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- Cortes Bratti, Ximena
(författare)
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Intoxication of mammalian cells by the cytolethal distending toxin of Haemophilus ducreyi : a novel mechanism of action
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 2. |
- Cortes-Bratti, Ximena, et al.
(författare)
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Thioredoxin 80-activated-monocytes (TAMs) inhibit the replication of intracellular pathogens
- 2011
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 6:2
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Guerra, Lina, et al.
(författare)
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The biology of the cytolethal distending toxins
- 2011
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Ingår i: Toxins. - : MDPI. - 2072-6651. ; 3:3, s. 172-190
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Forskningsöversikt (refereegranskat)abstract
- 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-B(2) 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.
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