| 1. |
- Bassères, Eugénie, et al.
(författare)
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The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton
- 2010
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Ingår i: Cellular Microbiology. - : Wiley-Blackwell. - 1462-5814 .- 1462-5822. ; 12:11, s. 1622-1633
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Tidskriftsartikel (refereegranskat)abstract
- Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S). These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH-L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria-based drug delivery systems.
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| 2. |
- Bergonzini, Anna, 1990-
(författare)
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Effects of bacterial genotoxins on immune modulation, chronic inflammation and cancer development
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The intestinal microbiome of Inflammatory Bowel Disease and colorectal cancer patients is enriched in genotoxin-producing bacteria, which cause DNA damage in the host cells.Genotoxins have recently been identified as a novel family of effectors produced by pathogenic and commensal bacteria. At present, only three types of bacterial genotoxins have been identified: colibactin, produced by some Escherichia coli strains; cytolethal distending toxins, produced by several Gram-negative pathogens; and the typhoid toxin, produced by Salmonella enterica serovar Typhi.Exposure to high toxin doses activates the classical DNA damage response, which consequently blocks proliferation and eventually induces death in mammalian cells. However, exposure to low toxin doses has shown to promote classical signs of carcinogenesis in vitro, such as cell survival and acquisition of genomic instability. Despite an extensive characterization of their mode of action in vitro, we have a poor understanding of genotoxins´ role in chronic infection and, considering the genotoxic potential, of their carcinogenic capacity. To investigate further the role played by the genotoxins, we focused specifically on Salmonella Typhi, since it is the only genotoxin-producing bacterium that induces a chronic infection associated with increased risk of tumor development in humans. The results presented in this thesis show that these unusual bacterial effectors are not classical toxins, but rather act as immunomodulators, highlighting a complex and tissue-specific crosstalk between two highly conserved stress responses: the immune response and the DNA damage response. Our data indicate that the impact of genotoxin-producing bacteria on the modulation of the host mucosal response is still poorly characterized and suggest that the host-microbe interaction and the tissue microenvironment are the key players in determining the outcome of the infection and the toxin carcinogenic potential.
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| 3. |
- Bergonzini, Anna, 1990-, et al.
(författare)
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The challenge of establishing immunocompetent human intestinal 3D models
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Expression of typhoid toxin in Salmonella Typhimurium causes DNA damage, activating the DNA damage response (DDR), in absence of an inflammatory response in the colonic mucosa of infected mice. The anti-inflammatory effect is tissue specific and is not observed in the liver, suggesting that the local immune microenvironment modulates the DDR outcome.To assess the role of the immune cells in the DDR outcome induced by the genotoxigenic Salmonella, we have initiated the development of an immunocompetent 3D colonic mucosal model based on a collagen matrix containing colonic fibroblasts and different subtypes of immune cells, overlayed with colonic epithelial cells.Embedding of peripheral blood mononuclear cells in the collagen matrix did not influenced either the tissue integrity or the activation of the DDR, observed exclusively upon infection with the genotoxigenic strain. However, embedding of T cells, monocytes, or non-polarized macrophages altered the pattern of the DDR and the toxin specific effect was lost. Presence of macrophages was further associated with alteration of the epithelial layer integrity. This effect was infection-dependent, but not toxin specific.Our data demonstrated that addition of immune cells to a 3D mucosal model altered the DDR induced by a genotoxigenic bacterium, highlighting the need to develop and optimize immunocompetent in vitro models.
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| 4. |
- Blazkova, Hana, et al.
(författare)
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Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling
- 2010
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Ingår i: Journal of Cellular and Molecular Medicine (Print). - : John Wiley & Sons. - 1582-1838 .- 1582-4934. ; 14:1-2, s. 357-367
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Tidskriftsartikel (refereegranskat)abstract
- Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram‐negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type‐dependent cell‐cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR‐90 and WI‐38 fibroblasts, HeLa and U2‐OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX+ foci), enhanced senescence‐associated β‐galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL‐6, IL‐8 and IL‐24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the ‘distended’ morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.
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| 5. |
- Brauner, Annelie, et al.
(författare)
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Is there a risk of cancer development after Campylobacter infection?
- 2010
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Ingår i: Scandinavian Journal of Gastroenterology. - : Taylor & Francis. - 0036-5521 .- 1502-7708. ; 45:7-8, s. 893-897
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: All Campylobacter jejuni species produce a genotoxin, which induce DNA double strand breaks, could lead to an increased risk of cancer especially in the gastro-intestinal tract.MATERIAL AND METHODS: All individuals in Stockholm County who tested positive with C. jejuni between 1989 and 2006 were included. The cohort was followed-up until December 31, 2007 for the occurrence of cancer, overall and site specific. Standard incidence ratios (SIR) with 95% confidence intervals (CI) were calculated by comparisons with the background population.RESULTS: There were 16,276 individuals who tested positive for C. jejuni generating 124,387 person years. Excluding the first year of follow-up the overall risk for cancer did neither differ from that expected SIR = 0.95 (95% CI 0.82-1.09) nor after 10 years or more of follow-up; SIR = 0.91 (95% CI 0.71-1.16). There was no increased risk for cancer in the gastro-intestinal tract, but there were significantly increased risks for melanomas SIR = 1.84 (95% CI 1.27-2.57) and squamous cell skin cancer SIR = 1.52 (95% CI 1.01-2.19) while a significantly decreased risk of respiratory cancers among males SIR = 0.32 (95% CI 0.12-0.70) was observed.CONCLUSIONS: Our results indicate no excess risks of malignancies following an infection by C. jejuni at least during the first decade. Furthermore, the finding of a decreased risk of respiratory cancers could be of interest, if the results are reproduced in future studies in other populations.
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| 6. |
- Chaves-Olarte, Esteban, et al.
(författare)
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A sensor histidine kinase from a plant-endosymbiont bacterium restores the virulence of a mammalian intracellular pathogen
- 2023
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Ingår i: Microbial Pathogenesis. - : Elsevier. - 0882-4010 .- 1096-1208. ; 185
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Tidskriftsartikel (refereegranskat)abstract
- Alphaproteobacteria include organisms living in close association with plants or animals. This interaction relies partly on orthologous two-component regulatory systems (TCS), with sensor and regulator proteins modulating the expression of conserved genes related to symbiosis/virulence. We assessed the ability of the exoS+Sm gene, encoding a sensor protein from the plant endosymbiont Sinorhizobium meliloti to substitute its orthologous bvrS in the related animal/human pathogen Brucella abortus. ExoS phosphorylated the B. abortus regulator BvrR in vitro and in cultured bacteria, showing conserved biological function. Production of ExoS in a B. abortus bvrS mutant reestablished replication in host cells and the capacity to infect mice. Bacterial outer membrane properties, the production of the type IV secretion system VirB, and its transcriptional regulators VjbR and BvrR were restored as compared to parental B. abortus. These results indicate that conserved traits of orthologous TCS from bacteria living in and sensing different environments are sufficient to achieve phenotypic plasticity and support bacterial survival. The knowledge of bacterial genetic networks regulating host interactions allows for an understanding of the subtle differences between symbiosis and parasitism. Rewiring these networks could provide new alternatives to control and prevent bacterial infection.
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| 7. |
- Chen, Xi, et al.
(författare)
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Type-I interferon signatures in SARS-CoV-2 infected Huh7 cells
- 2021
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Ingår i: Cell Death Discovery. - : Springer Nature. - 2058-7716. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) has caused a global health emergency. A key feature of COVID-19 is dysregulated interferon-response. Type-I interferon (IFN-I) is one of the earliest antiviral innate immune responses following viral infection and plays a significant role in the pathogenesis of SARS-CoV-2. In this study, using a proteomics-based approach, we identified that SARS-CoV-2 infection induces delayed and dysregulated IFN-I signaling in Huh7 cells. We demonstrate that SARS-CoV-2 is able to inhibit RIG-I mediated IFN-β production. Our results also confirm the recent findings that IFN-I pretreatment is able to reduce the susceptibility of Huh7 cells to SARS-CoV-2, but not post-treatment. Moreover, senescent Huh7 cells, in spite of showing accentuated IFN-I response were more susceptible to SARS-CoV-2 infection, and the virus effectively inhibited IFIT1 in these cells. Finally, proteomic comparison between SARS-CoV-2, SARS-CoV, and MERS-CoV revealed a distinct differential regulatory signature of interferon-related proteins emphasizing that therapeutic strategies based on observations in SARS-CoV and MERS-CoV should be used with caution. Our findings provide a better understanding of SARS-CoV-2 regulation of cellular interferon response and a perspective on its use as a treatment. Investigation of different interferon-stimulated genes and their role in the inhibition of SARS-CoV-2 pathogenesis may direct novel antiviral strategies.
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| 8. |
- 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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| 9. |
- 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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| 10. |
- Fahrer, Jörg, et al.
(författare)
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Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts
- 2014
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Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 18, s. 31-43
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Tidskriftsartikel (refereegranskat)abstract
- Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.
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| 11. |
- Frisan, Teresa, 1967-, et al.
(författare)
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A bacterial genotoxin causes virus reactivation and genomic instability in Epstein-Barr virus infected epithelial cells pointing to a role of co-infection in viral oncogenesis
- 2019
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Ingår i: International Journal of Cancer. - : John Wiley & Sons. - 0020-7136 .- 1097-0215. ; 144:1, s. 98-109
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Tidskriftsartikel (refereegranskat)abstract
- We have addressed the role of bacterial co-infection in viral oncogenesis using as model Epstein-Barr virus (EBV), a human herpesvirus that causes lymphoid malignancies and epithelial cancers. Infection of EBV carrying epithelial cells with the common oral pathogenic Gram-negative bacterium Aggregatibacter actinomycetemcomitans (Aa) triggered reactivation of the productive virus cycle. Using isogenic Aa strains that differ in the production of the cytolethal distending toxin (CDT) and purified catalytically active or inactive toxin, we found that the CDT acts via induction of DNA double strand breaks and activation of the Ataxia Telangectasia Mutated (ATM) kinase. Exposure of EBV-negative epithelial cells to the virus in the presence of sub-lethal doses of CDT was accompanied by the accumulation of latently infected cells exhibiting multiple signs of genomic instability. These findings illustrate a scenario where co-infection with certain bacterial species may favor the establishment of a microenvironment conducive to the EBV-induced malignant transformation of epithelial cells.
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| 12. |
- Frisan, Teresa, 1967-
(författare)
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Bacterial genotoxins : the long journey to the nucleus of mammalian cells
- 2016
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier. - 0006-3002 .- 1878-2434 .- 0005-2736. ; 1858:3, s. 567-575
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Forskningsöversikt (refereegranskat)abstract
- Bacterial protein genotoxins target the DNA of eukaryotic cells, causing DNA single and double strand breaks. The final outcome of the intoxication is induction of DNA damage responses and activation of DNA repair pathways. When the damage is beyond repair, the target cell either undergoes apoptosis or enters a permanent quiescent stage, known as cellular senescence. In certain instances, intoxicated cells can survive and proliferate. This event leads to accumulation of genomic instability and acquisition of malignant traits, underlining the carcinogenic potential of these toxins. The toxicity is dependent on the toxins' internalization and trafficking from the extracellular environment to the nucleus, and requires a complex interaction with several cellular membrane compartments: the plasma membrane, the endosomes, the trans Golgi network and the endoplasmic reticulum, and finally the nucleus. This review will discuss the current knowledge of the bacterial genotoxins internalization pathways and will highlight the issues that still remain unanswered. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.
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| 13. |
- Frisan, Teresa, 1967-
(författare)
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Co- and polymicrobial infections in the gut mucosa : the host-microbiota-pathogen perspective
- 2021
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Ingår i: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 23:2
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Forskningsöversikt (refereegranskat)abstract
- Infections in humans occur in the context of complex niches where the pathogen interacts with both the host microenvironment and immune response, and the symbiotic microbial community. The polymicrobial nature of many human infections adds a further layer of complexity. The effect of co- or polymicrobial infections can result in enhanced severity due to pathogens cooperative interaction or reduced morbidity because one of the pathogens affects the fitness of the other(s). In this review, the concept of co-infections and polymicrobial interactions in the context of the intestinal mucosa is discussed, focusing on the interplay between the host, the microbiota and the pathogenic organisms. Specifically, we will examine examples of pathogen-cooperative versus -antagonistic behaviour during co- and polymicrobial infections. We discuss: the infection-induced modulation of the host microenvironment and immune responses; the direct modulation of the microorganism's fitness; the potentiation of inflammatory/carcinogenic conditions by polymicrobial biofilms; and the promotion of co-infections by microbial-induced DNA damage. Open questions in this very exciting field are also highlighted.
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| 14. |
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| 15. |
- Frisan, Teresa, 1967-, et al.
(författare)
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Ubiquitin C-terminal hydrolase-L1 interacts with adhesion complexes and promotes cell migration, survival, and anchorage independent growth
- 2012
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Ingår i: The FASEB Journal. - : The Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 26:12, s. 5060-5070
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Tidskriftsartikel (refereegranskat)abstract
- Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, β-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.
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| 16. |
- Gilmore, Michael C., 1995-
(författare)
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Studies on cell wall recycling and modification in Gram-negative bacteria
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The bacterial cell wall is made from peptidoglycan (PG), a heteropolymer which forms a bag-like exoskeleton that envelopes the cell. PG is constantly remodelled during growth and division, and in response to environmental stimuli. Decades of study of this process have focused largely on a select few model organisms, leaving its diversity poorly understood. In this thesis, I present studies on different aspects of PG recycling and modification in several Gram-negative models, with a particular focus on the plant pathogen Agrobacterium tumefaciens, a model of the Hyphomicrobiales group of the Alphaproteobacteria which includes several species of medical and environmental interest. It is shown that A. tumefaciens encodes a novel PG transporter, which is vital for cell wall integrity and resistance to β- lactam antibiotics, and widely conserved in the Hyphomicrobiales and Rhodobacterales orders. Growth defects caused by the loss of the transporter are suppressed by mutations in a novel glycopolymer, which is hypothesized to play a role in sequestering metal ions and thereby lowering periplasmic oxidative stress. Next, in collaboration, it is shown that PG recycling in the best studied model, Escherichia coli, is more complicated than previously thought. Rather than depending mostly on the MFS-family transporter AmpG, E. coli uses an ABC transporter, MppA-OppBCDF or AmpG depending on the growth phase and conditions. Finally, two studies on modification of PG by deacetylation are presented. First, A. tumefaciens is shown to encode a novel anhydroMurNAc deacetylase, which specifically deacetylates the PG chain termini. Then, it is shown that the causative agent of Legionnaires’ disease, Legionella pneumophila, depends on deacetylation of its PG during infection for defence against host lysozyme and correct polar placement of its type IV secretion system.
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| 17. |
- Grasso, Francesca, et al.
(författare)
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Bacterial Genotoxins : Merging the DNA Damage Response into Infection Biology
- 2015
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Ingår i: Biomolecules. - : MDPI. - 2218-273X. ; 5:3, s. 1762-1782
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Forskningsöversikt (refereegranskat)abstract
- Bacterial genotoxins are unique among bacterial toxins as their molecular target is DNA. The consequence of intoxication or infection is induction of DNA breaks that, if not properly repaired, results in irreversible cell cycle arrest (senescence) or death of the target cells. At present, only three bacterial genotoxins have been identified. Two are protein toxins: the cytolethal distending toxin (CDT) family produced by a number of Gram-negative bacteria and the typhoid toxin produced by Salmonella enterica serovar Typhi. The third member, colibactin, is a peptide-polyketide genotoxin, produced by strains belonging to the phylogenetic group B2 of Escherichia coli. This review will present the cellular effects of acute and chronic intoxication or infection with the genotoxins-producing bacteria. The carcinogenic properties and the role of these effectors in the context of the host-microbe interaction will be discussed. We will further highlight the open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.
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| 18. |
- 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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| 19. |
- 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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| 20. |
- 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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| 21. |
- 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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| 22. |
- Guidi, R, et al.
(författare)
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Bacterial genotoxin functions as immune-modulator and promotes host survival
- 2016
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Ingår i: Microbial cell (Graz, Austria). - : Shared Science Publishers. - 2311-2638. ; 3:8, s. 355-357
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial genotoxins are effectors that cause DNA damage in target cells. Many aspects of the biology of these toxins have been characterised in vitro, such as structure, cellular internalisation pathways and effects on the target cells. However, little is known about their function in vivo. Salmonella enterica serovar Typhi (S. Typhi) is a Gram-negative, intracellular bacterium that causes typhoid fever, a debilitating disease infecting more than 20 million people every year. S. Typhiproduce a genotoxin named typhoid toxin (TT), but its role in the contest of host infection is poorly characterized. The major obstacle in addressing this issue is that S. Typhi is exclusively a human pathogen. To overcome this limitation, we have used as model bacterium S. Typhimurium, and engineered it to produce endogenous levels of an active and inactive typhoid toxin, hereby named as TT (or genotoxic) and cdtB (or control), respectively. To our surprise, infection with the genotoxin strain strongly suppressed intestinal inflammation, leading to a better survival of the host during the acute phase of infection, suggesting typhoid toxin may exert a protective role. The presence of a functional genotoxin was also associated with an increased frequency of asymptomatic carriers.
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| 23. |
- 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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| 24. |
- 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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| 25. |
- Humphreys, Daniel, et al.
(författare)
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Senescence and Host-Pathogen Interactions
- 2020
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Ingår i: Cells. - : MDPI. - 2073-4409. ; 9:7
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Forskningsöversikt (refereegranskat)abstract
- Damage to our genomes triggers cellular senescence characterised by stable cell cycle arrest and a pro-inflammatory secretome that prevents the unrestricted growth of cells with pathological potential. In this way, senescence can be considered a powerful innate defence against cancer and viral infection. However, damage accumulated during ageing increases the number of senescent cells and this contributes to the chronic inflammation and deregulation of the immune function, which increases susceptibility to infectious disease in ageing organisms. Bacterial and viral pathogens are masters of exploiting weak points to establish infection and cause devastating diseases. This review considers the emerging importance of senescence in the host-pathogen interaction: we discuss the pathogen exploitation of ageing cells and senescence as a novel hijack target of bacterial pathogens that deploys senescence-inducing toxins to promote infection. The persistent induction of senescence by pathogens, mediated directly through virulence determinants or indirectly through inflammation and chronic infection, also contributes to age-related pathologies such as cancer. This review highlights the dichotomous role of senescence in infection: an innate defence that is exploited by pathogens to cause disease.
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| 26. |
- Lagopati, Nefeli, et al.
(författare)
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Biological Effect of Silver-modified Nanostructured Titanium Dioxide in Cancer
- 2021
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Ingår i: Cancer Genomics & Proteomics. - : Anticancer Research USA Inc.. - 1109-6535 .- 1790-6245. ; 18:3, s. 425-439
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND/AIM: Nanomedicine is a promising scientific field that exploits the unique properties of innovative nanomaterials, providing alternative solutions in diagnostics, prevention and therapeutics. Titanium dioxide nanoparticles (TiO2 NPs) have a great spectrum of photocatalytic antibacterial and anticancer applications. The chemical modification of TiO2 optimizes its bioactive performance. The aim of this study was the development of silver modified NPs (Ag/TiO2 NPs) with anticancer potential.MATERIALS AND METHODS: Ag/TiO2 NPs were prepared through the sol-gel method, were fully characterized and were tested on cultured breast cancer epithelial cells (MCF-7 and MDA-MB-231). The MTT colorimetric assay was used to estimate cellular viability. Western blot analysis of protein expression along with a DNA-laddering assay were employed for apoptosis detection.RESULTS AND CONCLUSION: We show that photo-activated Ag/TiO2 NPs exhibited significant cytotoxicity on the highly malignant MDA-MB-231 cancer cells, inducing apoptosis, while MCF-7 cells that are characterized by low invasive properties were unaffected under the same conditions.
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| 27. |
- Levi, Laura, et al.
(författare)
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Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading
- 2015
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 10:4
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Tidskriftsartikel (refereegranskat)abstract
- Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin β1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.
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| 28. |
- Li, Jinlin, et al.
(författare)
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The Epstein-Barr virus deubiquitinating enzyme BPLF1 regulates the activity of topoisomerase II during productive infection
- 2021
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 17:9
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Tidskriftsartikel (refereegranskat)abstract
- Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 co-immunoprecipitated and deubiquitinated TOP2, and stabilized SUMOylated TOP2 trapped in cleavage complexes (TOP2cc), which halted the DNA damage response to TOP2-induced double strand DNA breaks and promoted cell survival. Induction of the productive virus cycle in epithelial and lymphoid cell line carrying recombinant EBV encoding the active enzyme was accompanied by TOP2 deubiquitination, accumulation of TOP2ccs and resistance to Etoposide toxicity. The protective effect of BPLF1 was dependent on the expression of tyrosyl-DNA phosphodiesterase 2 (TDP2) that releases DNA-trapped TOP2 and promotes error-free DNA repair. These findings highlight a previously unrecognized function of BPLF1 in supporting a non-proteolytic pathway for TOP2cc debulking that favors cell survival and virus production.
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| 29. |
- Lopez Chiloeches, Maria, et al.
(författare)
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Bacterial Toxins Are a Never-Ending Source of Surprises : From Natural Born Killers to Negotiators
- 2021
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Ingår i: Toxins. - : MDPI. - 2072-6651. ; 13:6
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Forskningsöversikt (refereegranskat)abstract
- The idea that bacterial toxins are not only killers but also execute more sophisticated roles during bacteria-host interactions by acting as negotiators has been highlighted in the past decades. Depending on the toxin, its cellular target and mode of action, the final regulatory outcome can be different. In this review, we have focused on two families of bacterial toxins: genotoxins and pore-forming toxins, which have different modes of action but share the ability to modulate the host's immune responses, independently of their capacity to directly kill immune cells. We have addressed their immuno-suppressive effects with the perspective that these may help bacteria to avoid clearance by the host's immune response and, concomitantly, limit detrimental immunopathology. These are optimal conditions for the establishment of a persistent infection, eventually promoting asymptomatic carriers. This immunomodulatory effect can be achieved with different strategies such as suppression of pro-inflammatory cytokines, re-polarization of the immune response from a pro-inflammatory to a tolerogenic state, and bacterial fitness modulation to favour tissue colonization while preventing bacteraemia. An imbalance in each of those effects can lead to disease due to either uncontrolled bacterial proliferation/invasion, immunopathology, or both.
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| 30. |
- Lopez Chiloeches, Maria, et al.
(författare)
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Characterization of macrophage infiltration and polarization by double fluorescence immunostaining in mouse colonic mucosa
- 2021
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Ingår i: STAR Protocols. - : Cell Press. - 2666-1667. ; 2:4
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Tidskriftsartikel (refereegranskat)abstract
- We recently characterized the association between DNA damage and immunoresponse in vivo in colonic mucosa of mice infected with a Salmonella Typhimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe how to assess the extent and features of infiltrating macrophages by double immunofluorescence. Total macrophage population was determined using an F4/80 antibody, whereas the specific M2-like population was assessed using a CD206 antibody. For complete details on the use and execution of this protocol, please refer to Martin et al. (2021).
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| 31. |
- Lopez Chiloeches, Maria, et al.
(författare)
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Genotoxin-producing Salmonella enterica induces tissue-specific types of DNA damage and DNA damage response outcomes
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
-
Tidskriftsartikel (refereegranskat)abstract
- Introduction: Typhoid toxin-expressing Salmonella enterica causes DNA damage in the intestinal mucosa in vivo, activating the DNA damage response (DDR) in the absence of inflammation. To understand whether the tissue microenvironment constrains the infection outcome, we compared the immune response and DDR patterns in the colon and liver of mice infected with a genotoxigenic strain or its isogenic control strain.Methods: In situ spatial transcriptomic and immunofluorescence have been used to assess DNA damage makers, activation of the DDR, innate immunity markers in a multiparametric analysis.Result: The presence of the typhoid toxin protected from colonic bacteria-induced inflammation, despite nuclear localization of p53, enhanced co-expression of type-I interferons (IfnbI) and the inflammasome sensor Aim2, both classic features of DNA-break-induced DDR activation. These effects were not observed in the livers of either infected group. Instead, in this tissue, the inflammatory response and DDR were associated with high oxidative stress-induced DNA damage.Conclusions: Our work highlights the relevance of the tissue microenvironment in enabling the typhoid toxin to suppress the host inflammatory response in vivo.
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| 32. |
- Martin, Oceane C. B., et al.
(författare)
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Bacterial Genotoxin-Induced DNA Damage and Modulation of the Host Immune Microenvironment
- 2020
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Ingår i: Toxins. - : MDPI. - 2072-6651. ; 12:2
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Forskningsöversikt (refereegranskat)abstract
- Bacterial genotoxins (BTGX) induce DNA damage, which results in senescence or apoptosis of the target cells if not properly repaired. Three BTGXs have been identified: the cytolethal distending toxin (CDT) family produced by several Gram-negative bacteria, the typhoid toxin produced by several Salmonella enterica serovars, and colibactin, a peptide-polyketide, produced mainly by the phylogenetic group B2 Escherichia coli. The cellular responses induced by BTGXs resemble those of well-characterized carcinogenic agents, and several lines of evidence indicate that bacteria carrying genotoxin genes can contribute to tumor development under specific circumstances. Given their unusual mode of action, it is still enigmatic why these effectors have been acquired by microbes and what is their role in the context of the biology of the producing bacterium, since it is unlikely that their primary purpose is to induce/promote cancer in the mammalian host. In this review, we will discuss the possibility that the DNA damage induced by BTGX modulates the host immune response, acting as immunomodulator, leading to the establishment of a suitable niche for the producing bacterium. We will further highlight open questions that remain to be solved regarding the biology of this unusual family of bacterial toxins.
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| 33. |
- Martin, Oceane C. B., et al.
(författare)
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Infection with genotoxin-producing Salmonella enterica synergises with loss of the tumour suppressor APC in promoting genomic instability via the PI3K pathway in colonic epithelial cells
- 2019
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Ingår i: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 21:12
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Tidskriftsartikel (refereegranskat)abstract
- Several commensal and pathogenic Gram-negative bacteria produce DNA-damaging toxins that are considered bona fide carcinogenic agents. The microbiota of colorectal cancer (CRC) patients is enriched in genotoxin-producing bacteria, but their role in the pathogenesis of CRC is poorly understood. The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in the majority of sporadic CRCs. We investigated whether the loss of APC alters the response of colonic epithelial cells to infection by Salmonella enterica, the only genotoxin-producing bacterium associated with cancer in humans. Using 2D and organotypic 3D cultures, we found that APC deficiency was associated with sustained activation of the DNA damage response, reduced capacity to repair different types of damage, including DNA breaks and oxidative damage, and failure to induce cell cycle arrest. The reduced DNA repair capacity and inability to activate adequate checkpoint responses was associated with increased genomic instability in APC-deficient cells exposed to the genotoxic bacterium. Inhibition of the checkpoint response was dependent on activation of the phosphatidylinositol 3-kinase pathway. These findings highlight the synergistic effect of the loss of APC and infection with genotoxin-producing bacteria in promoting a microenvironment conducive to malignant transformation.
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| 34. |
- Mathiasen, Sarah L., et al.
(författare)
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Bacterial genotoxins induce T cell senescence
- 2021
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Ingår i: Cell Reports. - : Elsevier. - 2211-1247. ; 35:10
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Tidskriftsartikel (refereegranskat)abstract
- Several types of pathogenic bacteria produce genotoxins that induce DNA damage in host cells. Accumulating evidence suggests that a central function of these genotoxins is to dysregulate the host's immune response, but the underlying mechanisms remain unclear. To address this issue, we investigated the effects of the most widely expressed bacterial genotoxin, the cytolethal distending toxin (CDT), on T cells—the key mediators of adaptive immunity. We show that CDT induces premature senescence in activated CD4 T cells in vitro and provide evidence suggesting that infection with genotoxin-producing bacteria promotes T cell senescence in vivo. Moreover, we demonstrate that genotoxin-induced senescent CD4 T cells assume a senescence-associated secretory phenotype (SASP) which, at least partly, is orchestrated by the ATM-p38 signaling axis. These findings provide insight into the immunomodulatory properties of bacterial genotoxins and uncover a putative link between bacterial infections and T cell senescence.
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| 35. |
- Papalampros, Alexandros, et al.
(författare)
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Unique Spatial Immune Profiling in Pancreatic Ductal Adenocarcinoma with Enrichment of Exhausted and Senescent T Cells and Diffused CD47-SIRP proportional to Expression
- 2020
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 12:7
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Pancreatic ductal adenocarcinoma (PDAC) is resistant to single-agent immunotherapies. To understand the mechanisms leading to the poor response to this treatment, a better understanding of the PDAC immune landscape is required. The present work aims to study the immune profile in PDAC in relationship to spatial heterogeneity of the tissue microenvironment (TME) in intact tissues. Methods: Serial section and multiplex in situ analysis were performed in 42 PDAC samples to assess gene and protein expression at single-cell resolution in the: (a) tumor center (TC), (b) invasive front (IF), (c) normal parenchyma adjacent to the tumor, and (d) tumor positive and negative draining lymph nodes (LNs). Results: We observed: (a) enrichment of T cell subpopulations with exhausted and senescent phenotype in the TC, IF and tumor positive LNs; (b) a dominant type 2 immune response in the TME, which is more pronounced in the TC; (c) an emerging role of CD47-SIRP a axis; and (d) a similar immune cell topography independently of the neoadjuvant chemotherapy. Conclusion: This study reveals the existence of dysfunctional T lymphocytes with specific spatial distribution, thus opening a new dimension both conceptually and mechanistically in tumor-stroma interaction in PDAC with potential impact on the efficacy of immune-regulatory therapeutic modalities.
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| 36. |
- Pateras, Ioannis S., et al.
(författare)
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Diagnostic challenges during inflammation and cancer : current biomarkers and future perspectives in navigating through the minefield of reactive versus dysplastic and cancerous lesions in the digestive system
- 2024
-
Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 25:2
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Forskningsöversikt (refereegranskat)abstract
- In the setting of pronounced inflammation, changes in the epithelium may overlap with neoplasia, often rendering it impossible to establish a diagnosis with certainty in daily clinical practice. Here, we discuss the underlying molecular mechanisms driving tissue response during persistent inflammatory signaling along with the potential association with cancer in the gastrointestinal tract, pancreas, extrahepatic bile ducts, and liver. We highlight the histopathological challenges encountered in the diagnosis of chronic inflammation in routine practice and pinpoint tissue-based biomarkers that could complement morphology to differentiate reactive from dysplastic or cancerous lesions. We refer to the advantages and limitations of existing biomarkers employing immunohistochemistry and point to promising new markers, including the generation of novel antibodies targeting mutant proteins, miRNAs, and array assays. Advancements in experimental models, including mouse and 3D models, have improved our understanding of tissue response. The integration of digital pathology along with artificial intelligence may also complement routine visual inspections. Navigating through tissue responses in various chronic inflammatory contexts will help us develop novel and reliable biomarkers that will improve diagnostic decisions and ultimately patient treatment.
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| 37. |
- Pateras, Ioannis S., et al.
(författare)
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Short term starvation potentiates the efficacy of chemotherapy in triple negative breast cancer via metabolic reprogramming
- 2023
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Ingår i: Journal of Translational Medicine. - : BioMed Central (BMC). - 1479-5876. ; 21:1
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Chemotherapy (CT) is central to the treatment of triple negative breast cancer (TNBC), but drug toxicity and resistance place strong restrictions on treatment regimes. Fasting sensitizes cancer cells to a range of chemotherapeutic agents and also ameliorates CT-associated adverse effects. However, the molecular mechanism(s) by which fasting, or short-term starvation (STS), improves the efficacy of CT is poorly characterized.Methods: The differential responses of breast cancer or near normal cell lines to combined STS and CT were assessed by cellular viability and integrity assays (Hoechst and PI staining, MTT or H2DCFDA staining, immunofluorescence), metabolic profiling (Seahorse analysis, metabolomics), gene expression (quantitative real-time PCR) and iRNA-mediated silencing. The clinical significance of the in vitro data was evaluated by bioinformatical integration of transcriptomic data from patient data bases: The Cancer Genome Atlas (TCGA), European Genome-phenome Archive (EGA), Gene Expression Omnibus (GEO) and a TNBC cohort. We further examined the translatability of our findings in vivo by establishing a murine syngeneic orthotopic mammary tumor-bearing model.Results: We provide mechanistic insights into how preconditioning with STS enhances the susceptibility of breast cancer cells to CT. We showed that combined STS and CT enhanced cell death and increased reactive oxygen species (ROS) levels, in association with higher levels of DNA damage and decreased mRNA levels for the NRF2 targets genes NQO1 and TXNRD1 in TNBC cells compared to near normal cells. ROS enhancement was associated with compromised mitochondrial respiration and changes in the metabolic profile, which have a significant clinical prognostic and predictive value. Furthermore, we validate the safety and efficacy of combined periodic hypocaloric diet and CT in a TNBC mouse model.Conclusions: Our in vitro, in vivo and clinical findings provide a robust rationale for clinical trials on the therapeutic benefit of short-term caloric restriction as an adjuvant to CT in triple breast cancer treatment.
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| 38. |
- Pons, Benoît J, et al.
(författare)
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Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not
- 2019
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Ingår i: PLOS ONE. - : Public Library Science. - 1932-6203. ; 14:3
-
Tidskriftsartikel (refereegranskat)abstract
- The Cytolethal Distending Toxin (CDT) is produced by many pathogenic bacteria. CDT is known to induce genomic DNA damage to host eukaryotic cells through its catalytic subunit, CdtB. CdtB is structurally homologous to DNase I and has a nuclease activity, dependent on several key residues. Yet some differences between various CdtB subunit activities, and discrepancies between biochemical and cellular data, have been observed. To better characterise the role of CdtB in the induction of DNA damage, we affinity-purified wild-type and mutants of CdtB, issued from E. coli and H. ducreyi, under native and denaturing conditions. We then compared their nuclease activity by a classic in vitro assay using plasmid DNA, and two different eukaryotic assays-the first assay where host cells were transfected with a plasmid encoding CdtB, the second assay where host cells were directly transfected with purified CdtB. We show here that in vitro nuclease activities are difficult to quantify, whereas CdtB activities in host cells can be easily interpreted and confirmed the loss of function of the catalytic mutant. Our results highlight the importance of performing multiple assays while studying the effects of bacterial genotoxins, and indicate that the classic in vitro assay should be complemented with cellular assays.
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| 39. |
- Robb Huhn, G., et al.
(författare)
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Endocytosis of the CdtA subunit from the Haemophilus ducreyi cytolethal distending toxin
- 2021
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Ingår i: Cellular Microbiology. - : John Wiley & Sons. - 1462-5814 .- 1462-5822. ; 23:11
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Tidskriftsartikel (refereegranskat)abstract
- Many Gram-negative pathogens produce a cytolethal distending toxin (CDT) with two cell-binding subunits (CdtA + CdtC) and a catalytic CdtB subunit. After adhesion to the plasma membrane of a target cell, CDT moves by retrograde transport to endoplasmic reticulum. CdtB then enters the nucleus where it generates DNA breaks that lead to cell cycle arrest and apoptosis or senescence. CdtA anchors the CDT holotoxin to the plasma membrane and is thought to remain on the cell surface after endocytosis of the CdtB/CdtC heterodimer. Here, we re-examined the potential endocytosis and intracellular transport of CdtA from the Haemophilus ducreyi CDT. We recorded the endocytosis of holotoxin-associated CdtA with a cell-based enzyme-linked immunoabsorbent assay (CELISA) and visualised its presence in the early endosomes by confocal microscopy 10 min after CDT binding to the cell surface. Western blot analysis documented the rapid degradation of internalised CdtA. Most of internalised CdtB and CdtC were degraded as well. The rapid rate of CDT internalisation and turnover, which could explain why CdtA endocytosis was not detected in previous studies, suggests only a minor pool of cell-associated CdtB reaches the nucleus. Our work demonstrates that CDT is internalised as an intact holotoxin and identifies the endosomes as the site of CdtA dissociation from CdtB/CdtC.Take Aways:During the endocytosis of CDT, CdtA is thought to remain at the cell surface.A cell-based ELISA documented the rapid endocytosis of CdtA.CdtA was visualised in the early endosomes by confocal microscopy.Intracellular CdtA was rapidly degraded, along with most of CdtB and CdtC.
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| 40. |
- Rolén, Ulrika, et al.
(författare)
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The ubiquitin C-terminal hydrolase UCH-L1 regulates B-cell proliferation and integrin activation
- 2009
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Ingår i: Journal of Cellular and Molecular Medicine (Print). - : John Wiley & Sons. - 1582-1838 .- 1582-4934. ; 13:8b, s. 1666-1678
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme that catalyses the hydrolysis of polyubiquitin precursors and small ubiquitin adducts. UCH-L1 has been detected in a variety of malignant and metastatic tumours but its biological function in these cells is unknown. We have previously shown that UCH-L1 is highly expressed in Burkitt's lymphoma (BL) and is up-regulated upon infection of B lymphocytes with Epstein-Barr virus (EBV). Here we show that knockdown of UCH-L1 by RNAi inhibits the proliferation of BL cells in suspension and semisolid agar and activates strong LFA-1-dependent homotypic adhesion. Induction of cell adhesion correlated with cation-induced binding to ICAM-1, clustering of LFA-1 into lipid rafts and constitutive activation of the Rap1 and Rac1 GTPases. Expression of a catalytically active UCH-L1 promoted the proliferation of a UCH-L1-negative EBV transformed lymphoblastoid cell line (LCL) and inhibited cell adhesion, whereas a catalytic mutant had no effect, confirming the requirement of UCH-L1 enzymatic activity for the regulation of these phenotypes. Our results identify UCH-L1 as a new player in the signalling pathways that promote the proliferation and invasive capacity of malignant B cells.
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| 41. |
- Saccon, Elisa, et al.
(författare)
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Cell-type-resolved quantitative proteomics map of interferon response against SARS-CoV-2
- 2021
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 24:5
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Tidskriftsartikel (refereegranskat)abstract
- The commonly used laboratory cell lines are the first line of experimental models to study the pathogenicity and performing antiviral assays for emerging viruses. Here, we assessed the tropism and cytopathogenicity of the first Swedish isolate of SARS-CoV-2 in six different human cell lines, compared their growth characteristics, and performed quantitative proteomics for the susceptible cell lines. Overall, Calu-3, Caco2, Huh7, and 293FT cell lines showed a high-to-moderate level of susceptibility to SARS-CoV-2. In Caco2 cells, the virus can achieve high titers in the absence of any prominent cytopathic effect. The protein abundance profile during SARS-CoV-2 infection revealed cell-type-specific regulation of cellular pathways. Type-I interferon signaling was identified as the common dysregulated cellular response in Caco2, Calu-3, and Huh7 cells. Together, our data show cell-type specific variability for cytopathogenicity, susceptibility, and cellular response to SARS-CoV-2 and provide important clues to guide future studies.
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| 42. |
- Seiwert, Nina, et al.
(författare)
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AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells
- 2017
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Ingår i: Cell Death and Disease. - : Nature Publishing Group. - 2041-4889. ; 8:8
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Tidskriftsartikel (refereegranskat)abstract
- DNA double-strand breaks (DSBs) are critical DNA lesions, which threaten genome stability and cell survival. DSBs are directly induced by ionizing radiation (IR) and radiomimetic agents, including the cytolethal distending toxin (CDT). This bacterial genotoxin harbors a unique DNase-I-like endonuclease activity. Here we studied the role of DSBs induced by CDT and IR as a trigger of autophagy, which is a cellular degradation process involved in cell homeostasis, genome protection and cancer. The regulatory mechanisms of DSB-induced autophagy were analyzed, focusing on the ATM-p53-mediated DNA damage response and AKT signaling in colorectal cancer cells. We show that treatment of cells with CDT or IR increased the levels of the autophagy marker LC3B-II. Consistently, an enhanced formation of autophagosomes and a decrease of the autophagy substrate p62 were observed. Both CDT and IR concomitantly suppressed mTOR signaling and stimulated the autophagic flux. DSBs were demonstrated as the primary trigger of autophagy using a DNase I-defective CDT mutant, which neither induced DSBs nor autophagy. Genetic abrogation of p53 and inhibition of ATM signaling impaired the autophagic flux as revealed by LC3B-II accumulation and reduced formation of autophagic vesicles. Blocking of DSB-induced apoptotic cell death by the pan-caspase inhibitor Z-VAD stimulated autophagy. In line with this, pharmacological inhibition of autophagy increased cell death, while ATG5 knockdown did not affect cell death after DSB induction. Interestingly, both IR and CDT caused AKT activation, which repressed DSB-triggered autophagy independent of the cellular DNA-PK status. Further knockdown and pharmacological inhibitor experiments provided evidence that the negative autophagy regulation was largely attributable to AKT2. Finally, we show that upregulation of CDT-induced autophagy upon AKT inhibition resulted in lower apoptosis and increased cell viability. Collectively, the findings demonstrate that DSBs trigger pro-survival autophagy in an ATM- and p53-dependent manner, which is curtailed by AKT2 signaling.
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- Toh, Eric, 1988-
(författare)
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Roles of secreted bacterial factors in modulation of host cell signalling
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Pathogenic bacteria employ several secretion systems to release or inject virulence factors that may alter host cell processes, generate a replicative niche, and aid bacterial survival in adverse environments. This thesis presents my investigations on how bacterial factors can modulate host cell signalling mechanisms. We investigated possible signalling pathways involved in targets of the Vibrio cholerae protein MakA that was found to mediate inhibition of tumour cell proliferation. Caenorhabditis elegans grazing on MakA-producing bacteria revealed that MakA may affect lipid-mediated signalling in the nematodes by affecting the level of PPK-1, a homologue of eukaryotic PIP5K1α. We studied the possible effects of MakA on eukaryotic PIP5K1α in human colon cancer cell lines and found decreased levels of PIP5K1α and pAkt in the lipid-signalling pathway. Immunoblot analyses demonstrated that MakA inhibited cyclin-dependent kinase 1 and increased p27 expression in the colon cancer cells, resulting in G2/M cell cycle arrest. MakA also caused downregulation of Ki67 and cyclin D1, limiting cancer cell proliferation. MakA is the first reported bacterial protein targeting the PIP5K1α lipid signalling pathway, thereby displaying anti-cancer capabilities. We discovered that phosphatidic acid (PA)-mediated MakA binding to host cell plasma membranes generated endomembrane-rich aggregates that caused host target cell autophagy and cytotoxicity. PA binding and cell toxicity by MakA required its N-terminal domain. The MakA genetic determinant is located within a novel pathogenicity island that also encodes the MakB, MakC, MakD, and MakE proteins. In most V. cholerae and Vibrio anguillarum genomes, mak genes form an operon, makCDBAE. The immunoblot analyses showed that wild-type V. cholerae A1552 released the MakA, MakB, and MakE proteins via the flagellum, while a flagellum-deficient mutant released very little or none. Structurally, MakA, MakB, and MakE belong to a superfamily of bacterial alpha-pore-forming toxins. Identification and structural analysis of V. cholerae Mak proteins revealed that the MakA/B/E toxin is common to several pathogenic Vibrionaceae strains, and this previously unrecognised tripartite toxin may increase their fitness and pathogenicity in various environments and host organisms. Bacteria release spherical lipid nanostructures, extracellular membrane vesicles, that may play many biological roles. Previously, Escherichia coli was shown to release physiologically active cytolysin A (ClyA) via outer membrane vesicles (OMVs). ClyA, the first recognised member of the bacterial alpha-pore-forming proteins, has become a model for how oligomerization and pore formation occur in membranes. The clyA gene is cryptic in commensal non-pathogenic E. coli bacteria displaying no cytolytic activity. We found that the sublytic concentration of ClyA released via OMVs by non-pathogenic E. coli profoundly affected host cells. The ClyA+ OMVs were rapidly internalised into colon cancer cells by macropinocytosis and clathrin-mediated, dynamin-dependent endocytosis. The OMV-associated ClyA caused reduced levels of cancer-activating proteins like EZH2, H3K27me3, CXCR4, STAT3, and MDM2 via the EZH2/H3K27me3/miR622/CXCR4 signalling axis. Evidently, sublytic levels of ClyA in OMVs from non-pathogenic E. coli can modulate epigenetics by targeting EZH2 protein stability and we hypothesised that E. coli in colorectal cancer microbiomes may preferentially lack this protein. Given our current understanding of ClyA interactions in cancer cell signalling, it will be intriguing to determine if and how the status of the clyA locus is involved in the aetiology of colorectal cancer.
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