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Cell-to-cell propagation of the bacterial toxin CNF1 via extracellular vesicles: potential impact on the therapeutic use of the toxin.

Fabbri A, Cori S, Zanetti C, Guidotti M, Sargiacomo M, Loizzo S, Fiorentini C - Toxins (Basel) (2015)

Bottom Line: We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1).Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1.Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome 00161, Italy. alessia.fabbri@iss.it.

ABSTRACT
Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

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NF-κB translocation in EV-CNF1-treated cells. (A) Fluorescence micrographs of HEp-2 cells untreated or treated for 4 h with CNF1 or with EV-CNF1, stained with an anti-p65 antibody. The illustrations are used to evidence the positively-stained nuclei (arrows) after CNF1 or EV-CNF1 exposure. The negative staining for rabbit antibody is shown. (B) The graph reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate), showing the time-dependent nuclear translocation of p65 NF-κB after the different experimental conditions. *p < 0.05; **p < 0.01; ***p < 0.005.
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toxins-07-04610-f002: NF-κB translocation in EV-CNF1-treated cells. (A) Fluorescence micrographs of HEp-2 cells untreated or treated for 4 h with CNF1 or with EV-CNF1, stained with an anti-p65 antibody. The illustrations are used to evidence the positively-stained nuclei (arrows) after CNF1 or EV-CNF1 exposure. The negative staining for rabbit antibody is shown. (B) The graph reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate), showing the time-dependent nuclear translocation of p65 NF-κB after the different experimental conditions. *p < 0.05; **p < 0.01; ***p < 0.005.

Mentions: The second step was to verify if EV-CNF1 could induce another typical activity stimulated by CNF1 in HEp-2 cells: that is, the nuclear translocation of the transcription factor NF-κB [21,22]. Such translocation occurs maximally between 4 and 10 h from the treatment with CNF1, with about 50% of positive nuclei for the p65 protein [21]. We herein investigated whether EV-CNF1 could trigger a response similar to that of CNF1. Immunofluorescence micrographs (Figure 2A) revealed that in both cells treated with the toxin or with EVs taken from cells previously intoxicated with CNF1 for 2 h, the p65 NF-κB subunit was translocated into the nucleus. Moreover, when a quantitative analysis was performed (Figure 2B), we found a nearly comparable percentage of cells with p65-positive nuclei between CNF1 and EV-CNF1. In particular, the percentage of cells with p65-positive nuclei was around 20% in EV-CNF1-treated cells, whereas cells exposed to CNF1 reached 26%–31% of positivity. The maximum nuclear translocation of p65 NF-κB was obtained between 2 and 4 h after treatment with CNF1 or EV-CNF1. Starting from 6 h of treatment, a general decrease in p65-positive nuclei was observed, which was consistent with the drop in Rho GTPase activation previously reported for this cell line [22]. The increment at 8 and 24 h of exposure with respect to 6 h was only apparent, since such an augmentation was not significant (p = 0.3355 by ANOVA). These results indicate that EVs are able to propagate the toxin activity, with an effect similar to that of CNF1.


Cell-to-cell propagation of the bacterial toxin CNF1 via extracellular vesicles: potential impact on the therapeutic use of the toxin.

Fabbri A, Cori S, Zanetti C, Guidotti M, Sargiacomo M, Loizzo S, Fiorentini C - Toxins (Basel) (2015)

NF-κB translocation in EV-CNF1-treated cells. (A) Fluorescence micrographs of HEp-2 cells untreated or treated for 4 h with CNF1 or with EV-CNF1, stained with an anti-p65 antibody. The illustrations are used to evidence the positively-stained nuclei (arrows) after CNF1 or EV-CNF1 exposure. The negative staining for rabbit antibody is shown. (B) The graph reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate), showing the time-dependent nuclear translocation of p65 NF-κB after the different experimental conditions. *p < 0.05; **p < 0.01; ***p < 0.005.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663523&req=5

toxins-07-04610-f002: NF-κB translocation in EV-CNF1-treated cells. (A) Fluorescence micrographs of HEp-2 cells untreated or treated for 4 h with CNF1 or with EV-CNF1, stained with an anti-p65 antibody. The illustrations are used to evidence the positively-stained nuclei (arrows) after CNF1 or EV-CNF1 exposure. The negative staining for rabbit antibody is shown. (B) The graph reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate), showing the time-dependent nuclear translocation of p65 NF-κB after the different experimental conditions. *p < 0.05; **p < 0.01; ***p < 0.005.
Mentions: The second step was to verify if EV-CNF1 could induce another typical activity stimulated by CNF1 in HEp-2 cells: that is, the nuclear translocation of the transcription factor NF-κB [21,22]. Such translocation occurs maximally between 4 and 10 h from the treatment with CNF1, with about 50% of positive nuclei for the p65 protein [21]. We herein investigated whether EV-CNF1 could trigger a response similar to that of CNF1. Immunofluorescence micrographs (Figure 2A) revealed that in both cells treated with the toxin or with EVs taken from cells previously intoxicated with CNF1 for 2 h, the p65 NF-κB subunit was translocated into the nucleus. Moreover, when a quantitative analysis was performed (Figure 2B), we found a nearly comparable percentage of cells with p65-positive nuclei between CNF1 and EV-CNF1. In particular, the percentage of cells with p65-positive nuclei was around 20% in EV-CNF1-treated cells, whereas cells exposed to CNF1 reached 26%–31% of positivity. The maximum nuclear translocation of p65 NF-κB was obtained between 2 and 4 h after treatment with CNF1 or EV-CNF1. Starting from 6 h of treatment, a general decrease in p65-positive nuclei was observed, which was consistent with the drop in Rho GTPase activation previously reported for this cell line [22]. The increment at 8 and 24 h of exposure with respect to 6 h was only apparent, since such an augmentation was not significant (p = 0.3355 by ANOVA). These results indicate that EVs are able to propagate the toxin activity, with an effect similar to that of CNF1.

Bottom Line: We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1).Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1.Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome 00161, Italy. alessia.fabbri@iss.it.

ABSTRACT
Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

Show MeSH
Related in: MedlinePlus