Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.     

Comparative structure-function analysis of cytolethal distending toxins

Cytolethal distending toxins (CDTs) constitute a family of bacterial proteins that enter eukaryotic cells with genotoxic activity leading to cell cycle arrest and apoptosis. CDTs are widespread, having been found in a variety of Gram-negative pathogens with a broad tissue tropism. The recently determined crystal structure of the Haemophilus ducreyi CDT provides a powerful starting point for analysis of the structure and function in this toxin family. In this study, we apply comparative modeling and structural analysis to extend the experimental structural information to multiple CDT toxins from a diverse species. Analysis of structurally and functionally important residues in the active subunit, CdtB, and putative cell delivery elements, CdtA and CdtC, begins to establish the fundamental, mechanistic elements of this unique holotoxin. The results reveal that key structural features with important functional consequences are highly conserved across different CDTs, providing a blueprint for directed examination of functional hypotheses in a variety of pathogenic contexts.     

In vivo virulence properties of bacterial cytolethal-distending toxin

Multiple pathogenic Gram-negative bacteria produce cytolethal-distending toxins (CDTs). CDT is typically composed of three subunits: the catalytic subunit CdtB has DNase I-like activity, whereas CdtA and CdtC are binding proteins for delivering CdtB into target cells. Translocation of CdtB to the nucleus induces genotoxic effects on host DNA, triggering DNA repair cascades that lead to cell cycle arrest and eventual cell death. Several lines of evidence indicate that this toxin contributes to the pathogenicity of CDT-producing bacteria in vivo . Helicobacter hepaticus and Campylobacter jejuni CDTs are essential for persistent infection of the gastrointestinal tract and increase the severity of mucosal inflammation or liver disease in susceptible mouse strains. Haemophilus ducreyi CDT may contribute to the pathogenesis of chancroid in rabbits. Recently, H. hepaticus CDT has been shown to play a crucial role in promoting the progression of infectious hepatitis to pre-malignant, dysplastic lesions via activation of a pro-inflammatory NF-κB pathway and increased proliferation of hepatocytes, providing the first evidence that CDT has carcinogenic potential in vivo . Thus, both in vitro and in vivo data indicate that CDT is a bacterial virulence factor.     

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

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.     

Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling

Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram-negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type-dependent cell-cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR-90 and WI-38 fibroblasts, HeLa and U2-OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX⁺ foci), enhanced senescence-associated β-galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL-6, IL-8 and IL-24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the 'distended' morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.     

Bacterial cyclostatin, or how do bacteria manipulate the eukaryotic cell cycle

Several bacterial proteins have been recently described that share the ability to inhibit the proliferation of cells in culture without causing early signs of cytotoxicity. Such observations suggest the existence of bacterial mechanisms of control of the eukaryotic cell cycle contributing to pathogenicity or adaptation to the host. This emerging concept of cellular microbiology is critically analyzed considering as a model the cytolethal distending toxins (CDT), a family of toxins whose mode of action on the cell cycle has been thoroughly studied over the last few years. CDTs activate a physiological G2 checkpoint in exposed cells, probably from an initial DNA alteration whose precise molecular nature has not yet been determined. Experimental data are lacking to extrapolate in vivo the antiproliferative effect of these bacterial proteins that we tentatively propose to call cyclostatins.     

The Haemophilus ducreyi cytolethal distending toxin induces DNA double-strand breaks and promotes ATM-dependent activation of RhoA

Among bacterial protein toxins, the cytolethal distending toxins (CDTs) are unique in their ability to activate the DNA damage checkpoint responses, causing cell cycle arrest or apoptosis in intoxicated cells. We provide direct evidence that natural intoxication of cells with the Haemophilus ducreyi CDT (HdCDT) holotoxin induces DNA double-strand breaks similarly to ionizing radiation. Upon DNA damage, epithelial cells and fibroblasts promote the formation of actin stress fibres via activation of the small GTPase RhoA. This phenomenon is not toxin specific, but is part of the ATM-induced cellular responses to genotoxic stresses, including ionizing radiation. Activation of RhoA is associated with prolonged cell survival, as HdCDT-treated epithelial cells expressing a dominant-negative form of RhoA detach and consequently die faster than cells expressing a functional RhoA. Our data highlight several novel aspects of CDT biology: (i) we show that a member of the CDT family causes DNA double-strand breaks in naturally intoxicated cells, acting as a true genotoxic agent; and (ii) we disclose the existence of a novel signalling pathway for intracellularly triggered activation of the RhoA GTPase via the ATM kinase in response to DNA damage, possibly required to prolong cell survival.     

Cellular internalization of cytolethal distending toxin: a new end to a known pathway

The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin A-treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.     

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.     

A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4 h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli , Campylobacter jejuni and Shigella dysenteriae . Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt . Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.     

The Haemophilus ducreyi cytolethal distending toxin activates sensors of DNA damage and repair complexes in proliferating and non-proliferating cells

Cytolethal distending toxins (CDTs) block proliferation of mammalian cells by activating DNA damage-induced checkpoint responses. We demonstrate that the Haemophilus ducreyi CDT (HdCDT) induces phosphorylation of the histone H2AX as early as 1 h after intoxication and re-localization of the DNA repair complex Mre11 in HeLa cells with kinetics similar to those observed upon ionizing radiation. Early phosphorylation of H2AX was dependent on a functional Ataxia Telangiectasia mutated (ATM) kinase. Microinjection of a His-tagged HdCdtB subunit, homologous to the mammalian DNase I, was sufficient to induce re-localization of the Mre11 complex 1 h post treatment. However, the enzymatic potency was much lower than that exerted by bovine DNase I, which caused marked chromatin changes at 106 times lower concentrations than HdCdtB. H2AX phosphorylation and Mre11 re-localization were induced also in HdCDT-treated, non-proliferating dendritic cells (DCs) in a differentiation dependent manner, and resulted in cell death. The data highlight several novel aspects of CDTs biology. We demonstrate that the toxin activates DNA damage-associated molecules in an ATM-dependent manner, both in proliferating and non-proliferating cells, acting as other DNA damaging agents. Induction of apoptotic death of immature DCs by HdCDT may represent a previously unknown mechanism of immune evasion by CDT-producing microbes.     

Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

The Haemophilus ducreyi cytolethal distending toxin induces cell cycle arrest and apoptosis via the DNA damage checkpoint pathways

The cytolethal distending toxins (CDTs) induce cell cycle arrest by a mechanism still not well characterized. We demonstrate that the effect of the Haemophilus ducreyi CDT (HdCDT) is cell type-specific: B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G(2), whereas normal fibroblasts arrested both in G(1) and G(2). We studied normal keratinocytes and fibroblasts, which are relevant for understanding the pathogenicity of H. ducreyi. The response to HdCDT resembles the checkpoint response activated by ionizing radiation. Both responses were characterized by an early induction of the p53 gene and the cyclin-dependent kinase inhibitor p21 in fibroblasts, and activation of the chk2 kinase in epithelial cells. In the Ataxia Telangiectasia-mutated gene (ATM)-deficient lymphoblastoid cell lines, intoxication was significantly delayed compared with ATM wild type cells, and was associated with a slower kinetic of p53 stabilization, suggesting that the early response to HdCDT is ATM-dependent. Activation of ATM-dependent pathways was further confirmed by the ability of caffeine to partially override the HdCDT-mediated cell cycle arrest. Our data shed new light on the mechanism of action of this novel family of bacterial toxins, limiting the target candidates to DNA or molecules directly involved in activation of checkpoint responses.     

Cytolethal Distending Toxins Require Components of the ER-Associated Degradation Pathway for Host Cell Entry

Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.     

Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts

Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.     

Cytolethal distending toxin generates cell death by inducing a bottleneck in the cell cycle

Cytolethal distending toxin (CDT) is a bacterial protein that is widely distributed among gram-negative bacteria including Escherichia coli, Campylobacter spp., enterohepatic Helicobacter spp., Actinobacillus actinomycetemcomitans and Haemophilus ducreyi. In vitro studies demonstrated that it is able to stop proliferation of various cell lines. The toxin is composed of three subunits designated CDTs A, B and C. The B subunit targets the eukaryotic DNA and triggers a signalling pathway involving different protein kinases which results in a cell block before entering into mitosis. To date, the individual role of the A and C subunits has not been totally elucidated. There are indications that the CDT is also produced in vivo. Its exact role in pathogenesis is not yet clear, but possible actions include inhibition of epithelial cell proliferation, apoptosis of immune cells and inhibition of a fibrotic response.     

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing

Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.     

Cytolethal distending toxin: limited damage as a strategy to modulate cellular functions

The coevolution of bacterial pathogens and their hosts has contributed to the development of very complex and sophisticated functional pathogen--host interfaces. Thus, well-adapted pathogens have evolved a variety of strategies to manipulate host cell functions precisely. For example, a group of unrelated Gram-negative pathogenic bacteria have evolved a toxin, known as cytolethal distending toxin (CDT), that has the ability to control cell cycle progression in eukaryotic cells. Recent studies have identified CdtB as the active subunit of the CDT holotoxin. Through its nuclease activity, CdtB causes limited DNA damage, thereby triggering the DNA-damage response that ultimately results in the observed arrest of the cell cycle. In addition, it has been established that CDT is a tripartite AB toxin in which CdtB is the active 'A' subunit and CdtA and CdtC constitute the heterodimeric 'B' subunit required for the delivery of CdtB into the target cell. The mechanism of action of CDT suggests that the infliction of limited damage could be a strategy used by pathogenic bacteria to modulate host cell functions.