Abstract
The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.
Highlights
The Cytolethal Distending Toxin (CDT) is a genotoxic virulence factor found in severalGram-negative pathogenic bacteria, such as Escherichia coli (E. coli) [1], Campylobacter spp. [2], Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) [3,4], and Haemophilus ducreyi (H. ducreyi) [5]
Our results indicate that CDT toxicity is induction, mediated by CdtB nuclease activity, but this depends on the proper CdtB transport to the strongly linked to DNA damage induction, mediated by CdtB nuclease activity, but this depends on nucleus, in which phosphatase activity is probably involved
Our results indicated that 1 ng/mL of WT CDT induced a the CdtB phosphatase activity
Summary
The Cytolethal Distending Toxin (CDT) is a genotoxic virulence factor found in severalGram-negative pathogenic bacteria, such as Escherichia coli (E. coli) [1], Campylobacter spp. [2], Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) [3,4], and Haemophilus ducreyi (H. ducreyi) [5]. CDT-producing bacteria are associated with many inflammatory diseases such as chancroid, enterocolitis, and periodontitis (reviewed in [6]). In such contexts, CDT has been shown to favor bacterial long-term persistence and inflammation [7,8,9,10,11,12], and to promote colorectal carcinogenesis [13,14,15] in mice models. CDT is a tripartite toxin presenting an AB2 structure [24,25,26]: The CdtA and CdtC (B moieties) subunits are responsible for the delivery of the catalytic CdtB (A moiety) subunit in host cells, which in turn employs retrograde trafficking across different subcellular compartment to reach the nucleus [27,28]. The typhoid toxin produced by Salmonella enterica serovar Typhi presents an A2 B5 structure: Five regulatory PltB subunits and two catalytic subunits, PltA and CdtB [29]
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