Abstract
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. Here we demonstrate that CDTs from Haemophilus ducreyi, Aggregatibacter actinomycetemcomitans, Escherichia coli, and Campylobacter jejuni differ in their abilities to intoxicate host cells with defined defects in host factors previously implicated in CDT binding, including glycoproteins, and glycosphingolipids. The absence of cell surface sialic acid sensitized cells to intoxication by three of the four CDTs tested. Surprisingly, fucosylated N-linked glycans and glycolipids, previously implicated in CDT-host interactions, were not required for intoxication by any of the CDTs tested. Finally, altering host-cellular cholesterol, also previously implicated in CDT binding, affected intoxication by only a subset of CDTs tested. The findings presented here provide insight into the molecular and cellular basis of CDT-host interactions.
Highlights
These data combined with the fact that the CdtB subunits from these four Cytolethal distending toxins (CDTs) display similar enzymatic activities in vitro7 support a model whereby Campylobacter jejuni CDT (Cj-CDT) utilizes a receptor and/or entry pathway that is distinct from the three other toxins (Aa, Hd, and Ec-CDTs)
We tested whether host cellular factors previously implicated in CDT-host interactions were required for cellular intoxication by CDTs derived from four bacterial pathogens that cause periodontal disease, gastroenteritis or chancroid
These four CDTs could be categorized into three groups based on their relative abilities to intoxicate a variety of wild-type and mutant host-cell types
Summary
Unknown, because mutants that failed to bind thyroglobulin retained near wild-type activity in intoxication assays. In addition to a proposed role for host glycans in CDT binding, Aa-CdtC was recently demonstrated to possess a functional cholesterol recognition/interaction amino acid consensus (CRAC) motif important for binding of toxin to cholesterol-rich microdomains (28). It is not clear how CdtC binding to cholesterol relates to the previously proposed roles for glycolipids or glycoproteins. To determine if CDTs from various pathogens utilize similar host factors for intoxication, we set out to determine the role of several classes of cell surface biomolecules (i.e. glycoproteins, glycosphingolipids, cholesterol, and others) in intoxication by four CDTs. We chose to investigate two highly conserved CDTs, Aa-CDT and Haemophilus ducreyi CDT (Hd-CDT), which share 91.9% and 93.5% amino acid identity in their CdtA and CdtC subunits, respectively (5) (Fig. 1). We propose that individual CDTs utilize distinct host factors to efficiently intoxicate target cells
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