| 1. |
- Guerra, Lina, et al.
(author)
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Bacterial genotoxin triggers FEN1-dependent RhoA activation, cytoskeleton remodeling and cell survival
- 2011
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 124:16, s. 2735-2742
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Journal article (peer-reviewed)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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| 2. |
- Guerra, Lina, et al.
(author)
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Cellular internalization of cytolethal distending toxin : a new end to a known pathway
- 2005
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In: Cellular Microbiology. - : Hindawi Limited. - 1462-5814 .- 1462-5822. ; 7:7, s. 921-34
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Journal article (peer-reviewed)abstract
- The cytolethal distending toxins (CDTs) are unique in their ability to induce DNA damage, activate checkpoint responses and cause cell cycle arrest or apoptosis in intoxicated cells. However, little is known about their cellular internalization pathway. We demonstrate that binding of the Haemophilus ducreyi CDT (HdCDT) on the plasma membrane of sensitive cells was abolished by cholesterol extraction with methyl-beta-cyclodextrin. The toxin was internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER), as assessed by N-linked glycosylation. Further translocation from the ER did not require the ER-associated degradation (ERAD) pathway, and was Derlin-1 independent. The genotoxic activity of HdCDT was dependent on its internalization and its DNase activity, as induction of DNA double-stranded breaks was prevented in Brefeldin A-treated cells and in cells exposed to a catalytically inactive toxin. Our data contribute to a better understanding of the CDT mode of action and highlight two important aspects of the biology of this bacterial toxin family: (i) HdCDT translocation from the ER to the nucleus does not involve the classical pathways followed by other retrogradely transported toxins and (ii) toxin internalization is crucial for execution of its genotoxic activity.
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| 3. |
- Guerra, Lina
(author)
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Cellular responses to the DNA damaging cytolethal distending toxin
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Cytolethal distending toxin (CDT) is a genotoxin, which belongs to a group of bacterial protein toxins called cyclomodulins. These are characterized by their interference with the eukaryotic cell cycle. CDT causes DNA damage, which induces cell cycle arrest and apoptosis. The active holotoxin consists of three subunits CdtA, CdtB and CdtC, where CdtB is the active subunit and has structural and functional similarities with DNase I. We demonstrated that CDT uses the same internalization pathway as several other bacterial toxins do, such as cholera toxin and Shiga toxin. The binding on the plasma membrane is dependent on cholesterol. The toxin is internalized via the Golgi complex, and retrogradely transported to the endoplasmic reticulum (ER) and found in the nucleoplasmic reticulum. The translocation from the ER to the nucleus does not require either the ER-associated (ERAD) pathway or the Derlin-1 protein. Additionally, we showed that CDT is not farnesylated, a modification known to occur in the cytosol. In contrast, to other AB toxins, CdtB was demonstrated to have heat-stable properties and is not degraded by the 20S proteasome. All these evidence suggest that the toxin is translocated directly from the ER to the nucleus. In adherent cells the cellular response to the CDT-induced DNA damage involved activation of the RhoA GTPase. We showed that the RhoA-specific Guanine nucleotide exchange factor (GEF) Net1 is dephosphorylated and translocated from the nucleus to the cytosol upon DNA damage. Knock down of Net1 by RNAi prevents RhoA activation, inhibits the formation of stress fibers, and enhances cell death. This indicates that Net1 activation is required for RhoAmediated response to genotoxic stress. The Net1 and the RhoA dependent signals converge the activation of mitogen-activated protein kinase p38 (p38 MAPK) and its downstream target MAPK-activated protein kinase 2 (MK2). To further investigate this novel cell survival pathway in response to CDT we screened a yeast deletion library for CdtB-sensitive strains. Approximately 4500 yeast deletion strains were transformed with a plasmid containing CdtB. The screen shows that 78 mutated strains were hypersensitive to CdtB. Twenty of the human ortholog genes were found to interact with the actin cytoskeleton regulation network. Our analysis focused on TSG101, FEN1 and Vinculin (VCL). We demonstrated that they are all required to induce actin stress fiber formation in response to DNA damage. FEN1 and VCL also regulate the RhoA GTPase and p38 MAPK activation, and delay cell death in response to CDT intoxication. In response to DNA damage, Ataxia-telangiectasia mutated (ATM) and ATM and Rad-3-related kinases (ATR) are activated and orchestrate DNA damage response. The transcription factor Myc has multi-functions such as inducing apoptosis in response to DNA damage. The Mycregulated effectors acting upstream of the mitochorial apoptotic pathway are still unknown. We demonstrated that Myc is required for activation of the ATM-dependent DNA damage checkpoint response in cells exposed to ionizing radiation or CDT. Activation of ATM effectors, such as histone H2AX and the nuclear foci formation of the Nijmegen Berakage Syndrome (Nbs)1 protein, were abolished in the absence of Myc. The cellular response to UV irradiation, known to activate an ATR-dependent checkpoint, was not delayed in the absent of the Myc expression. This data demonstrate that Myc is required for activation of the ATM-dependent pathway. Our studies highlight the importance of understanding the CDT biology and its mode of action. This knowledge could provide new tools to elucidate the putative involvement of bacteria in carcinogenesis.
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| 4. |
- Guerra, Lina, et al.
(author)
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Do bacterial genotoxins contribute to chronic inflammation, genomic instability and tumor progression?
- 2011
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In: The FEBS Journal. - : John Wiley & Sons. - 1742-464X .- 1742-4658. ; 278:23, s. 4577-4588
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Research review (peer-reviewed)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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| 5. |
- Guerra, Lina, et al.
(author)
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Myc is required for activation of the ATM-dependent checkpoints in response to DNA damage
- 2010
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In: PLOS ONE. - : Public Library Science. - 1932-6203. ; 5:1
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Journal article (peer-reviewed)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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| 6. |
- Guerra, Lina, et al.
(author)
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The biology of the cytolethal distending toxins
- 2011
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In: Toxins. - : MDPI. - 2072-6651. ; 3:3, s. 172-190
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Research review (peer-reviewed)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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| 7. |
- Guidi, Riccardo, et al.
(author)
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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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In: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 15:1, s. 98-113
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Journal article (peer-reviewed)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. |
- Nguyen, Thanh N, et al.
(author)
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Global Impact of the COVID-19 Pandemic on Stroke Volumes and Cerebrovascular Events: A 1-Year Follow-up.
- 2023
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In: Neurology. - 1526-632X. ; 100:4
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Journal article (peer-reviewed)abstract
- Declines in stroke admission, IV thrombolysis (IVT), and mechanical thrombectomy volumes were reported during the first wave of the COVID-19 pandemic. There is a paucity of data on the longer-term effect of the pandemic on stroke volumes over the course of a year and through the second wave of the pandemic. We sought to measure the effect of the COVID-19 pandemic on the volumes of stroke admissions, intracranial hemorrhage (ICH), IVT, and mechanical thrombectomy over a 1-year period at the onset of the pandemic (March 1, 2020, to February 28, 2021) compared with the immediately preceding year (March 1, 2019, to February 29, 2020).We conducted a longitudinal retrospective study across 6 continents, 56 countries, and 275 stroke centers. We collected volume data for COVID-19 admissions and 4 stroke metrics: ischemic stroke admissions, ICH admissions, IVT treatments, and mechanical thrombectomy procedures. Diagnoses were identified by their ICD-10 codes or classifications in stroke databases.There were 148,895 stroke admissions in the 1 year immediately before compared with 138,453 admissions during the 1-year pandemic, representing a 7% decline (95% CI [95% CI 7.1-6.9]; p < 0.0001). ICH volumes declined from 29,585 to 28,156 (4.8% [5.1-4.6]; p < 0.0001) and IVT volume from 24,584 to 23,077 (6.1% [6.4-5.8]; p < 0.0001). Larger declines were observed at high-volume compared with low-volume centers (all p < 0.0001). There was no significant change in mechanical thrombectomy volumes (0.7% [0.6-0.9]; p = 0.49). Stroke was diagnosed in 1.3% [1.31-1.38] of 406,792 COVID-19 hospitalizations. SARS-CoV-2 infection was present in 2.9% ([2.82-2.97], 5,656/195,539) of all stroke hospitalizations.There was a global decline and shift to lower-volume centers of stroke admission volumes, ICH volumes, and IVT volumes during the 1st year of the COVID-19 pandemic compared with the prior year. Mechanical thrombectomy volumes were preserved. These results suggest preservation in the stroke care of higher severity of disease through the first pandemic year.This study is registered under NCT04934020.
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| 9. |
- Potapov, Anton M., et al.
(author)
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Globally invariant metabolism but density-diversity mismatch in springtails
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Journal article (peer-reviewed)abstract
- Soil life supports the functioning and biodiversity of terrestrial ecosystems. Springtails (Collembola) are among the most abundant soil arthropods regulating soil fertility and flow of energy through above- and belowground food webs. However, the global distribution of springtail diversity and density, and how these relate to energy fluxes remains unknown. Here, using a global dataset representing 2470 sites, we estimate the total soil springtail biomass at 27.5 megatons carbon, which is threefold higher than wild terrestrial vertebrates, and record peak densities up to 2 million individuals per square meter in the tundra. Despite a 20-fold biomass difference between the tundra and the tropics, springtail energy use (community metabolism) remains similar across the latitudinal gradient, owing to the changes in temperature with latitude. Neither springtail density nor community metabolism is predicted by local species richness, which is high in the tropics, but comparably high in some temperate forests and even tundra. Changes in springtail activity may emerge from latitudinal gradients in temperature, predation and resource limitation in soil communities. Contrasting relationships of biomass, diversity and activity of springtail communities with temperature suggest that climate warming will alter fundamental soil biodiversity metrics in different directions, potentially restructuring terrestrial food webs and affecting soil functioning.
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