Cytolethal Distending Toxin Family Members Are Differentially Affected by Alterations in Host Glycans and Membrane Cholesterol

Abstract

Cytolethal distending toxins (CDTs) 6 are members of a group of bacterial toxins and effectors called "cyclomodulins" that interfere with the eukaryotic cell cycle rather than inducing overt cytotoxicity (1, 2). Inhibiting cell cycle disrupts many of the normal functions of rapidly dividing eukaryotic cells, including lymphocytes and epithelial cells, which provide immunity and physical barriers to microbial pathogens (3-5). Thus, it is not surprising that cdt genes are found in a diverse group of Gram-negative pathogens that colonize different niches within the host. Although a growing body of evidence supports the importance of CDTs in bacterial virulence and host-pathogen interactions (6), the manner in which individual CDTs interact with and intoxicate host cells remains poorly understood.CDTs are AB 2 toxins, consisting of a hetero-trimeric complex of three proteins (CdtA, CdtB, and CdtC) at a 1:1:1 molar ratio (5,7,8). The current model is that CdtA and CdtC are the binding "B" moieties that collaborate to facilitate binding and entry of the catalytic "A" subunit, CdtB, into mammalian cells. CdtB shares a common tertiary structure with DNase I and phosphatidylinositol 3,4,5-triphosphate phosphatase enzymes and displays both activities in cell-free systems (9 -13). It is not currently known which activity is of greater importance, and this may depend on the specific toxin and/or the host target cell type (12,14). CdtB enzymatic activity induces cell cycle arrest predominantly at the G 2 /M transition, resulting in cellular distension and ultimately cell death (5,15,16).Consistent with their proposed roles as binding subunits, CdtA and/or CdtC increase the ability of CdtB to associate with host cells and greatly enhance intoxication (7,(17)(18)(19)(20)(21)(22)(23)(24)(25). The identification of ricin-like lectin domains in CdtA and CdtC from structural and biochemical data first suggested that these subunits may interact with carbohydrates on the cell surface (13,26,27). Consistent with this hypothesis, CDT produced by Escherichia coli (Ec-CDT) was reported to require N-linked glycoproteins for binding and subsequent intoxication of HeLa cells (23). Moreover, Ec-CDT bound fucose in vitro, and fucose-specific lectins blocked Ec-CDT-mediated cell cycle arrest, presumably by preventing binding of toxin to its receptor. These findings suggested that fucose might serve as a binding determinant for Ec-CDT. Similarly, host glycans were reported to support Aggregatibacter actinomycetemcomitans (Aa-CDT) intoxication. Specifically, Aa-CDT bound three glycosphingolipids, GM1, GM2, and GM3, and intoxication of human monocytic U937 cells was blocked by preincubation of toxin with liposomes that contained G M3 (24). In addition, the CdtA subunit of Aa-CDT bound to the glycoprotein thyroglobulin (19). However, the functional significance of this binding is * This work was supported, in whole or in part, by National Institutes of Health Grants T32DE007296 (to A. E.), F31AI061837 (to F. J. M.-A.), and AI59095 (to S. R. B. ...

Molecular Characterizations of Cytolethal Distending Toxin Produced by Providencia alcalifaciens Strains Isolated from Patients with Diarrhea

“…These observations indicate that the receptor for PaCDT may be different from that of other CDTs, including Ec-CDT-I. Similar findings that CDTs produced from different bacteria, e.g., A. actinomycetemcomitans, H. ducreyi, C. jejuni, and E. coli, may display variable target cell tropism and may have different receptors as reported by Eshraghi et al (16).…”

“…The nuclear localization signals (NLS1 and NLS2) detected in EcCDT-II were also almost conserved in PaCdtB, except for two amino acid substitutions in each of the regions (NLS1, A198D and R210N; NLS2, F262Y and S267F). The RR(X) [10][11][12][13][14][15][16][17][18][19][20] RR motif (33) in NLS2 was found to be completely conserved, suggesting that PaCDT may enter into the nucleus for its genotoxic activity. Distribution of cdt genes among Providencia spp.…”

Cytolethal distending toxin A, B and C subunit proteins are necessary for the genotoxic effect of Escherichia coli CDT-V

“…However, there is increasing evidence suggesting that despite the similar genotoxic activity, there are differences between the molecular mechanisms used by the different CDT types in interaction with the host cell (McSweeney and Dreyfus, 2005;Eshraghi et al, 2010;Gargi et al, 2013). The exact role of the CdtA and CdtC subunits is not well understood in the case of E. coli CDT, and no data are available regarding their roles in CDT type V.…”

Cytolethal distending toxin: a conserved bacterial genotoxin that blocks cell cycle progression, leading to apoptosis of a broad range of mammalian cell lineages

“…Although CDT is a broad range genotoxin (Ge et al, 2005;Pratt et al, 2006;Shenker et al, 2007), it remains to be shown whether this apparent cell type specificity is in part attributable to inherent differences in cell surface receptor binding of CdtA and CdtC to host cell membrane based on: (i) host cell surface biomolecule chemical composition or density, (ii) intrinsic differences in CdtB uptake and nuclear translocation or (iii) variable target cell lineage DDR competence (Carette et al, 2009;Eshraghi et al, 2010). However, other factors that can determine the outcome of CDT interactions with susceptible cells, including differential receptor intrinsic affinity and also amino acid sequence divergence of CdtA and CdtC binding subunits encoded by different bacteria, need further detailed analysis (Carette et al, 2009;Eshraghi et al, 2010).The mutational status of individual cell lines is another critical factor that can determine activation of specific checkpoint and apoptotic pathways, and thus the stage of cell cycle arrest and kinetics of progression to apoptosis in response to genotoxic injury. As described earlier, two of the most important mediators of CDT-induced DDR are ATM and p53, both of which play critical roles in determining the stage of cell cycle arrest and pathways and efficiency of apoptosis execution.…”

“…et al, 2005). More recent studies examining the susceptibility of target cell lines expressing a wide range of surface membrane biomolecules to CDT representing each of the three distinct CdtA-CdtC sequence clusters described earlier suggest that a cell surface polypeptide component rather than glycoconjugates is the receptor that is most likely to be responsible for CDT-host cell specificity (Eshraghi et al, 2010). These findings are consistent with a requirement for a putative G protein-coupled transmembrane protein, designated TMEM181, which localizes to membrane lipid rafts in induction of EcolCdtB-I cytotoxicity by a novel loss-of-function haploid genetic screen using a highly sensitive myeloid leukaemia cell line (Carette et al, 2009).…”