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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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Actin cytoskeleton modification and Rac1 activation induced by extracellular vesicles (EVs) in cells. (A) Fluorescence micrographs of HEp-2 and Me-665 cells stained with fluorescein- or Tetramethylrhodamine (TRITC)-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EVs derived from CNF1-treated cells for 24 h (EV-CNF1) show a rearrangement of the actin cytoskeleton in stress fibers (arrowheads) and ruffles/spikes (arrows) similar to that obtained in CNF1-treated cells. (B) Fluorescence micrographs of HEp-2 cells stained with TRITC-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EV-CNF1 derived from trypsin-treated cells show a rearrangement of the actin cytoskeleton in stress fibers and ruffles/spikes similar to that obtained in cells exposed to EV-CNF1. (C) Western blot analysis of the pull-down assay of HEp-2 cells showing the increase in Rac1-GTP following treatment with CNF1, as well as with EV-CNF1. The blot in the left panel shows one representative experiment, whereas the graph in the right panel reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate). * p < 0.05; *** p < 0.001.
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toxins-07-04610-f001: Actin cytoskeleton modification and Rac1 activation induced by extracellular vesicles (EVs) in cells. (A) Fluorescence micrographs of HEp-2 and Me-665 cells stained with fluorescein- or Tetramethylrhodamine (TRITC)-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EVs derived from CNF1-treated cells for 24 h (EV-CNF1) show a rearrangement of the actin cytoskeleton in stress fibers (arrowheads) and ruffles/spikes (arrows) similar to that obtained in CNF1-treated cells. (B) Fluorescence micrographs of HEp-2 cells stained with TRITC-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EV-CNF1 derived from trypsin-treated cells show a rearrangement of the actin cytoskeleton in stress fibers and ruffles/spikes similar to that obtained in cells exposed to EV-CNF1. (C) Western blot analysis of the pull-down assay of HEp-2 cells showing the increase in Rac1-GTP following treatment with CNF1, as well as with EV-CNF1. The blot in the left panel shows one representative experiment, whereas the graph in the right panel reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate). * p < 0.05; *** p < 0.001.

Mentions: In order to verify if exosomes and/or ectosomes could carry and propagate CNF1 (or its specific signals) from cell to cell, we first verified, by fluorescence microscopy, the actin cytoskeleton organization in cells challenged either with CNF1, or with EVs from control cells (EV-control), or derived from cells pre-exposed to CNF1 for 2 h (EV-CNF1). As shown in Figure 1A, incubation of cells with EV-CNF1 drove actin cytoskeleton changes that morphologically resemble those induced by CNF1 itself, mainly consisting of the formation of stress fibers (arrowheads) and ruffling/spikes (arrows). This result supports the hypothesis that EV-CNF1 can carry the CNF1 activity. Furthermore, we also tested the EV-CNF1 in an additional cell type, the human metastatic cell line 665 (Me-665) that has been shown to be suitable for studies on toxins and exosomes [16]. The cytoskeletal changes induced by EV-CNF1 were comparable to that provoked by the toxin (Figure 1A), indicating that EV-CNF1 could represent a general route of toxin propagation not restricted to a specific cell type. When an activity assay was performed, by comparing titration of CNF1 with EV-CNF1 effects on actin in HEp-2 cells, we found that the response obtained with EV-CNF1 corresponded to that obtained with 0.7 × 10−12 M CNF1. Furthermore, to eliminate the possibility that a small amount of CNF1, eventually present in the medium used to collect the EVs, could be responsible for the observed activity, we briefly treated cells with trypsin before overnight incubation. As shown in Figure 1B, treatment of cells producing EVs with trypsin did not inhibit the capability of EV-CNF1 to rearrange the actin cytoskeleton, proving that CNF1 is inside the EVs.


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)

Actin cytoskeleton modification and Rac1 activation induced by extracellular vesicles (EVs) in cells. (A) Fluorescence micrographs of HEp-2 and Me-665 cells stained with fluorescein- or Tetramethylrhodamine (TRITC)-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EVs derived from CNF1-treated cells for 24 h (EV-CNF1) show a rearrangement of the actin cytoskeleton in stress fibers (arrowheads) and ruffles/spikes (arrows) similar to that obtained in CNF1-treated cells. (B) Fluorescence micrographs of HEp-2 cells stained with TRITC-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EV-CNF1 derived from trypsin-treated cells show a rearrangement of the actin cytoskeleton in stress fibers and ruffles/spikes similar to that obtained in cells exposed to EV-CNF1. (C) Western blot analysis of the pull-down assay of HEp-2 cells showing the increase in Rac1-GTP following treatment with CNF1, as well as with EV-CNF1. The blot in the left panel shows one representative experiment, whereas the graph in the right panel reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate). * p < 0.05; *** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04610-f001: Actin cytoskeleton modification and Rac1 activation induced by extracellular vesicles (EVs) in cells. (A) Fluorescence micrographs of HEp-2 and Me-665 cells stained with fluorescein- or Tetramethylrhodamine (TRITC)-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EVs derived from CNF1-treated cells for 24 h (EV-CNF1) show a rearrangement of the actin cytoskeleton in stress fibers (arrowheads) and ruffles/spikes (arrows) similar to that obtained in CNF1-treated cells. (B) Fluorescence micrographs of HEp-2 cells stained with TRITC-phalloidin to detect the actin cytoskeleton organization. Cells exposed to EV-CNF1 derived from trypsin-treated cells show a rearrangement of the actin cytoskeleton in stress fibers and ruffles/spikes similar to that obtained in cells exposed to EV-CNF1. (C) Western blot analysis of the pull-down assay of HEp-2 cells showing the increase in Rac1-GTP following treatment with CNF1, as well as with EV-CNF1. The blot in the left panel shows one representative experiment, whereas the graph in the right panel reports the mean ± SEM from three different experiments (n = 3 experiments, with each experiment performed in duplicate). * p < 0.05; *** p < 0.001.
Mentions: In order to verify if exosomes and/or ectosomes could carry and propagate CNF1 (or its specific signals) from cell to cell, we first verified, by fluorescence microscopy, the actin cytoskeleton organization in cells challenged either with CNF1, or with EVs from control cells (EV-control), or derived from cells pre-exposed to CNF1 for 2 h (EV-CNF1). As shown in Figure 1A, incubation of cells with EV-CNF1 drove actin cytoskeleton changes that morphologically resemble those induced by CNF1 itself, mainly consisting of the formation of stress fibers (arrowheads) and ruffling/spikes (arrows). This result supports the hypothesis that EV-CNF1 can carry the CNF1 activity. Furthermore, we also tested the EV-CNF1 in an additional cell type, the human metastatic cell line 665 (Me-665) that has been shown to be suitable for studies on toxins and exosomes [16]. The cytoskeletal changes induced by EV-CNF1 were comparable to that provoked by the toxin (Figure 1A), indicating that EV-CNF1 could represent a general route of toxin propagation not restricted to a specific cell type. When an activity assay was performed, by comparing titration of CNF1 with EV-CNF1 effects on actin in HEp-2 cells, we found that the response obtained with EV-CNF1 corresponded to that obtained with 0.7 × 10−12 M CNF1. Furthermore, to eliminate the possibility that a small amount of CNF1, eventually present in the medium used to collect the EVs, could be responsible for the observed activity, we briefly treated cells with trypsin before overnight incubation. As shown in Figure 1B, treatment of cells producing EVs with trypsin did not inhibit the capability of EV-CNF1 to rearrange the actin cytoskeleton, proving that CNF1 is inside the EVs.

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