Abstract
Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.
Highlights
Cytolethal distending toxins (CDTs) are produced by a variety of Gram-negative pathogens including the oral pathogen Aggregatibacter actinomycetemcomitans, the sexually transmitted pathogen Haemophilus ducreyi, and the gastrointestinal pathogens, Escherichia coli and Campylobacter jejuni
Each pool of 16106 cells was selected with 20 nM A. actinomycetemcomitans CDT (Aa-CDT), a toxin concentration high enough to cause death in parental cells
CHO-CDTRA2 cells were resistant to the highest dose of H. ducreyi CDT (Hd-CDT) tested (Fig. 1b) and more modestly resistant to CDTs from E. coli (Ec-CDT; Fig. 1c) and C. jejuni (Cj-CDT; Fig. 1d)
Summary
Cytolethal distending toxins (CDTs) are produced by a variety of Gram-negative pathogens including the oral pathogen Aggregatibacter actinomycetemcomitans, the sexually transmitted pathogen Haemophilus ducreyi, and the gastrointestinal pathogens, Escherichia coli and Campylobacter jejuni. These toxins belong to a larger, emerging group of intracellular-acting ‘‘cyclomodulins’’ whose expression is associated with increased persistence, invasiveness and severity of disease [1,2,3,4,5,6,7]. CDTs cause DNA damage in susceptible host cells, resulting in the induction of DNA repair signaling mechanisms including phosphorylation of the histone H2AX, cell cycle arrest at the G2/M interface and disruption of cytokinesis [8]. The cellular response to CDTs is well characterized [8,12], the mechanism by which CDTs bind to host cells and gain access to their nuclear target is less clear
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