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The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

Valeri M, Zurli V, Ayala I, Colanzi A, Lapazio L, Corda D, Soriani M, Pizza M, Rossi Paccani S - PLoS ONE (2015)

Bottom Line: It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins.Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins.Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Vaccines & Diagnostics s.r.l.-a GSK company- Via Fiorentina 1, Siena, Italy.

ABSTRACT
Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

No MeSH data available.


Related in: MedlinePlus

NarE impacts on actin cytoskeleton.(A) Actin-staining using TRITC-conjugated phalloidin (red) in Chang cells. The nucleus was stained with DAPI (blue). 1 μg/ml NarE, as well as 100 ng/ml TcdA, used as positive control, causes disruption of the actin cytoskeleton. Scale bar represents 20 μm. (B) Flow cytometric analysis of relative unpolymerized actin (fold increase vs untreated control, gray bar) in Chang cells treated with 1–10 μg/ml NarE or 100 ng/ml TcdA, as positive control. Data in columns are given from 3 independent experiments. **P≤0.01;*P≤0.05. Error bars, SD.
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pone.0127614.g005: NarE impacts on actin cytoskeleton.(A) Actin-staining using TRITC-conjugated phalloidin (red) in Chang cells. The nucleus was stained with DAPI (blue). 1 μg/ml NarE, as well as 100 ng/ml TcdA, used as positive control, causes disruption of the actin cytoskeleton. Scale bar represents 20 μm. (B) Flow cytometric analysis of relative unpolymerized actin (fold increase vs untreated control, gray bar) in Chang cells treated with 1–10 μg/ml NarE or 100 ng/ml TcdA, as positive control. Data in columns are given from 3 independent experiments. **P≤0.01;*P≤0.05. Error bars, SD.

Mentions: The observation that one of the ADP-ribosylated substrates could be actin (Fig 3, panel B), along with the phenotype of intoxication (cell rounding), lead us to further assess NarE effect on actin cytoskeleton of Chang cells. To this end, after 4 hours of incubation of cells with the wild-type or the mutant protein, morphological alterations were analyzed by confocal microscopy using fluorochrome-conjugated phalloidin. F-actin depolymerization and dissolution of cytoskeleton was observed in cells treated with NarE, suggesting that actin could be, directly or indirectly, a target of NarE-ADP-ribosylating activity. Representative micrographs are shown in Fig 5A. These results were further corroborated by monitoring global changes in actin dynamics using the DNase I inhibition assay, a selective analysis in which G-actin is detected with the fluorophore-derivatized Dnase I. Fig 5B displays a significant increase in the amount of unpolymerized actin after incubation with NarE. Similar results were obtained when TcdA was used. Notably, any effect on actin cytoskeleton was detected in cells treated with NarE-R7K.


The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity.

Valeri M, Zurli V, Ayala I, Colanzi A, Lapazio L, Corda D, Soriani M, Pizza M, Rossi Paccani S - PLoS ONE (2015)

NarE impacts on actin cytoskeleton.(A) Actin-staining using TRITC-conjugated phalloidin (red) in Chang cells. The nucleus was stained with DAPI (blue). 1 μg/ml NarE, as well as 100 ng/ml TcdA, used as positive control, causes disruption of the actin cytoskeleton. Scale bar represents 20 μm. (B) Flow cytometric analysis of relative unpolymerized actin (fold increase vs untreated control, gray bar) in Chang cells treated with 1–10 μg/ml NarE or 100 ng/ml TcdA, as positive control. Data in columns are given from 3 independent experiments. **P≤0.01;*P≤0.05. Error bars, SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440719&req=5

pone.0127614.g005: NarE impacts on actin cytoskeleton.(A) Actin-staining using TRITC-conjugated phalloidin (red) in Chang cells. The nucleus was stained with DAPI (blue). 1 μg/ml NarE, as well as 100 ng/ml TcdA, used as positive control, causes disruption of the actin cytoskeleton. Scale bar represents 20 μm. (B) Flow cytometric analysis of relative unpolymerized actin (fold increase vs untreated control, gray bar) in Chang cells treated with 1–10 μg/ml NarE or 100 ng/ml TcdA, as positive control. Data in columns are given from 3 independent experiments. **P≤0.01;*P≤0.05. Error bars, SD.
Mentions: The observation that one of the ADP-ribosylated substrates could be actin (Fig 3, panel B), along with the phenotype of intoxication (cell rounding), lead us to further assess NarE effect on actin cytoskeleton of Chang cells. To this end, after 4 hours of incubation of cells with the wild-type or the mutant protein, morphological alterations were analyzed by confocal microscopy using fluorochrome-conjugated phalloidin. F-actin depolymerization and dissolution of cytoskeleton was observed in cells treated with NarE, suggesting that actin could be, directly or indirectly, a target of NarE-ADP-ribosylating activity. Representative micrographs are shown in Fig 5A. These results were further corroborated by monitoring global changes in actin dynamics using the DNase I inhibition assay, a selective analysis in which G-actin is detected with the fluorophore-derivatized Dnase I. Fig 5B displays a significant increase in the amount of unpolymerized actin after incubation with NarE. Similar results were obtained when TcdA was used. Notably, any effect on actin cytoskeleton was detected in cells treated with NarE-R7K.

Bottom Line: It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins.Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins.Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway.

View Article: PubMed Central - PubMed

Affiliation: Vaccines & Diagnostics s.r.l.-a GSK company- Via Fiorentina 1, Siena, Italy.

ABSTRACT
Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.

No MeSH data available.


Related in: MedlinePlus