The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors

Campylobacter jejuni is a foodborne bacterial pathogen that is common in the developed world. However, we know less about its biology and pathogenicity than we do about other less prevalent pathogens. Interest in C. jejuni has increased in recent years as a result of the growing appreciation of its importance as a pathogen and the availability of new model systems and genetic and genomic technologies. C. jejuni establishes persistent, benign infections in chickens and is rapidly cleared by many strains of laboratory mouse, but causes significant inflammation and enteritis in humans. Comparing the different host responses to C. jejuni colonization should increase our understanding of this organism.

Campylobacter jejuni : molecular biology and pathogenesis

Many bacterial pathogens encode a multisubunit toxin, termed cytolethal distending toxin (CDT), that induces cell cycle arrest, cytoplasm distention, and, eventually, chromatin fragmentation and cell death. In one such pathogen, Campylobacter jejuni, one of the subunits of this toxin, CdtB, was shown to exhibit features of type I deoxyribonucleases. Transient expression of this subunit in cultured cells caused marked chromatin disruption. Microinjection of low amounts of CdtB induced cytoplasmic distention and cell cycle arrest. CdtB mutants with substitutions in residues equivalent to those required for catalysis or magnesium binding in type I deoxyribonucleases did not cause chromatin disruption. CDT holotoxin containing these mutant forms of CdtB did not induce morphological changes or cell cycle arrest.

https://science.sciencemag.org/content/sci/290/5490/354.full.pdf

A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein.

Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G2/M transition phase of the cell cycle Highly related toxins are also produced by other important bacterial pathogens CDT activity requires the function of three genes: cdtA, cdtB, and cdtC Recent studies have established that CdtB is the active subunit of CDT, exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest Microinjection of CdtB into target cells led to G2/M arrest and cytoplasmic distention, in a manner indistinguishable from that caused by CDT treatment Despite this progress, nothing is known about the composition of the CDT holotoxin or the function of CdtA and CdtC We show here that, when applied individually, purified CdtA, CdtB, or CdtC does not exhibit toxic activity In contrast, CdtA, CdtB, and CdtC when combined, interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity CdtA has a domain that shares similarity with the B chain of ricin-related toxins We therefore proposed that CDT is a tripartite toxin composed of CdtB as the enzymatically active subunit and of CdtA and CdtC as the heterodimeric B subunit required for the delivery of CdtB

CdtA, CdtB, and CdtC Form a Tripartite Complex That Is Required for Cytolethal Distending Toxin Activity

The tripartite cytolethal distending toxin (CDT) induces cell cycle arrest and apoptosis in eukaryotic cells1,2. The subunits CdtA and CdtC associate with the nuclease CdtB to form a holotoxin that translocates CdtB into the host cell, where it acts as a genotoxin by creating DNA lesions3,4,5,6,7. Here we show that the crystal structure of the holotoxin from Haemophilus ducreyi reveals that CDT consists of an enzyme of the DNase-I family, bound to two ricin-like lectin domains. CdtA, CdtB and CdtC form a ternary complex with three interdependent molecular interfaces, characterized by globular, as well as extensive non-globular, interactions. The lectin subunits form a deeply grooved, highly aromatic surface that we show to be critical for toxicity. The holotoxin possesses a steric block of the CdtB active site by means of a non-globular extension of the CdtC subunit, and we identify putative DNA binding residues in CdtB that are essential for toxin activity.

Assembly and function of a bacterial genotoxin

The Haemophilus ducreyi Cytolethal Distending Toxin Induces Cell Cycle Arrest and Apoptosis via the DNA Damage Checkpoint Pathways

Articulo cientifico -- Universidad de Costa Rica. Instituto de Investigaciones en Salud, 2003. Este documento es privado debido a limitaciones de derechos de autor.

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

The potent cytolethal distending toxin produced by Haemophilus ducreyi is a putative virulence factor in the pathogenesis of chancroid. We studied its action on eukaryotic cells, with the long-term goal of understanding the pathophysiology of the disease. Intoxication of cultured human epithelial-like cells, human keratinocytes, and hamster fibroblasts was irreversible, and appeared as a gradual distention of three- to fivefold the size of control cells. Organized actin assemblies appeared concomitantly with cell enlargement, promoted by a mechanism that probably does not involve small GTPases of the Rho protein family. Intoxicated cells did not proliferate. Similar to cells treated with other cytolethal distending toxins, these cells accumulated in the G2 phase of the cell cycle, demonstrating an increased level of the tyrosine phosphorylated (inactive) form of the cyclin-dependent kinase p34cdc2. DNA synthesis was not affected until several hours after this increase, suggesting that the toxin acts directly on some kinase/phosphatase in the signaling network controlling the p34cdc2 activity. We propose that this toxin has an important role both in the generation of chancroid ulcers and in their slow healing. The toxin may also be an interesting new tool for molecular studies of the eukaryotic cell- cycle machinery.

/pdf/the-cytolethal-distending-toxin-from-the-chancroid-bacterium-1wnaxbgqgs.pdf

The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase

The cytolethal distending toxins (CDTs), produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

The biology of the cytolethal distending toxins.

The chancroid bacterium Haemophilus ducreyi produces a toxin (HdCDT) which is a member of the recently discovered family of cytolethal distending toxins (CDTs). These protein toxins prevent the cyclin-dependent kinase cdc2 from being activated, thus blocking the transition of cells from the G(2) phase into mitosis, with the consequent arrest of intoxicated cells in G(2). It is not known whether these toxins act by signaling from the cell surface or intracellularly only. Here we report that HdCDT has to undergo at least internalization before being able to act. Cellular intoxication was inhibited (i) by removal of clathrin coats via K(+) depletion, (ii) by treatment with drugs that inhibit receptor clustering into coated pits, and (iii) in cells genetically manipulated to fail in clathrin-dependent endocytosis. Intoxication was also completely inhibited in cells treated with bafilomycin A1 or nocodazole and in cells incubated at 18 degrees C, i.e., under conditions known to block the fusion of early endosomes with downstream compartments. Moreover, disruption of the Golgi complex by treatment with brefeldin A or ilimaquinone blocked intoxication. In conclusion, our data indicate that HdCDT enters cells via clathrin-coated pits and has to be transported via the Golgi complex in order to intoxicate cells. This is the first member of the family of CDTs for which cellular internalization and some details of the pathway have been demonstrated.

https://iai.asm.org/content/iai/68/12/6903.full.pdf

Ximena Cortes-Bratti

Papers

The Haemophilus ducreyi Cytolethal Distending Toxin Induces Cell Cycle Arrest and Apoptosis via the DNA Damage Checkpoint Pathways

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

The cytolethal distending toxin from the chancroid bacterium Haemophilus ducreyi induces cell-cycle arrest in the G2 phase

The biology of the cytolethal distending toxins.

Cellular internalization of cytolethal distending toxin from Haemophilus ducreyi.