UCLA

Cytolethal distending toxins (CDTs) are tripartite protein exotoxins produced by a diverse group of pathogenic Gram-negative bacteria. Based on their ability to induce DNA damage, cell cycle arrest and apoptosis of cultured cells, CDTs are proposed to enhance virulence by blocking cellular division and/or directly killing epithelial and immune cells. Despite the widespread distribution of CDTs among several important human pathogens, our understanding of how these toxins interact with host cells is limited. This dissertation identifies and characterizes host factors that confer sensitivity to CDTs from Aggregatibacter actinomycetemcomitans, Haemophilus ducreyi, Escherichia coli, and Campylobacter jejuni. Host plasma membrane cholesterol supported intoxication and was found to be limiting for sensitivity to CDT in CHO-K1 cells. In contrast, a role for host glycans and the membrane protein TMEM181, which were previously implicated as receptors for binding of CDT to host cells, were found to be dispensable or play a negative role in sensitivity to CDT. Characterization of CDT-resistant mutants from two independent forward genetic screens identified a series of genes that play a role in CDT intoxication. One of these genes, Derlin-2 (Derl2), is a central component of endoplasmic reticulum associated degredation (ERAD) pathway, suggesting that CDT utilizes ERAD to escape from the lumen of the ER. Derl2 deficient cells are resistant to CDT due to decreased retrotranslocation of CDT from the lumen of the ER. Further, the mechanism of Derl2-dependent of escape of CDTs from the ER is distinct from previously described Derl2-dependent retrotranslocation of ERAD substrates. Specifically, two independent requirements for Derl2-mediated ERAD of misfolded proteins, a conserved WR motif and interaction with the AAA-ATPase p97, are dispensable for retrotranslocation of CDT and another retrograde trafficking toxin, ricin. This previously undescribed mechanism demonstrates a novel Derl2-dependent ERAD pathway exploited by retrograde trafficking toxins. In total, the findings presented here provide insight into the molecular and cellular basis of CDT-host interactions.