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Frisan, T., Nagy, N., Chioureas, D., Terol, M., Grasso, F. & Masucci, M. G. (2019). 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. International Journal of Cancer, 144(1), 98-109
Open this publication in new window or tab >>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
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2019 (English)In: International Journal of Cancer, ISSN 0020-7136, E-ISSN 1097-0215, Vol. 144, no 1, p. 98-109Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
DNA damage, Epstein-Barr virus, Gram-negative bacteria, cytolethal distending toxin, virus reactivation
National Category
Cell and Molecular Biology Microbiology in the medical area
Research subject
Microbiology; Infectious Diseases
Identifiers
urn:nbn:se:umu:diva-154014 (URN)10.1002/ijc.31652 (DOI)000451479900010 ()29978480 (PubMedID)
Available from: 2018-12-11 Created: 2018-12-11 Last updated: 2018-12-12Bibliographically approved
Pons, B. J., Bezine, E., Hanique, M., Guillet, V., Mourey, L., Chicher, J., . . . Mirey, G. (2019). Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not. PLoS ONE, 14(3), Article ID e0214313.
Open this publication in new window or tab >>Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not
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2019 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 3, article id e0214313Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library Science, 2019
National Category
Cancer and Oncology Immunology Microbiology Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-165070 (URN)10.1371/journal.pone.0214313 (DOI)000462594000053 ()30921382 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Martin, O. C. B., Bergonzini, A., D'Amico, F., Chen, P., Shay, J. W., Dupuy, J., . . . Frisan, T. (2019). 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. Cellular Microbiology, Article ID e13099.
Open this publication in new window or tab >>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
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2019 (English)In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, article id e13099Article in journal (Refereed) Epub ahead of print
Abstract [en]

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.

Place, publisher, year, edition, pages
WILEY, 2019
Keywords
APC, bacteria and cancer, bacterial genotoxin, DNA damage response, DNA repair, organotypic del, tumour-suppressor gene
National Category
Microbiology
Identifiers
urn:nbn:se:umu:diva-163202 (URN)10.1111/cmi.13099 (DOI)000482652700001 ()31414579 (PubMedID)
Available from: 2019-09-13 Created: 2019-09-13 Last updated: 2019-09-13
Seiwert, N., Neitzel, C., Stroh, S., Frisan, T., Audebert, M., Toulany, M., . . . Fahrer, J. (2017). AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells. Cell Death and Disease, 8(8), Article ID e3019.
Open this publication in new window or tab >>AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells
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2017 (English)In: Cell Death and Disease, ISSN 2041-4889, E-ISSN 2041-4889, Vol. 8, no 8, article id e3019Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Infectious Medicine Cancer and Oncology Cell Biology Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-165068 (URN)10.1038/cddis.2017.418 (DOI)000409550500057 ()28837154 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Guidi, R., Belluz, L. D. & Frisan, T. (2016). Bacterial genotoxin functions as immune-modulator and promotes host survival. Microbial cell (Graz, Austria), 3(8), 355-357
Open this publication in new window or tab >>Bacterial genotoxin functions as immune-modulator and promotes host survival
2016 (English)In: Microbial cell (Graz, Austria), ISSN 2311-2638, Vol. 3, no 8, p. 355-357Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Shared Science Publishers, 2016
Keywords
genotoxin, Salmonella, typhoid toxin, DNA damage, inflammation, persistent infection
National Category
Cell and Molecular Biology Infectious Medicine Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-165071 (URN)10.15698/mic2016.08.520 (DOI)000384679300005 ()28357372 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Frisan, T. (2016). Bacterial genotoxins: the long journey to the nucleus of mammalian cells. Biochimica et Biophysica Acta, 1858(3), 567-575, Article ID S0005-2736(15)00267-9.
Open this publication in new window or tab >>Bacterial genotoxins: the long journey to the nucleus of mammalian cells
2016 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1858, no 3, p. 567-575, article id S0005-2736(15)00267-9Article, review/survey (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier, 2016
Keywords
Bacterial genotoxins, Cytolethal distending toxin, Endoplasmic reticulum, Intracellular trafficking, Lipid rafts, Nucleus, Retrograde transport, Toxin receptor, Trans Golgi network, Typhoid toxin
National Category
Cell and Molecular Biology Infectious Medicine Immunology Microbiology
Identifiers
urn:nbn:se:umu:diva-165074 (URN)10.1016/j.bbamem.2015.08.016 (DOI)000370307700012 ()26299818 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Frisan, T. & Sebo, P. (2016). Editorial: Why still study bacterial toxins in the third millennium?. Pathogens and Disease, 74(3), Article ID ftw009.
Open this publication in new window or tab >>Editorial: Why still study bacterial toxins in the third millennium?
2016 (English)In: Pathogens and Disease, E-ISSN 2049-632X, Vol. 74, no 3, article id ftw009Article in journal, Editorial material (Refereed) Published
Place, publisher, year, edition, pages
Oxford University Press, 2016
National Category
Cell and Molecular Biology Infectious Medicine Cancer and Oncology Microbiology Immunology
Identifiers
urn:nbn:se:umu:diva-165073 (URN)10.1093/femspd/ftw009 (DOI)000374475700013 ()26818621 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Del Bel Belluz, L., Guidi, R., Pateras, I. S., Levi, L., Mihaljevic, B., Rouf, S. F., . . . Frisan, T. (2016). The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection. PLoS Pathogens, 12(4), Article ID e1005528.
Open this publication in new window or tab >>The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection
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2016 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 12, no 4, article id e1005528Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library Science, 2016
National Category
Cell and Molecular Biology Infectious Medicine Cancer and Oncology Immunology Microbiology
Identifiers
urn:nbn:se:umu:diva-165072 (URN)10.1371/journal.ppat.1005528 (DOI)000378156900023 ()27055274 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Grasso, F. & Frisan, T. (2015). Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology. Biomolecules, 5(3), 1762-1782
Open this publication in new window or tab >>Bacterial Genotoxins: Merging the DNA Damage Response into Infection Biology
2015 (English)In: Biomolecules, E-ISSN 2218-273X, Vol. 5, no 3, p. 1762-1782Article, review/survey (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
MDPI, 2015
Keywords
DNA damage response, bacterial genotoxins, cancer, chronic infection, probiotics
National Category
Cell and Molecular Biology Cancer and Oncology Infectious Medicine Immunology Microbiology
Identifiers
urn:nbn:se:umu:diva-165079 (URN)10.3390/biom5031762 (DOI)000362504200028 ()26270677 (PubMedID)
Available from: 2019-11-08 Created: 2019-11-08 Last updated: 2019-11-08Bibliographically approved
Levi, L., Toyooka, T., Patarroyo, M. & Frisan, T. (2015). Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading. PLoS ONE, 10(4), Article ID e0124119.
Open this publication in new window or tab >>Bacterial genotoxins promote inside-out integrin β1 activation, formation of focal adhesion complexes and cell spreading
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 4, article id e0124119Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Public Library Science, 2015
National Category
Cell Biology Immunology Microbiology Cell and Molecular Biology Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-165080 (URN)10.1371/journal.pone.0124119 (DOI)000352845100240 ()25874996 (PubMedID)
Available from: 2019-11-12 Created: 2019-11-12 Last updated: 2019-11-12Bibliographically approved
Projects
Bacterial genotoxins: an interface between infection and cancer biology [2015-02896_VR]; Umeå UniversityPathophysiology of genotoxin producing bacteria in inflammation and cancer [2018-02521_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-1209-0942

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