Pharmacological inhibition of host cell neddylation reduces intoxication of cells by diphtheria toxin and clostridial enterotoxins TcdB and C2.

NEDD8 is a ubiquitin-like molecule that is conjugated to lysine residues of protein substrates in a process known as neddylation, which affects their stability, degradation, and interactions. It was recently found that the neddylation inhibitor MLN4924 prevents endocytic uptake of herpes simplex virus (HSV) into target cells, implicating a broader role for neddylation in viral entry and infection. Because AB-type bacterial toxins share similar cell entry mechanisms with viruses, we studied the impact of neddylation in the cellular internalization of a prototypical, well-established model of bacterial AB-type toxins, namely the diphtheria toxin (DT). We found that pretreatment of HeLa cells with MLN4924 reduced morphological changes and protein biosynthesis inhibition induced by DT. Arctigenin, another neddylation inhibitor acting at a later step of the neddylation cascade, was also capable of reducing DT intoxication in HeLa cells. In combination, both neddylation inhibitors synergistically increased their inhibitory potential against DT. Further mechanistic studies with MLN4924 revealed that the neddylation inhibitor neither interfered with cell surface binding, proteolytic activation, enzymatic activity, and membrane translocation of DT, nor with endosomal acidification in HeLa cells. Finally, we could demonstrate that pharmacological inhibition of neddylation also affects further bacterial protein toxins, namely single-chain toxin