A Bacterial Cytotoxin Identifies the RhoA Exchange Factor Net1 as a Key Effector in the Response to DNA Damage

Background: Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown. Principal Findings: We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoAdependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPKactivated protein kinase 2. Significance: Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin ma

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

Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G(2)/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 G(2)/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

Cytolethal Distending Toxin Demonstrates Genotoxic Activity in a Yeast Model

Cytolethal distending toxins (CDTs) are multisubunit proteins produced by a variety of bacterial pathogens that cause enlargement, cell cycle arrest, and apoptosis in mammalian cells. While their function remains uncertain, recent studies suggest that they can act as intracellular DNases in mammalian cells. Here we establish a novel yeast model for understanding CDT-associated disease. Expression of the CdtB subunit in yeast causes a G(2)/M arrest, as seen in mammalian cells. CdtB toxicity is not circumvented in yeast genetically altered to lack DNA damage checkpoint control or that constitutively promote cell cycle progression via mutant Cdk1, because CdtB causes a permanent type of damage that results in loss of viability. Finally, we establish that CDTs are likely to be potent genotoxins, as indicated by in vivo degradation of chromosomal DNA associated with expression of CdtB—suggesting that the varied distribution of CDT in bacteria implicates many human pathogens as possessors of genotoxic activity

Haemophilus ducreyi Inhibits Phagocytosis by U-937 Cells, a Human Macrophage-Like Cell Line

Haemophilus ducreyi is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live H. ducreyi can be isolated from these lesions. To analyze the interaction of H. ducreyi with macrophages, we investigated the ability of H. ducreyi strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although H. ducreyi strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that H. ducreyi avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that H. ducreyi inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to H. ducreyi, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine H. ducreyi strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which H. ducreyi avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes