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Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus

Expression of JHP0290 homologues in different H. pylori strains.Whole cell lysate (A) and culture broth (Supernatant) from equal number of cells (B) of various H. pylori strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. (C) Sequence homology analysis of JHP0290 homologues in H. pylori strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.
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pone.0124407.g001: Expression of JHP0290 homologues in different H. pylori strains.Whole cell lysate (A) and culture broth (Supernatant) from equal number of cells (B) of various H. pylori strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. (C) Sequence homology analysis of JHP0290 homologues in H. pylori strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.

Mentions: Sequence homology analysis indicated that JHP0290 is highly conserved among H. pylori strains. We first determined the expression of JHP0290 homologues in the whole cell extracts of several strains using the antibody raised against rJHP0290. Western blot analysis revealed that the protein was expressed by all of the strains that were tested in this study, although the expression level varied in different strains (Fig 1A). The same lysates were immunoblotted with antibodies against Urease and AhpC, which are abundantly expressed in H. pylori. The expression level of either Urease (data not shown) or AhpC (S1 Fig) did not vary significantly in the tested strains. The presence of a signal peptide (1–17 aa) in the sequence suggests that the protein is released by the bacterium. In a previous study, we reported that the JHP0290 protein of H. pylori strain J99 is released into the culture broth by the bacterium [42]. Kim et al. reported the secretion of a JHP0290 homolog (HP0305) by another H. pylori strain (NCTC 11637) [37]. To confirm the release of JHP0290 homologues by other strains of H. pylori, culture supernatants from various strains were immunoblotted with the anti-JHP0290 antibody. The release of JHP0290 homologues into the culture broth was observed for all strains (Fig 1B). Interestingly, the level of JHP0290 homologues released into the culture broth varied among strains; release did not correlate with the observed differences in expression levels. As shown in Fig 1A, the expression of the JHP0290 homologue in H. pylori strain TN2GF4 (Fig 1A, lane 3) was lower than that of the strain J99 (Fig 1A, lane 2); however, the level of protein released into the culture broth by TN2GF4 (Fig 1B, lane 3) was higher than that released by J99 (Fig 1B, lane 2). The same pattern was observed for strains P12 and HPAG1. The precise relationship between the amount of released JHP0290 protein homologues and the virulence of the bacterium requires further investigation.


Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Expression of JHP0290 homologues in different H. pylori strains.Whole cell lysate (A) and culture broth (Supernatant) from equal number of cells (B) of various H. pylori strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. (C) Sequence homology analysis of JHP0290 homologues in H. pylori strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.
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pone.0124407.g001: Expression of JHP0290 homologues in different H. pylori strains.Whole cell lysate (A) and culture broth (Supernatant) from equal number of cells (B) of various H. pylori strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. (C) Sequence homology analysis of JHP0290 homologues in H. pylori strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.
Mentions: Sequence homology analysis indicated that JHP0290 is highly conserved among H. pylori strains. We first determined the expression of JHP0290 homologues in the whole cell extracts of several strains using the antibody raised against rJHP0290. Western blot analysis revealed that the protein was expressed by all of the strains that were tested in this study, although the expression level varied in different strains (Fig 1A). The same lysates were immunoblotted with antibodies against Urease and AhpC, which are abundantly expressed in H. pylori. The expression level of either Urease (data not shown) or AhpC (S1 Fig) did not vary significantly in the tested strains. The presence of a signal peptide (1–17 aa) in the sequence suggests that the protein is released by the bacterium. In a previous study, we reported that the JHP0290 protein of H. pylori strain J99 is released into the culture broth by the bacterium [42]. Kim et al. reported the secretion of a JHP0290 homolog (HP0305) by another H. pylori strain (NCTC 11637) [37]. To confirm the release of JHP0290 homologues by other strains of H. pylori, culture supernatants from various strains were immunoblotted with the anti-JHP0290 antibody. The release of JHP0290 homologues into the culture broth was observed for all strains (Fig 1B). Interestingly, the level of JHP0290 homologues released into the culture broth varied among strains; release did not correlate with the observed differences in expression levels. As shown in Fig 1A, the expression of the JHP0290 homologue in H. pylori strain TN2GF4 (Fig 1A, lane 3) was lower than that of the strain J99 (Fig 1A, lane 2); however, the level of protein released into the culture broth by TN2GF4 (Fig 1B, lane 3) was higher than that released by J99 (Fig 1B, lane 2). The same pattern was observed for strains P12 and HPAG1. The precise relationship between the amount of released JHP0290 protein homologues and the virulence of the bacterium requires further investigation.

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus