The Biology of the Cytolethal Distending Toxins

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

A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus

Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.Universidad de Costa Rica/[74l-93-301]/UCR/Costa RicaInternational Foundation for Science/[F/0883-4]/IFS/SueciaSwedish Agency for Research Cooperation with Developing Countries//SAREC/SueciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

BACKGROUND: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). PRINCIPAL FINDINGS: In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. SIGNIFICANCE: Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria

Intoxication of mammalian cells by the cytolethal distending toxin of Haemophilus ducreyi : A novel mechanism of action

The cytolethal distending toxins (CDTs) are a newly described family of bacterial protein toxins with a novel mechanism of action: DNA damage. Haemophilus ducreyi produces a cytotoxin that belongs to this family. This bacterium causes chancroid, a sexually transmitted disease, characterised by mucocutaneous, slowly healing genital ulcers. As all genital ulcerative diseases, chancroid is a predisposing factor in the transmission of HIV. Since ale pathogenesis of the disease is not known the Haemophilus ducreyi cytolethal distending toxin (HdCDT) represents a putative virulence factor. At the beginning of our studies with HdCDT little was known about CDTs. To understand the mode of action of this toxin, we studied how it intoxicates mammalian cells. The morphological effect induced by the toxin was studied in epithelial-like cells and hamster fibroblasts. The intoxication was irreversible and appeared as a gradual cell distention, followed by cell death. A promotion of actin stress fibers was observed concomitantly with the cell enlargement. As shown for other CDTs, we found that HdCDT-intoxicated HEp-2 cells were arrested in the G2 phase of the cell cycle due to an accumulation of the tyrosine phosphorylated (inactive) form of the cyclin dependent kinase cdc2. To characterize better the mode of action of HdCDT we tested a broad panel of human cell lines. We could demonstrate that the HdCDT effect is cell type specific and not exclusively related to G2 arrest. B cell lines underwent apoptosis, epithelial cells and keratinocytes arrested exclusively in G2 whereas normal fibroblasts arrested both in G1 and G2. Moreover, we showed that the response induced by HdCDT is similar to the checkpoint response activated by ionizing radiation (IR). Both responses were characterized by an early induction of the p53 gene and the cyclin dependent kinase inhibitor p21 in human fibroblasts and activation of chk2 kinase in HeLa cells. Our work also suggested that ATM, a key molecule in sensing DNA damage, is needed for the early response to HdCDT. However, in the absence of functional ATM the checkpoint was activated after a delay, probably by a homologue such as ATR. We also demonstrated that the promotion of actin stress fibers induced by HdCDT is dependent on Rho activation. Our observations made a link between Rho activation and DNA damage. Finally, we demonstrated that these effects can occur only after cellular internalization of the toxin, and we have clarified some steps of the intracellular pathway followed by HdCDT. The toxin was found to enter HEp-2 cells via clathrin coated pits and to need an intact Golgi complex in order to induce intoxication. In conclusion, this work, using HdCDT as a model, has improved our understanding of the mode of action of CDTs

Growth curve of untreated control monocytes and TAMs after <i>L. monocytogenes</i> infection.

<p>Cells were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. After the infection, samples were taken at the indicated periods of time, and number of viable cells was assayed by trypan blue exclusion. The data are presented as number of viable cells (panel A) and as percentage of cell recovery, being 100% of recovery at time 0 (panel B). Mean ± SEM of six independent experiments.</p

TAMs control replication of <i>Listeria monocytogenes.</i>

<p><b>A</b>. Intracellular growth curve of <i>L. monocytogenes</i>. Cells were infected for 30 minutes at 37°C with a multiplicity of infection of 25∶1 bacteria per cell, and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and RPMI supplemented with 5 µg/ml gentamicin was added. At the indicated periods of time, 3×10⁵ cells were lysed and colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. A representative experiment is shown. <b>B</b>. Summary of the growth curves of <i>L. monocytogenes</i>. The data are presented as percentage of the number of CFU recovered in TAMs relative to the number of CFU recovered in control monocytes (mean ± SEM of five independent experiments). <b>C</b>. Cells were infected with <i>L. monocytogenes</i> for 30 minutes at 37°C with a MOI 25∶1 and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria. Intracellular bacteria were detected by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a> (mean ± SEM of five independent experiments).</p

Localization of GFP-tagged <i>L. monocytogenes</i> in acidic compartments.

<p><b>A</b>. Cells were infected with the GFP-tagged <i>L. monocytogenes</i> at MOI 50∶1 for 4 hours, and treated with the acidotropic dye Lysotracker Red. Cytospin centrifugation was performed and cells were fixed with 4% paraformaldehyde. Lysotracker Red positive vesicles containing GFP-labeled bacteria are indicated with arrows. Bar represents 5 µm. <b>B</b>. The percentage of Lysotracker Red positive vesicles containing GFP-labeled bacteria was quantified in two independent experiments. A minimum of 200 cells were analyzed. <b>C</b>. Scatter plot analysis showing the correlation between the GFP and Lysotracker Red fluorescence intensities in control cells and TAMs quantified by the Volocity software. A correlation coefficient value of r² equal to 1 indicates 100% bacterial localization with the lysosomal compartments.</p

Escape of <i>L. monocytogenes</i> NF-L327 into the cytosol of infected monocytes.

<p><b>A</b>. Cells were infected with the <i>L. monocytogenes</i> strain NF-L327, that expresses GFP when translocated into the cytosol, at MOI 50∶1 for 30 minutes and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and maintained in RPMI supplemented with 5 µg/ml gentamicin. Cells were lysed after infection (0 h) and 24 h in culture and the colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#pone-0016960-g003" target="_blank">Figure 3A</a>. A representative experiment is shown. <b>B</b>. Cells were infected with the <i>L. monocytogenes</i> NF-L327 at MOI 50∶1 as described in A and samples were taken 8 hours post-infection. Cytospin and fluorescence imaging were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. Bar represents 5 µm. <b>C</b>. Quantification of GFP-labeled <i>Listeria</i> was performed using the Volocity software. The data are presented as number of bacteria in 200 cells (SEM of two independent experiments).</p