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 Clostridioides difficile TcdB and binary toxin Clostridium botulinum C2. In conclusion, our study is the first to support a connection between neddylation and the cellular uptake of bacterial AB-type toxins, thereby establishing a foundation for future investigations into these molecular mechanisms and for the development of anti-toxin strategies.IMPORTANCENeddylation, a ubiquitin-like protein modification, is emerging as a critical regulator of viral entry into cells. Here, we demonstrate for the first time that pharmacological inhibition of neddylation impairs the intoxication of target cells with diverse bacterial AB-type toxins. Mechanistic studies suggest that neddylation most likely reduces toxin internalization, thus supporting the connection between neddylation and endocytic processes. Our study offers new opportunities to explore anti-toxin interventions and highlights the use of bacterial toxins as molecular probes to further study the role of neddylation in endocytic trafficking.