| 1. |
- Del Bel Belluz, Lisa, et al.
(författare)
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The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection
- 2016
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Ingår i: PLoS Pathogens. - : Public Library Science. - 1553-7366 .- 1553-7374. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied in vitro, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the Salmonella enterica genotoxin, known as typhoid toxin, in in vivo models of murine infection. Immunocompetent mice were infected with isogenic S. enterica, serovar Typhimurium (S. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.
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| 2. |
- Guerra, Lina, et al.
(författare)
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Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival
- 2011
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Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 124:16, s. 2735-2742
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Tidskriftsartikel (refereegranskat)abstract
- The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.
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| 3. |
- Guerra, Lina, et al.
(författare)
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Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?
- 2011
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Ingår i: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 278:23, s. 4577-4588
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Forskningsöversikt (refereegranskat)abstract
- Cytolethal distending toxin, produced by several Gram-negative bacteria, and colibactin, secreted by several commensal and extraintestinal pathogenic Escherichia coli strains, are the first bacterial genotoxins to be described to date. Exposure to cytolethal distending toxin and colibactin induces DNA damage, and consequently activates the DNA damage response, resulting in cell cycle arrest of the intoxicated cells and DNA repair. Irreversible DNA damage will lead to cell death by apoptosis or to senescence. It is well established that chronic exposure to DNA damaging agents, either endogenous (reactive oxygen species) or exogenous (ionizing radiation), may cause genomic instability as a result of the alteration of genes coordinating the DNA damage response, thus favoring tumor initiation and progression. In this review, we summarize the state of the art of the biology of cytolethal distending toxin and colibactin, focusing on the activation of the DNA damage response and repair pathways, and discuss the cellular responses induced in intoxicated cells, as well as how prolonged intoxication may lead to chronic inflammation, the accumulation of genomic instability, and tumor progression in both in vitro and in vivo models.
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| 4. |
- Guerra, Lina, et al.
(författare)
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Myc is required for activation of the ATM-dependent checkpoints in response to DNA damage
- 2010
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The MYC protein controls cellular functions such as differentiation, proliferation, and apoptosis. In response to genotoxic agents, cells overexpressing MYC undergo apoptosis. However, the MYC-regulated effectors acting upstream of the mitochondrial apoptotic pathway are still unknown.PRINCIPAL FINDINGS: In this study, we demonstrate that expression of Myc is required to activate the Ataxia telangiectasia mutated (ATM)-dependent DNA damage checkpoint responses in rat cell lines exposed to ionizing radiation (IR) or the bacterial cytolethal distending toxin (CDT). Phosphorylation of the ATM kinase and its downstream effectors, such as histone H2AX, were impaired in the myc null cell line HO15.19, compared to the myc positive TGR-1 and HOmyc3 cells. Nuclear foci formation of the Nijmegen Breakage Syndrome (Nbs) 1 protein, essential for efficient ATM activation, was also reduced in absence of myc. Knock down of the endogenous levels of MYC by siRNA in the human cell line HCT116 resulted in decreased ATM and CHK2 phosphorylation in response to irradiation. Conversely, cell death induced by UV irradiation, known to activate the ATR-dependent checkpoint, was similar in all the cell lines, independently of the myc status.CONCLUSION: These data demonstrate that MYC contributes to the activation of the ATM-dependent checkpoint responses, leading to cell death in response to specific genotoxic stimuli.
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| 5. |
- 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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| 6. |
- Guidi, Riccardo
(författare)
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Bacteria and cancer : from toxin delivery to carcinogenesis
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epidemiological evidence link certain chronic bacterial infections to a higher risk of cancer development. Induction of an inflammatory circuit and the accumulation of genomic instability are considered mechanisms by which bacteria contribute to malignant transformation. Whether production of toxins, that directly induce DNA damage, enhances the tumor promoting effects of chronic inflammation is still unknown. This thesis investigates the role of the cytolethal distending toxin (CDT), the first bacterial genotoxin identified, in carcinogenesis. We have studied the cellular responses to acute and chronic CDT intoxication, as well as the toxin production and secretion during bacterial infection. Acute CDT intoxication triggers the activation of the DNA damage response and induction of survival signals in the target cells, which may favor cancer growth. Through a screening of a Saccharomyces cerevisiaelibrary, we identified 78 genes whose deletion confers hypersensitivity to CDT exposure (paper I). Bioinformatics analysis revealed that DNA repair and endocytosis were the two most represented signaling pathways among the genes identified in the screening. We further demonstrated that in response to DNA damage, the flap-endonuclease 1 (FEN1) regulated the RHOAdependent activation of the actin cytoskeleton and cell survival via the ROCK and MAPK p38 kinases, respectively, revealing a complex and previously unrecognized crosstalk between DNA damage, cell survival and cytoskeleton dynamics. As chronic exposure to DNA damaging agents is a well-characterized risk for cancer development, we assessed the effects of chronic CDT exposure in vitro(paper II). Cells grown for more than six months in the presence of sub-lethal toxin doses showed an altered DNA damage response, genomic instability, and acquisition of several hallmarks of tumor progression, such as enhanced oxidative stress and capacity of anchorage independent growth. Cell survival of the chronically intoxicated cells was dependent on sustained activation of the MAPK p38 pathway. To dissect the role of CDT in tumor development in vivo, we produced aSalmonella typhimuriumstrain that encode for the Salmonella typhiCDT-like toxin, known as typhoid toxin (TT). As control, we used an isogenic strain carrying an inactive toxin. Both strains successfully infected the immunocompetent sv129 mice for more than 2 months, however only the bacteria expressing the active genotoxin caused an enhanced inflammation in liver and spleen. To understand how this potential bacterial carcinogen is delivered to the target cells, we studied the secretion of the Salmonella TT (paper III). We demonstrated that TT is secreted from the bacterium via outer membrane vesicles (OMVs). These vesicles are further released into the extracellular environment via an exocytosis-like process. The paracrine internalization of TT-loaded OMVs by bystander cells was dependent on dynamin-1-mediated endocytosis. Taken together, our studies contribute to elucidate the survival strategy of cancer cells in response to CDT, its role in cancer progression and its secreting mechanisms.
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| 7. |
- Guidi, Riccardo, et al.
(författare)
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Chronic exposure to the cytolethal distending toxins of Gram-negative bacteria promotes genomic instability and altered DNA damage response
- 2013
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Ingår i: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 15:1, s. 98-113
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Tidskriftsartikel (refereegranskat)abstract
- Epidemiological evidence links chronic bacterial infections to the increased incidence of certain types of cancer but the molecular mechanisms by which bacteria contribute to tumour initiation and progression are still poorly characterized. Here we show that chronic exposure to the genotoxin cytolethal distending toxin (CDT) of Gram-negative bacteria promotes genomic instability and acquisition of phenotypic properties of malignancy in fibroblasts and colon epithelial cells. Cells grown for more than 30 weeks in the presence of sublethal doses of CDT showed increased mutation frequency, and accumulation of chromatin and chromosomal aberrations in the absence of significant alterations of cell cycle distribution, decreased viability or senescence. Cell survival was dependent on sustained activity of the p38 MAP kinase. The ongoing genomic instability was associated with impaired activation of the DNA damage response and failure to efficiently activate cell cycle checkpoints upon exposure to genotoxic stress. Independently selected sublines showed enhanced anchorage-independent growth as assessed by the formation of colonies in semisolid agarose. These findings support the notion that chronic infection by CDT-producing bacteria may promote malignant transformation, and point to the impairment of cellular control mechanisms associated with the detection and repair of DNA damage as critical events in the process.
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| 8. |
- Guidi, Riccardo, et al.
(författare)
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Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells
- 2013
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Ingår i: Cellular Microbiology. - : Wiley-Blackwell. - 1462-5814 .- 1462-5822. ; 15:12, s. 2034-2050
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Scandale, Walter, et al.
(författare)
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Apparatus to study crystal channeling and volume reflection phenomena at the SPS H8 beamline
- 2008
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Ingår i: Review of Scientific Instruments. - : AIP Publishing. - 0034-6748 .- 1089-7623. ; 79:2
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Tidskriftsartikel (refereegranskat)abstract
- A high performance apparatus has been designed and built by the H8-RD22 collaboration for the study of channeling and volume reflection phenomena in the interaction of 400 GeVc protons with bent silicon crystals, during the 2006 data taking in the external beamline H8 of the CERN SPS. High-quality silicon short crystals were bent by either anticlastic or quasimosaic effects. Alignment with the highly parallel (8 μrad divergence) proton beam was guaranteed through a submicroradian goniometric system equipped with both rotational and translational stages. Particle tracking was possible by a series of silicon microstrip detectors with high-resolution and a parallel plate gas chamber, triggered by various scintillating detectors located along the beamline. Experimental observation of volume reflection with 400 GeVc protons proved true with a deflection angle of (10.4±0.5) μrad with respect to the unperturbed beam, with a silicon crystal whose (111) planes were parallel to the beam. © 2008 American Institute of Physics.
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| 10. |
- Scandale, Walter, et al.
(författare)
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Deflection of 400GeV/c proton beam with bent silicon crystals at the CERN Super Proton Synchrotron
- 2008
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Ingår i: Physical Review Special Topics - Accelerators and Beams. - 1098-4402. ; 11:6
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Tidskriftsartikel (refereegranskat)abstract
- This paper presents a detailed study of the deflection phenomena of a 400GeV/c proton beam impinging on a new generation of bent silicon crystals; the tests have been performed at the CERN Super Proton Synchrotron H8 beam line. Channeling and volume reflection angles are measured with an extremely precise goniometer and with high resolution silicon microstrip detectors. Volume reflection has been observed and measured for the first time at this energy, with a single-pass efficiency as large as 98%, in good agreement with the simulation results. This efficiency makes volume reflection a possible candidate for collimation with bent crystals at the CERN Large Hadron Collider. © 2008 The American Physical Society.
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