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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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CNF1 colocalizes with Tsg101-positive vesicles and is carried by CNF1-EVs. (A) Fluorescence micrographs of HEp-2 cells double stained with anti-Tsg101, a marker of exosomes, and anti-CNF1. In control cells, Tsg101 is distributed inside the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining is negative. In CNF1-treated cells, a clear staining with the anti-CNF1 is observable inside the cytoplasm, as well as at the periphery of the cells. Interestingly, Tsg101 was co-localized with CNF1 in cytoplasmic punctuated structures, as well as in vesicle-like structures detected at the cell periphery (insets). Negative staining for mouse and rabbit antibodies is shown. (B) Fluorescence micrographs of HEp-2 cells incubated with EV and EV-CNF1 for 24 h and stained with the CNF1 antibody. In cells incubated with EV-CNF1, a positive staining is clearly evident. (C) Western blot analysis of EV and EV-CNF1 showing the presence of CNF1 inside EV-CNF1. Purified CNF1 was loaded as a positive marker.
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toxins-07-04610-f003: CNF1 colocalizes with Tsg101-positive vesicles and is carried by CNF1-EVs. (A) Fluorescence micrographs of HEp-2 cells double stained with anti-Tsg101, a marker of exosomes, and anti-CNF1. In control cells, Tsg101 is distributed inside the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining is negative. In CNF1-treated cells, a clear staining with the anti-CNF1 is observable inside the cytoplasm, as well as at the periphery of the cells. Interestingly, Tsg101 was co-localized with CNF1 in cytoplasmic punctuated structures, as well as in vesicle-like structures detected at the cell periphery (insets). Negative staining for mouse and rabbit antibodies is shown. (B) Fluorescence micrographs of HEp-2 cells incubated with EV and EV-CNF1 for 24 h and stained with the CNF1 antibody. In cells incubated with EV-CNF1, a positive staining is clearly evident. (C) Western blot analysis of EV and EV-CNF1 showing the presence of CNF1 inside EV-CNF1. Purified CNF1 was loaded as a positive marker.

Mentions: The third step was to verify whether CNF1 could be found in EVs. To address this question, HEp-2 cells were treated with CNF1 for 24 h, and the presence of the toxin was verified by fluorescence microscopy, by double staining cells with anti-Tumor Susceptibility Gene (Tsg) 101, a marker for exosomes, and anti-CNF1. As shown in Figure 3, in control cells, Tsg101 was distributed in the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining was negative. In CNF1-challenged cells, the anti-CNF1 staining clearly showed the presence of the toxin inside the cytoplasm, as well as at the cell periphery. Interestingly, Tsg101 co-localized with CNF1 both in cytoplasmic punctuated structures and in the vesicle-like structures detected at the cell periphery (see the insets in Figure 3). Thus, we wondered whether CNF1 could be found in EVs derived from cells intoxicated with CNF1. To address this question, HEp-2 cells were treated with EVs or EV-CNF1 for 24 h and then stained with an anti-CNF1 antibody. As shown in Figure 3B, cells treated with EV-CNF1 were clearly positive for the anti-CNF1 staining, whereas EV-treated cells were negative. We thus verified, by Western blot analysis, whether CNF1 could be found in EVs derived from CNF1-intoxicated cells. As shown in Figure 3C, whereas EVs derived from control cells were negative for CNF1, EV-CNF1 were clearly positive for the toxin. Furthermore, we have quantified the amount of CNF1 protein alone and CNF1 protein contained in EVs that is delivered to the untreated cells. A densitometric quantification of CNF1 bands in Figure 3C revealed the presence of about 1.17 × 10−2 ng of CNF1 per μg of EVs. These results demonstrate that CNF1 can be found in EV derived from CNF1-intoxicated cells and strongly support the hypothesis that CNF1 can travel from cell to cell also via extracellular vesicles.


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)

CNF1 colocalizes with Tsg101-positive vesicles and is carried by CNF1-EVs. (A) Fluorescence micrographs of HEp-2 cells double stained with anti-Tsg101, a marker of exosomes, and anti-CNF1. In control cells, Tsg101 is distributed inside the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining is negative. In CNF1-treated cells, a clear staining with the anti-CNF1 is observable inside the cytoplasm, as well as at the periphery of the cells. Interestingly, Tsg101 was co-localized with CNF1 in cytoplasmic punctuated structures, as well as in vesicle-like structures detected at the cell periphery (insets). Negative staining for mouse and rabbit antibodies is shown. (B) Fluorescence micrographs of HEp-2 cells incubated with EV and EV-CNF1 for 24 h and stained with the CNF1 antibody. In cells incubated with EV-CNF1, a positive staining is clearly evident. (C) Western blot analysis of EV and EV-CNF1 showing the presence of CNF1 inside EV-CNF1. Purified CNF1 was loaded as a positive marker.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04610-f003: CNF1 colocalizes with Tsg101-positive vesicles and is carried by CNF1-EVs. (A) Fluorescence micrographs of HEp-2 cells double stained with anti-Tsg101, a marker of exosomes, and anti-CNF1. In control cells, Tsg101 is distributed inside the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining is negative. In CNF1-treated cells, a clear staining with the anti-CNF1 is observable inside the cytoplasm, as well as at the periphery of the cells. Interestingly, Tsg101 was co-localized with CNF1 in cytoplasmic punctuated structures, as well as in vesicle-like structures detected at the cell periphery (insets). Negative staining for mouse and rabbit antibodies is shown. (B) Fluorescence micrographs of HEp-2 cells incubated with EV and EV-CNF1 for 24 h and stained with the CNF1 antibody. In cells incubated with EV-CNF1, a positive staining is clearly evident. (C) Western blot analysis of EV and EV-CNF1 showing the presence of CNF1 inside EV-CNF1. Purified CNF1 was loaded as a positive marker.
Mentions: The third step was to verify whether CNF1 could be found in EVs. To address this question, HEp-2 cells were treated with CNF1 for 24 h, and the presence of the toxin was verified by fluorescence microscopy, by double staining cells with anti-Tumor Susceptibility Gene (Tsg) 101, a marker for exosomes, and anti-CNF1. As shown in Figure 3, in control cells, Tsg101 was distributed in the cytoplasm in a polarized way close to the nucleus, whereas CNF1 staining was negative. In CNF1-challenged cells, the anti-CNF1 staining clearly showed the presence of the toxin inside the cytoplasm, as well as at the cell periphery. Interestingly, Tsg101 co-localized with CNF1 both in cytoplasmic punctuated structures and in the vesicle-like structures detected at the cell periphery (see the insets in Figure 3). Thus, we wondered whether CNF1 could be found in EVs derived from cells intoxicated with CNF1. To address this question, HEp-2 cells were treated with EVs or EV-CNF1 for 24 h and then stained with an anti-CNF1 antibody. As shown in Figure 3B, cells treated with EV-CNF1 were clearly positive for the anti-CNF1 staining, whereas EV-treated cells were negative. We thus verified, by Western blot analysis, whether CNF1 could be found in EVs derived from CNF1-intoxicated cells. As shown in Figure 3C, whereas EVs derived from control cells were negative for CNF1, EV-CNF1 were clearly positive for the toxin. Furthermore, we have quantified the amount of CNF1 protein alone and CNF1 protein contained in EVs that is delivered to the untreated cells. A densitometric quantification of CNF1 bands in Figure 3C revealed the presence of about 1.17 × 10−2 ng of CNF1 per μg of EVs. These results demonstrate that CNF1 can be found in EV derived from CNF1-intoxicated cells and strongly support the hypothesis that CNF1 can travel from cell to cell also via extracellular vesicles.

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