous substrate levels modified by PMT in cells pretreated with auranofin was reduced compared to cells treated with PMT alone. Auranofin had no inhibitory effect on the activity of the catalytic domain of constitutively active PMT in vitro, demonstrating that auranofin did not directly inhibit PMT activity, but interferes with the mode of action of PMT in cells. In conclusion, the results show that TrxR is crucial for the mode of action of PMT in mammalian cells, and that the drug auranofin can serve as an efficient inhibitor, which might be a starting point for novel therapeutic options against toxin-associated diseases.

Role of AIP56 disulphide bond and its reduction by cytosolic redox systems for efficient intoxication

“…In conclusion, here, we provide evidence that the AIP56 disulphide bond is required for toxicity and that the TrxR-Trx redox system plays a role intoxication, by reducing that bond at the cytosol, similarly to what has been reported for DT, BoNT, and TeNT (Carle et al, 2017;Pirazzini et al, 2013;Pirazzini et al, 2014). The…”

“…A growing body of evidence suggests that cellular redox factors play essential roles in intoxication by AB toxins, by mediating the reduction of their disulphide bonds (Carle, Brink, Orth, Aktories, & Barth, 2017;Pirazzini et al, 2013;Ratts et al, 2003;Schnell et al, 2016;Sun, 2012). The maintenance of the redox potential of the cell cytosol requires a network of redox systems.…”

“…The reductive cleavage of the disulphide bond in AB toxins by cytosolic redox factors, particularly the TrxR-Trx system has been described for several bacterial and plant toxins including DT, BoNT, TeNT, Pasteurella multocida toxin, and ricin (Bellisola et al, 2004;Carle et al, 2017;Falnes & Olsnes, 1995;Kistner & Habermann, 1992;Moskaug, Sandvig, & Olsnes, 1987;Papini, Rappuoli, Murgia, & Montecucco, 1993;Pirazzini et al, 2013;Schiavo et al, 1990;Schnell et al, 2016). To clarify if AIP56 intoxication also required the activity of cellular redox systems, we used pharmacological inhibitors of those systems and assessed if they affected toxicity.…”