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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 impairs human epithelial monolayer integrity and induces cell rounding.(A) Top left, Chang monolayer was treated either with NarE or NarE-R7K (1μg/ml) and barrier resistance was continuously measured using an electric cell-substrate impedance sensing (xCELLigence) system. TcdA (100 ng/ml) was used as positive control, while saline was used as negative control. CI, Cell Index (arbitrary unit for electric impedance measurement). Top right, Variation of Chang cells CI after 6 h of treatment with NarE or NarE-R7K (1μg/ml). The results are expressed as the difference between normalized CI before agents addition (CI = 1) and CI value after 6 h of incubation averaged from three independent experiments, each performed on duplicate samples. Error bars represent the SD. **, P ≤ 0.01. (B) Effect of NarE treatment on the morphology of Chang cells. Chang cells were incubated at 37°C with NarE, NarE-R7K (1μg/ml), TcdA as positive control (100 ng/ml) or left untreated as control. After 4 hours, pictures were taken to assess the change in morphology. Bar, 20 μm.
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pone.0127614.g004: NarE impairs human epithelial monolayer integrity and induces cell rounding.(A) Top left, Chang monolayer was treated either with NarE or NarE-R7K (1μg/ml) and barrier resistance was continuously measured using an electric cell-substrate impedance sensing (xCELLigence) system. TcdA (100 ng/ml) was used as positive control, while saline was used as negative control. CI, Cell Index (arbitrary unit for electric impedance measurement). Top right, Variation of Chang cells CI after 6 h of treatment with NarE or NarE-R7K (1μg/ml). The results are expressed as the difference between normalized CI before agents addition (CI = 1) and CI value after 6 h of incubation averaged from three independent experiments, each performed on duplicate samples. Error bars represent the SD. **, P ≤ 0.01. (B) Effect of NarE treatment on the morphology of Chang cells. Chang cells were incubated at 37°C with NarE, NarE-R7K (1μg/ml), TcdA as positive control (100 ng/ml) or left untreated as control. After 4 hours, pictures were taken to assess the change in morphology. Bar, 20 μm.

Mentions: Since NarE ADP-ribosylates Chang substrates, we then examined whether it could alter essential functions of eukaryotic cells. To this end electrical cell-substrate impedance sensing (xCELLigence) was used to measure trans-epithelial electrical resistance. Impedance reflects the status of the cell monolayer, including confluence, viability, and junction functionality [24, 25]. Chang cells were grown to confluence on xCELLigence inserts and the experiments were started when trans-epithelial resistance (TEER) was found constant between two measures taken 24 hours apart. Cells were then incubated with NarE or NarE-R7K. Vehicle was used as negative control. Changes in monolayer integrity were monitored every 15 min. As shown in Fig 4A Chang monolayer treated with 1μg/ml NarE demonstrated a rapid and progressive loss of resistance. Similar results were obtained when Clostridium difficile toxin, TcdA, a well-known cytotoxin affecting actin cytoskeleton [7, 26], was used. Neither vehicle nor NarER-7K altered monolayer resistance.


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 impairs human epithelial monolayer integrity and induces cell rounding.(A) Top left, Chang monolayer was treated either with NarE or NarE-R7K (1μg/ml) and barrier resistance was continuously measured using an electric cell-substrate impedance sensing (xCELLigence) system. TcdA (100 ng/ml) was used as positive control, while saline was used as negative control. CI, Cell Index (arbitrary unit for electric impedance measurement). Top right, Variation of Chang cells CI after 6 h of treatment with NarE or NarE-R7K (1μg/ml). The results are expressed as the difference between normalized CI before agents addition (CI = 1) and CI value after 6 h of incubation averaged from three independent experiments, each performed on duplicate samples. Error bars represent the SD. **, P ≤ 0.01. (B) Effect of NarE treatment on the morphology of Chang cells. Chang cells were incubated at 37°C with NarE, NarE-R7K (1μg/ml), TcdA as positive control (100 ng/ml) or left untreated as control. After 4 hours, pictures were taken to assess the change in morphology. Bar, 20 μm.
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pone.0127614.g004: NarE impairs human epithelial monolayer integrity and induces cell rounding.(A) Top left, Chang monolayer was treated either with NarE or NarE-R7K (1μg/ml) and barrier resistance was continuously measured using an electric cell-substrate impedance sensing (xCELLigence) system. TcdA (100 ng/ml) was used as positive control, while saline was used as negative control. CI, Cell Index (arbitrary unit for electric impedance measurement). Top right, Variation of Chang cells CI after 6 h of treatment with NarE or NarE-R7K (1μg/ml). The results are expressed as the difference between normalized CI before agents addition (CI = 1) and CI value after 6 h of incubation averaged from three independent experiments, each performed on duplicate samples. Error bars represent the SD. **, P ≤ 0.01. (B) Effect of NarE treatment on the morphology of Chang cells. Chang cells were incubated at 37°C with NarE, NarE-R7K (1μg/ml), TcdA as positive control (100 ng/ml) or left untreated as control. After 4 hours, pictures were taken to assess the change in morphology. Bar, 20 μm.
Mentions: Since NarE ADP-ribosylates Chang substrates, we then examined whether it could alter essential functions of eukaryotic cells. To this end electrical cell-substrate impedance sensing (xCELLigence) was used to measure trans-epithelial electrical resistance. Impedance reflects the status of the cell monolayer, including confluence, viability, and junction functionality [24, 25]. Chang cells were grown to confluence on xCELLigence inserts and the experiments were started when trans-epithelial resistance (TEER) was found constant between two measures taken 24 hours apart. Cells were then incubated with NarE or NarE-R7K. Vehicle was used as negative control. Changes in monolayer integrity were monitored every 15 min. As shown in Fig 4A Chang monolayer treated with 1μg/ml NarE demonstrated a rapid and progressive loss of resistance. Similar results were obtained when Clostridium difficile toxin, TcdA, a well-known cytotoxin affecting actin cytoskeleton [7, 26], was used. Neither vehicle nor NarER-7K altered monolayer resistance.

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