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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 gains access to the cytoplasm and targets host cell proteins.(A) Chang cells, grown to confluence, were exposed to NarE (1μg/ml) or saline (control) for 1 h washed and further incubated for 1, 3 and 6h. At the end of the incubation period cytoplasmic (CE), membrane (ME), nuclear soluble (NE) and cytoskeletal (PE) fractions were isolated and processed for western blot analysis. A mouse polyclonal anti-NarE antibody was used. Polyclonal antibodies anti-GAPDH, anti-ADAM10, anti-Lamin and anti-Cytokeratin were used to check the purity of each fraction. HRP-conjugated secondary antibodies were used before developing in chemiluminescence. The molecular weights of the single proteins are in brackets. (B) Immunoblot analysis of the ADP-ribosylation state of NarE substrates in whole-cell extracts from Chang cells treated with NarE (1μg/ml) for 30 min at 37°C. Biotin-ADP-ribose labelled proteins were identified using an anti-streptavidin HRP antibody. The results of a representative experiment are shown. Anti-actin was use as loading control. The position of the molecular mass markers are indicated on the left.
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pone.0127614.g003: NarE gains access to the cytoplasm and targets host cell proteins.(A) Chang cells, grown to confluence, were exposed to NarE (1μg/ml) or saline (control) for 1 h washed and further incubated for 1, 3 and 6h. At the end of the incubation period cytoplasmic (CE), membrane (ME), nuclear soluble (NE) and cytoskeletal (PE) fractions were isolated and processed for western blot analysis. A mouse polyclonal anti-NarE antibody was used. Polyclonal antibodies anti-GAPDH, anti-ADAM10, anti-Lamin and anti-Cytokeratin were used to check the purity of each fraction. HRP-conjugated secondary antibodies were used before developing in chemiluminescence. The molecular weights of the single proteins are in brackets. (B) Immunoblot analysis of the ADP-ribosylation state of NarE substrates in whole-cell extracts from Chang cells treated with NarE (1μg/ml) for 30 min at 37°C. Biotin-ADP-ribose labelled proteins were identified using an anti-streptavidin HRP antibody. The results of a representative experiment are shown. Anti-actin was use as loading control. The position of the molecular mass markers are indicated on the left.

Mentions: We then hypothesized that NarE internalization and consequent association to the endocytic route, may allow this ADP-ribosyl transferase to gain access to the cytosol. To this end we isolated cytoplasmic, membrane, nuclear soluble and cytoskeletal fractions from Chang cells incubated with NarE. Briefly, Chang cells were exposed to NarE for 1 h, washed, to remove unbound NarE, and further incubated for 1, 3 and 6h before being processed. As shown in Fig 3A, Western blotting analysis revealed that already at 2h, NarE accumulated in the cytosolic fraction. To identify putative cellular substrate/s ADP-ribosylated by NarE, whole-cell lysates were incubated with NarE or NarE-R7K in presence of biotinylated NAD+ (or with 32P-NAD+, data not shown). Interestingly, as shown in Fig 3B, incubation of cell extracts with NarE, but not with the genetically inactivated mutant NarE-R7K, resulted in the increase of ADP-ribosylated proteins, indicating that NarE targets Chang cell proteins. Of note, we detected bands of approximately 42–45 kDa, compatible with the molecular weight of actin, or of α-subunit of heterotrimeric G proteins, both well-known targets for modification of many bacterial toxins [8, 7].


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 gains access to the cytoplasm and targets host cell proteins.(A) Chang cells, grown to confluence, were exposed to NarE (1μg/ml) or saline (control) for 1 h washed and further incubated for 1, 3 and 6h. At the end of the incubation period cytoplasmic (CE), membrane (ME), nuclear soluble (NE) and cytoskeletal (PE) fractions were isolated and processed for western blot analysis. A mouse polyclonal anti-NarE antibody was used. Polyclonal antibodies anti-GAPDH, anti-ADAM10, anti-Lamin and anti-Cytokeratin were used to check the purity of each fraction. HRP-conjugated secondary antibodies were used before developing in chemiluminescence. The molecular weights of the single proteins are in brackets. (B) Immunoblot analysis of the ADP-ribosylation state of NarE substrates in whole-cell extracts from Chang cells treated with NarE (1μg/ml) for 30 min at 37°C. Biotin-ADP-ribose labelled proteins were identified using an anti-streptavidin HRP antibody. The results of a representative experiment are shown. Anti-actin was use as loading control. The position of the molecular mass markers are indicated on the left.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440719&req=5

pone.0127614.g003: NarE gains access to the cytoplasm and targets host cell proteins.(A) Chang cells, grown to confluence, were exposed to NarE (1μg/ml) or saline (control) for 1 h washed and further incubated for 1, 3 and 6h. At the end of the incubation period cytoplasmic (CE), membrane (ME), nuclear soluble (NE) and cytoskeletal (PE) fractions were isolated and processed for western blot analysis. A mouse polyclonal anti-NarE antibody was used. Polyclonal antibodies anti-GAPDH, anti-ADAM10, anti-Lamin and anti-Cytokeratin were used to check the purity of each fraction. HRP-conjugated secondary antibodies were used before developing in chemiluminescence. The molecular weights of the single proteins are in brackets. (B) Immunoblot analysis of the ADP-ribosylation state of NarE substrates in whole-cell extracts from Chang cells treated with NarE (1μg/ml) for 30 min at 37°C. Biotin-ADP-ribose labelled proteins were identified using an anti-streptavidin HRP antibody. The results of a representative experiment are shown. Anti-actin was use as loading control. The position of the molecular mass markers are indicated on the left.
Mentions: We then hypothesized that NarE internalization and consequent association to the endocytic route, may allow this ADP-ribosyl transferase to gain access to the cytosol. To this end we isolated cytoplasmic, membrane, nuclear soluble and cytoskeletal fractions from Chang cells incubated with NarE. Briefly, Chang cells were exposed to NarE for 1 h, washed, to remove unbound NarE, and further incubated for 1, 3 and 6h before being processed. As shown in Fig 3A, Western blotting analysis revealed that already at 2h, NarE accumulated in the cytosolic fraction. To identify putative cellular substrate/s ADP-ribosylated by NarE, whole-cell lysates were incubated with NarE or NarE-R7K in presence of biotinylated NAD+ (or with 32P-NAD+, data not shown). Interestingly, as shown in Fig 3B, incubation of cell extracts with NarE, but not with the genetically inactivated mutant NarE-R7K, resulted in the increase of ADP-ribosylated proteins, indicating that NarE targets Chang cell proteins. Of note, we detected bands of approximately 42–45 kDa, compatible with the molecular weight of actin, or of α-subunit of heterotrimeric G proteins, both well-known targets for modification of many bacterial toxins [8, 7].

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