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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 induces apoptosis in Chang cells.(A) Top panel, Immunoblot analysis, using a specific antibody to detect caspases-3 cleavage, in post-nuclear supernatants from Chang cells incubated for the indicated times in the presence or absence of either 1 μg/ml NarE or NarE-R7K. Bottom panel, Quantification by laser densitometry of the relative levels of caspase-3 cleavage (fold activation vs untreated controls) in Chang cells in the presence of 1 μg/ml NarE or NarE-R7K (n = 3). CHX, cycloheximide, 10 μg/ml for 24h, (as positive control); ctr, control. *P≤0.05, ***P≤0.001. Error bars, SD. (B) Fluorescence microscopy analysis of apoptotic cells treated with NarE for 24 hours. The data are expressed as percentages of TUNEL-positive cells (n = 2; *P≤0.05; percentages were calculated on 100 cells/sample).
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pone.0127614.g006: NarE induces apoptosis in Chang cells.(A) Top panel, Immunoblot analysis, using a specific antibody to detect caspases-3 cleavage, in post-nuclear supernatants from Chang cells incubated for the indicated times in the presence or absence of either 1 μg/ml NarE or NarE-R7K. Bottom panel, Quantification by laser densitometry of the relative levels of caspase-3 cleavage (fold activation vs untreated controls) in Chang cells in the presence of 1 μg/ml NarE or NarE-R7K (n = 3). CHX, cycloheximide, 10 μg/ml for 24h, (as positive control); ctr, control. *P≤0.05, ***P≤0.001. Error bars, SD. (B) Fluorescence microscopy analysis of apoptotic cells treated with NarE for 24 hours. The data are expressed as percentages of TUNEL-positive cells (n = 2; *P≤0.05; percentages were calculated on 100 cells/sample).

Mentions: We next investigated whether NarE-induced cell damages correlate with the activation of cell death pathways. Among a wide range of factors controlling apoptotic cell death, caspase-3 activation plays a key role and its activation requires proteolytic processing of its inactive zymogene into activated p17/19 and p12 fragments [27]. Therefore in order to assess NarE effect on the apoptotic pathway, Chang cells were incubated with NarE, NarE-R7K or cycloheximide, CHX, (as positive control) for various times (2h to 24h) and then caspase-3 cleavage, which is an indication of its activation state, was evaluated. As reported in Fig 6, 2h and 6 h of incubation with the toxin were sufficient to induce caspase 3 cleavage. However, there was a peak activity after 24 h of treatment. As expected, NarE-R7K, which is devoid of the ADP-ribosylation activity, did not induce caspase-3 activation.


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 induces apoptosis in Chang cells.(A) Top panel, Immunoblot analysis, using a specific antibody to detect caspases-3 cleavage, in post-nuclear supernatants from Chang cells incubated for the indicated times in the presence or absence of either 1 μg/ml NarE or NarE-R7K. Bottom panel, Quantification by laser densitometry of the relative levels of caspase-3 cleavage (fold activation vs untreated controls) in Chang cells in the presence of 1 μg/ml NarE or NarE-R7K (n = 3). CHX, cycloheximide, 10 μg/ml for 24h, (as positive control); ctr, control. *P≤0.05, ***P≤0.001. Error bars, SD. (B) Fluorescence microscopy analysis of apoptotic cells treated with NarE for 24 hours. The data are expressed as percentages of TUNEL-positive cells (n = 2; *P≤0.05; percentages were calculated on 100 cells/sample).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440719&req=5

pone.0127614.g006: NarE induces apoptosis in Chang cells.(A) Top panel, Immunoblot analysis, using a specific antibody to detect caspases-3 cleavage, in post-nuclear supernatants from Chang cells incubated for the indicated times in the presence or absence of either 1 μg/ml NarE or NarE-R7K. Bottom panel, Quantification by laser densitometry of the relative levels of caspase-3 cleavage (fold activation vs untreated controls) in Chang cells in the presence of 1 μg/ml NarE or NarE-R7K (n = 3). CHX, cycloheximide, 10 μg/ml for 24h, (as positive control); ctr, control. *P≤0.05, ***P≤0.001. Error bars, SD. (B) Fluorescence microscopy analysis of apoptotic cells treated with NarE for 24 hours. The data are expressed as percentages of TUNEL-positive cells (n = 2; *P≤0.05; percentages were calculated on 100 cells/sample).
Mentions: We next investigated whether NarE-induced cell damages correlate with the activation of cell death pathways. Among a wide range of factors controlling apoptotic cell death, caspase-3 activation plays a key role and its activation requires proteolytic processing of its inactive zymogene into activated p17/19 and p12 fragments [27]. Therefore in order to assess NarE effect on the apoptotic pathway, Chang cells were incubated with NarE, NarE-R7K or cycloheximide, CHX, (as positive control) for various times (2h to 24h) and then caspase-3 cleavage, which is an indication of its activation state, was evaluated. As reported in Fig 6, 2h and 6 h of incubation with the toxin were sufficient to induce caspase 3 cleavage. However, there was a peak activity after 24 h of treatment. As expected, NarE-R7K, which is devoid of the ADP-ribosylation activity, did not induce caspase-3 activation.

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