Lawrence A. Dreyfus

DNase I homologous residues in CdtB are critical for cytolethal distending toxin‐mediated cell cycle arrest

Cytolethal distending toxins (CDTs) block cell division by arresting the eukaryotic cell cycle at G2/M. Although previously not recognized in standard blast searches, a position‐specific iterated (psi) blast search of the protein data bank using CDT polypeptides as query sequences indicated that CdtB bears significant position‐specific homology to type I mammalian DNases. The psiblast sequence alignment reveals that residues of DNase I involved in phosphodiester bond hydrolysis (His134 and His252) are conserved in CdtB as well as their respective hydrogen bond pairs (Glu78 and Asp212). CdtB also contains a pentapeptide motif found in all DNase I enzymes. Further, crude CDT preparations possess detectable DNase activity not associated with identical preparations from control cells. Five CdtB mutations in amino acids corresponding to DNase I active site residues were prepared and expressed together with wild‐type CdtA and CdtC polypeptides. Mutation in four of the five DNase‐specific active site residues resulted in CDT preparations that lacked DNase activity and failed to induce cellular distension or arrest division of HeLa cells. The fifth mutation, Glu86 (Glu78 in DNase I), retained the ability to induce a moderate level of cell cycle arrest and displayed reduced DNase activity relative to wild‐type CDT. Together, these data suggest that the CDT holotoxin has intrinsic DNase activity that is associated with the CdtB polypeptide and that this DNase activity may be responsible for the CDT‐induced cell cycle arrest.

Escherichia coli CdtB Mediates Cytolethal Distending Toxin Cell Cycle Arrest

ABSTRACT We previously reported that the CdtB polypeptide ofEscherichia coli cytolethal distending toxin (CDT) shares significant pattern-specific homology with mammalian type I DNases. In addition, the DNase-related residues of CdtB are required for cellular toxicity. Here we demonstrate that purified CdtB converts supercoiled plasmid DNA to relaxed and linear forms and promotes cell cycle arrest when combined with an E. coli extract containing CdtA and CdtC. CdtB alone had no effect on HeLa cells, however; introduction of the polypeptide into HeLa cells by electroporation resulted in cellular distension, chromatin fragmentation, and cell cycle arrest, all of which are consequences of CDT action. In contrast to these findings, purified CdtBH154A lacked both DNA-nicking and cell cycle arrest activities. These results suggest a functional relationship between DNase-related residues in CdtB and CDT biological activity.

Nuclear localization of the Escherichia coli cytolethal distending toxin CdtB subunit

Cytolethal distending toxin (CDT) is a heterotrimeric protein toxin produced by several bacterial pathogens. Cells exposed to CDT die from either activation of the mitotic checkpoint cascade or apoptosis. Introduction of the purified CdtB subunit, a homologue of mammalian type I DNase, into cells mimics the action of the CDT holotoxin. Mutant CdtBs lacking DNase activity are devoid of biological activity. Chromosomal DNA appears to be the CDT target; thus, nuclear translocation of CdtB must precede cytolethal activity. Examination of the CdtB sequence indicates the presence of putative candidate bipartite nuclear localization signals (NLS). Here, we examine the functionality of the two potential NLS sequences found in the Escherichia coli CdtB‐II. Nuclear translocation of EcCdtB‐II was examined by monitoring the localization of an EcCdtB‐II–EGFP fusion in Cos‐7 cells. Our results indicated that EGFP‐EcCdtB‐II localized to the nucleus. The candidate EcCdtB‐II‐II NLS sequences were modified by site‐directed mutagenesis such that tandem arginine residues were changed to threonine and serine respectively. Mutation of both putative NLS sequences had no effect on EcCdtB‐II‐associated DNase activity; however, cell cycle arrest and nuclear localization were significantly impaired in cells that received CDT reconstituted from the EcCdtB‐II‐ΔNLS mutants. When HeLa cells were electroporated with the EcCdtB‐II‐ΔNLS1 and the EcCdtB‐II‐NLS double mutants, toxicity was not observed, whereas the activity of EcCdtB‐II‐ΔNLS2 was similar to that of wild‐type EcCdtB‐II. These data indicate that the putative NLS sequences are important for CDT‐mediated action arrest and that they are likely to function in the nuclear translocation of EcCdtB‐II.

Carbohydrate-Binding Specificity of the Escherichia coli Cytolethal Distending Toxin CdtA-II and CdtC-II Subunits

ABSTRACT Intoxication by cytolethal distending toxin depends on assembly of CdtB, the active A component of this AB toxin, with the cell surface-binding (B) component, composed of the CdtA-CdtC heterodimer, to form the active holotoxin. Here we examine the cell surface binding properties of Escherichia coli-derived CdtA-II (CdtA-IIEc) and CdtC-IIEc and their capacity to provide a binding platform for CdtB-IIEc. Using a flow cytometry-based binding assay, we demonstrate that CdtB-IIEc binds to the HeLa cell surface in a CdtA-IIEc- and CdtC-IIEc-dependent manner and that CdtA-IIEc and CdtC-IIEc compete for the same structure on the HeLa cell surface. Preincubation of cells with glycoproteins (thyroglobulin and fetuin), but not simple sugars, blocks surface binding of CdtA-IIEc and CdtC-IIEc. Moreover, CdtA-IIEc and CdtC-IIEc bind immobilized fetuin and thyroglobulin as well as fucose and to a lesser degree N-acetylgalactoseamine and N-acetylglucoseamine. Removal of N- but not O-linked carbohydrates from fetuin and thyroglobulin prevents binding of CdtA-IIEc and CdtC-IIEc to these glycoproteins. In addition, removal of N- but not O-linked surface sugar attachments prevents CDT-IIEc intoxication. To characterize the cell surface ligand recognized by CdtA-IIEc and CdtC-IIEc, lectins having various carbohydrate specificities were used to block CDT activity and the cell surface binding of CdtA-IIEc and CdtC-IIEc. Pretreatment of cells with AAA, SNA-I, STA, UEA-I, GNA, and NPA partially or completely blocked CDT activity. AAA, EEA, and UEA-I lectins, all having specificity for fucose, blocked surface binding of CdtA-IIEc and CdtC-IIEc. Together, our data indicate that CdtA-IIEc and CdtC-IIEc bind an N-linked fucose-containing structure on HeLa cells.

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.

Release of serotonin from RBL-2H3 cells by the Escherichia coli peptide toxin STb

Heat-stable enterotoxin b (STb) of Escherichia coli is a 48-amino acid basic, disulfide-bonded peptide that causes intestinal secretion in experimental animal models. Recent evidence suggests that the in vivo mechanism of STb action involves release of 5-hydroxytryptamine (5-HT) and production of prostaglandin E2 (PGE2). Here we show STb-mediated release of 5-HT from rat basophilic leukemic cells (RBL-2H3), a mast cell line model used extensively to study 5-HT release. Increasing concentrations of biologically active STb resulted in a dose-dependent release of 5-HT from RBL-2H3 cells. In contrast to these results, reduced and alkylated STb had no effect on 5-HT release. Release of 5-HT from RBL-2H3 cells was independent of extracellular calcium ions and did not involve changes in the intracellular concentration of free Ca2+. In addition, pertussis toxin treatment completely blocked 5-HT release, indicating a role for a pertussis toxin-sensitive G-protein in the mechanism of 5-HT release from this cell type.

Differences in crystal and solution structures of the cytolethal distending toxin B subunit : Relevance to nuclear translocation and functional activation

Cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells, which are mediated by the DNA-damaging CdtB subunit. Here we report the first x-ray structure of an isolated CdtB subunit (Escherichia coli-II CdtB, EcCdtB). In conjunction with previous structural and biochemical observations, active site structural comparisons between free and holotoxin-assembled CdtBs suggested that CDT intoxication is contingent upon holotoxin disassembly. Solution NMR structural and 15N relaxation studies of free EcCdtB revealed disorder in the interface with the CdtA and CdtC subunits (residues Gly233–Asp242). Residues Leu186–Thr209 of EcCdtB, which encompasses tandem arginine residues essential for nuclear translocation and intoxication, were also disordered in solution. In stark contrast, nearly identical well defined α-helix and β-strand secondary structures were observed in this region of the free and holotoxin CdtB crystallographic models, suggesting that distinct changes in structural ordering characterize subunit disassembly and nuclear localization factor binding functions.

Crystallization of Escherichia coli CdtB, the biologically active subunit of cytolethal distending toxin

Cytolethal distending toxin (CDT) is a secreted protein toxin produced by several bacterial pathogens. The biologically active CDT subunit CdtB is an active homolog of mammalian type I DNase. Internalization of CdtB and subsequent translocation into the nucleus of target cells results in DNA-strand breaks, leading to cell-cycle arrest and apoptosis. CdtB crystals were grown using microbatch methods with polyethylene glycol 8000 as the precipitant. The CdtB crystals contain one molecule of MW 30.5 kDa per asymmetric unit, belong to space group P2(1)2(1)2(1) and diffract to 1.72 A.