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Dr. Benoît PONS
University of Exeter

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0 Biochemistry
0 genotoxicity
0 In Vitro Assays
0 Phage ecology
0 Protein biochemistry

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Journal article
Published: 19 August 2020 in Toxins
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The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.

ACS Style

Benoît J. Pons; Nicolas Loiseau; Saleha Hashim; Soraya Tadrist; Gladys Mirey; Julien Vignard. Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B. Toxins 2020, 12, 530 .

AMA Style

Benoît J. Pons, Nicolas Loiseau, Saleha Hashim, Soraya Tadrist, Gladys Mirey, Julien Vignard. Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B. Toxins. 2020; 12 (9):530.

Chicago/Turabian Style

Benoît J. Pons; Nicolas Loiseau; Saleha Hashim; Soraya Tadrist; Gladys Mirey; Julien Vignard. 2020. "Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B." Toxins 12, no. 9: 530.

Preprint content
Published: 12 June 2020 in bioRxiv
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The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin that activates the DNA damage response and induces inflammatory signatures in host cells, but the precise relationship between these outcomes has not been addressed so far. CDT induces a singular time-dependent increase of DNA damage and cell cycle defects, questioning on possible impaired response to this toxin over the cell cycle. Here, we identify mitosis as a crucial phase during CDT intoxination. Despite active cell cycle checkpoints and in contrast to other DNA damaging agents, CDT-exposed cells reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation. These micronuclei are recognized by cGAS that elicits an inflammatory signature resulting in cell distention and senescence. Our results unravel for the first time the mitotic consequences of CDT genotoxic activity and relate them to pro-inflammatory cellular response. These findings may have important implications during bacterial infection regarding CDT-mediated immunomodulatory and tumorigenic processes.

ACS Style

Benoît Pons; Aurélie Pettes-Duler; Claire Naylies; Frédéric Taieb; Saleha Hashim; Soraya Tadrist; Yannick Lippi; Gladys Mirey; Julien Vignard. Cytolethal Distending Toxin: from mitotic DNA damage to cGAS-dependent pro-inflammatory response. bioRxiv 2020, 1 .

AMA Style

Benoît Pons, Aurélie Pettes-Duler, Claire Naylies, Frédéric Taieb, Saleha Hashim, Soraya Tadrist, Yannick Lippi, Gladys Mirey, Julien Vignard. Cytolethal Distending Toxin: from mitotic DNA damage to cGAS-dependent pro-inflammatory response. bioRxiv. 2020; ():1.

Chicago/Turabian Style

Benoît Pons; Aurélie Pettes-Duler; Claire Naylies; Frédéric Taieb; Saleha Hashim; Soraya Tadrist; Yannick Lippi; Gladys Mirey; Julien Vignard. 2020. "Cytolethal Distending Toxin: from mitotic DNA damage to cGAS-dependent pro-inflammatory response." bioRxiv , no. : 1.

Review
Published: 12 October 2019 in Toxins
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The Cytolethal Distending Toxin (CDT) is a bacterial virulence factor produced by several Gram-negative pathogenic bacteria. These bacteria, found in distinct niches, cause diverse infectious diseases and produce CDTs differing in sequence and structure. CDTs have been involved in the pathogenicity of the associated bacteria by promoting persistent infection. At the host-cell level, CDTs cause cell distension, cell cycle block and DNA damage, eventually leading to cell death. All these effects are attributable to the catalytic CdtB subunit, but its exact mode of action is only beginning to be unraveled. Sequence and 3D structure analyses revealed similarities with better characterized proteins, such as nucleases or phosphatases, and it has been hypothesized that CdtB exerts a biochemical activity close to those enzymes. Here, we review the relationships that have been established between CdtB structure and function, particularly by mutation experiments on predicted key residues in different experimental systems. We discuss the relevance of these approaches and underline the importance of further study in the molecular mechanisms of CDT toxicity, particularly in the context of different pathological conditions.

ACS Style

Benoît Pons; Julien Vignard; Gladys Mirey. Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship. Toxins 2019, 11, 595 .

AMA Style

Benoît Pons, Julien Vignard, Gladys Mirey. Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship. Toxins. 2019; 11 (10):595.

Chicago/Turabian Style

Benoît Pons; Julien Vignard; Gladys Mirey. 2019. "Cytolethal Distending Toxin Subunit B: A Review of Structure–Function Relationship." Toxins 11, no. 10: 595.

Research article
Published: 28 March 2019 in PLoS ONE
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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.

ACS Style

Benoît J. Pons; Elisabeth Bezine; Mélissa Hanique; Valérie Guillet; Lionel Mourey; Johana Chicher; Teresa Frisan; Julien Vignard; Gladys Mirey. Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not. PLoS ONE 2019, 14, e0214313 .

AMA Style

Benoît J. Pons, Elisabeth Bezine, Mélissa Hanique, Valérie Guillet, Lionel Mourey, Johana Chicher, Teresa Frisan, Julien Vignard, Gladys Mirey. Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not. PLoS ONE. 2019; 14 (3):e0214313.

Chicago/Turabian Style

Benoît J. Pons; Elisabeth Bezine; Mélissa Hanique; Valérie Guillet; Lionel Mourey; Johana Chicher; Teresa Frisan; Julien Vignard; Gladys Mirey. 2019. "Cell transfection of purified cytolethal distending toxin B subunits allows comparing their nuclease activity while plasmid degradation assay does not." PLoS ONE 14, no. 3: e0214313.

Gi cancer
Published: 30 October 2018 in Gut
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ObjectiveCampylobacter jejuni produces a genotoxin, cytolethal distending toxin (CDT), which has DNAse activity and causes DNA double-strand breaks. Although C. jejuni infection has been shown to promote intestinal inflammation, the impact of this bacterium on carcinogenesis has never been examined.DesignGerm-free (GF) ApcMin/+mice, fed with 1% dextran sulfate sodium, were used to test tumorigenesis potential of CDT-producing C. jejuni. Cells and enteroids were exposed to bacterial lysates to determine DNA damage capacity via γH2AX immunofluorescence, comet assay and cell cycle assay. To examine the interplay of CDT-producing C. jejuni, gut microbiome and host in tumorigenesis, colonic RNA-sequencing and faecal 16S rDNA sequencing were performed. Rapamycin was administrated to investigate the prevention of CDT-producing C. jejuni-induced tumorigenesis.ResultsGF ApcMin/+mice colonised with human clinical isolate C. jejuni81–176 developed significantly more and larger tumours when compared with uninfected mice. C. jejuni with a mutated cdtB subunit, mutcdtB, attenuated C. jejuni-induced tumorigenesis in vivo and decreased DNA damage response in cells and enteroids. C. jejuni infection induced expression of hundreds of colonic genes, with 22 genes dependent on the presence of cdtB. The C. jejuni-infected group had a significantly different microbial gene expression profile compared with the mutcdtB group as shown by metatranscriptomic data, and different microbial communities as measured by 16S rDNA sequencing. Finally, rapamycin could diminish the tumorigenic capability of C. jejuni.ConclusionHuman clinical isolate C. jejuni 81–176 promotes colorectal cancer and induces changes in microbial composition and transcriptomic responses, a process dependent on CDT production.

ACS Style

Zhen He; Raad Z Gharaibeh; Rachel C Newsome; Jllian L Pope; Michael Dougherty; Sarah Tomkovich; Benoit Pons; Gladys Mirey; Julien Vignard; David R Hendrixson; Christian Jobin. Campylobacter jejuni promotes colorectal tumorigenesis through the action of cytolethal distending toxin. Gut 2018, 68, 289 -300.

AMA Style

Zhen He, Raad Z Gharaibeh, Rachel C Newsome, Jllian L Pope, Michael Dougherty, Sarah Tomkovich, Benoit Pons, Gladys Mirey, Julien Vignard, David R Hendrixson, Christian Jobin. Campylobacter jejuni promotes colorectal tumorigenesis through the action of cytolethal distending toxin. Gut. 2018; 68 (2):289-300.

Chicago/Turabian Style

Zhen He; Raad Z Gharaibeh; Rachel C Newsome; Jllian L Pope; Michael Dougherty; Sarah Tomkovich; Benoit Pons; Gladys Mirey; Julien Vignard; David R Hendrixson; Christian Jobin. 2018. "Campylobacter jejuni promotes colorectal tumorigenesis through the action of cytolethal distending toxin." Gut 68, no. 2: 289-300.