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Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

Moon DC, Choi CH, Lee SM, Lee JH, Kim SI, Kim DS, Lee JC - PLoS ONE (2012)

Bottom Line: A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells.Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation.In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea.

ABSTRACT
Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

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A. baumannii OMVs deliver transposase to the nucleus of host cells.(A) TEM observation of OMVs from A. baumannii ATCC 17978. (B) Detection of A. baumannii transposase in the bacterial culture supernatant. Bacteria were cultured in LB broth and proteins in the culture supernatants were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (C) Secretion of A. baumannii transposase from bacteria via OMVs. Bacterial cell lysates (lane 1), OMVs (lane 2), and recombinant A. baumannii transposase (lane 3) were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (D) COS-7 cells were treated with A. baumannii OMVs (20 µg/ml of protein concentrations) for 24 h. Cells were fixed, permeabilized with Triton X-100, and stained with a mouse anti-A. baumannii transposase polyclonal immune sera, followed by Alexa Fluor 488-conjugated mouse immunoglobulin G (green). DAPI was used to stain the nuclei (blue). Subcellular distribution of A. baumannii transposase was analyzed by confocal microscopy. Analytical sectioning was performed from the top to the bottom of the cells. The figure represents all projections of the sections in one picture.
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pone-0038974-g002: A. baumannii OMVs deliver transposase to the nucleus of host cells.(A) TEM observation of OMVs from A. baumannii ATCC 17978. (B) Detection of A. baumannii transposase in the bacterial culture supernatant. Bacteria were cultured in LB broth and proteins in the culture supernatants were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (C) Secretion of A. baumannii transposase from bacteria via OMVs. Bacterial cell lysates (lane 1), OMVs (lane 2), and recombinant A. baumannii transposase (lane 3) were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (D) COS-7 cells were treated with A. baumannii OMVs (20 µg/ml of protein concentrations) for 24 h. Cells were fixed, permeabilized with Triton X-100, and stained with a mouse anti-A. baumannii transposase polyclonal immune sera, followed by Alexa Fluor 488-conjugated mouse immunoglobulin G (green). DAPI was used to stain the nuclei (blue). Subcellular distribution of A. baumannii transposase was analyzed by confocal microscopy. Analytical sectioning was performed from the top to the bottom of the cells. The figure represents all projections of the sections in one picture.

Mentions: Translocation to the cytoplasm of host cells is an essential step for nuclear targeting of bacterial proteins. We previously demonstrated that A. baumannii ATCC 19606T and a clinical isolate DU202 secreted OMVs [32], [33]. Since OMVs derived from A. baumannii and E. coli contained many bacterial proteins that originated from the outer membrane, periplasmic space, inner membrane, and cytoplasm [32]–[34], we determined whether A. baumannii Tnp was secreted from bacteria via OMVs. A. baumannii ATCC 17978 was cultured in Luria-Bertani (LB) broth and OMVs were purified from the culture supernatants. Transmission electron microscopic (TEM) analysis showed that A. baumannii ATCC 17978 secreted OMVs during in vitro culture (Fig. 2A). To verify the presence of OMVs, bacterial lysates, culture supernatants, and OMVs were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein profiles were different between three samples (data not shown), suggesting that OMVs purified from A. baumannii ATCC 17978 were not bacterial lysates or artifacts. To determine whether A. baumannii ATCC 17978 could secrete Tnp in vitro culture, bacterial culture supernatants were subjected to Western blot analysis using polyclonal anti-mouse A. baumannii Tnp antibody. A. baumannii Tnp, which has a molecular mass of 39.8 kDa, was detected in the bacterial culture supernatant (Fig. 2B). To verify the presence of Tnp in the A. baumannii OMVs, bacterial cell lysates and OMVs were subjected to Western blot analysis. As a control, recombinant A. baumannii Tnp, which has a molecular mass of 44.5 kDa, was used. A. baumannii Tnp with a molecular mass of 39.8 kDa was detected in both the bacterial cell lysates and OMVs (Fig. 2C). Next, to determine whether OMVs could deliver A. baumannii Tnp to host cells, COS-7 cells were treated with A. baumannii OMVs and the subcellular distribution of A. baumannii Tnp was analyzed by confocal laser microscopy. A. baumannii Tnp was mainly distributed in the nuclei of COS-7 cells (Fig. 2D). These results suggest that A. baumannii OMVs deliver their component Tnp to the cytoplasm of host cells.


Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

Moon DC, Choi CH, Lee SM, Lee JH, Kim SI, Kim DS, Lee JC - PLoS ONE (2012)

A. baumannii OMVs deliver transposase to the nucleus of host cells.(A) TEM observation of OMVs from A. baumannii ATCC 17978. (B) Detection of A. baumannii transposase in the bacterial culture supernatant. Bacteria were cultured in LB broth and proteins in the culture supernatants were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (C) Secretion of A. baumannii transposase from bacteria via OMVs. Bacterial cell lysates (lane 1), OMVs (lane 2), and recombinant A. baumannii transposase (lane 3) were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (D) COS-7 cells were treated with A. baumannii OMVs (20 µg/ml of protein concentrations) for 24 h. Cells were fixed, permeabilized with Triton X-100, and stained with a mouse anti-A. baumannii transposase polyclonal immune sera, followed by Alexa Fluor 488-conjugated mouse immunoglobulin G (green). DAPI was used to stain the nuclei (blue). Subcellular distribution of A. baumannii transposase was analyzed by confocal microscopy. Analytical sectioning was performed from the top to the bottom of the cells. The figure represents all projections of the sections in one picture.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369853&req=5

pone-0038974-g002: A. baumannii OMVs deliver transposase to the nucleus of host cells.(A) TEM observation of OMVs from A. baumannii ATCC 17978. (B) Detection of A. baumannii transposase in the bacterial culture supernatant. Bacteria were cultured in LB broth and proteins in the culture supernatants were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (C) Secretion of A. baumannii transposase from bacteria via OMVs. Bacterial cell lysates (lane 1), OMVs (lane 2), and recombinant A. baumannii transposase (lane 3) were subjected to 12% SDS-PAGE and Western blot analysis using the polyclonal anti-mouse transposase antibody. (D) COS-7 cells were treated with A. baumannii OMVs (20 µg/ml of protein concentrations) for 24 h. Cells were fixed, permeabilized with Triton X-100, and stained with a mouse anti-A. baumannii transposase polyclonal immune sera, followed by Alexa Fluor 488-conjugated mouse immunoglobulin G (green). DAPI was used to stain the nuclei (blue). Subcellular distribution of A. baumannii transposase was analyzed by confocal microscopy. Analytical sectioning was performed from the top to the bottom of the cells. The figure represents all projections of the sections in one picture.
Mentions: Translocation to the cytoplasm of host cells is an essential step for nuclear targeting of bacterial proteins. We previously demonstrated that A. baumannii ATCC 19606T and a clinical isolate DU202 secreted OMVs [32], [33]. Since OMVs derived from A. baumannii and E. coli contained many bacterial proteins that originated from the outer membrane, periplasmic space, inner membrane, and cytoplasm [32]–[34], we determined whether A. baumannii Tnp was secreted from bacteria via OMVs. A. baumannii ATCC 17978 was cultured in Luria-Bertani (LB) broth and OMVs were purified from the culture supernatants. Transmission electron microscopic (TEM) analysis showed that A. baumannii ATCC 17978 secreted OMVs during in vitro culture (Fig. 2A). To verify the presence of OMVs, bacterial lysates, culture supernatants, and OMVs were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein profiles were different between three samples (data not shown), suggesting that OMVs purified from A. baumannii ATCC 17978 were not bacterial lysates or artifacts. To determine whether A. baumannii ATCC 17978 could secrete Tnp in vitro culture, bacterial culture supernatants were subjected to Western blot analysis using polyclonal anti-mouse A. baumannii Tnp antibody. A. baumannii Tnp, which has a molecular mass of 39.8 kDa, was detected in the bacterial culture supernatant (Fig. 2B). To verify the presence of Tnp in the A. baumannii OMVs, bacterial cell lysates and OMVs were subjected to Western blot analysis. As a control, recombinant A. baumannii Tnp, which has a molecular mass of 44.5 kDa, was used. A. baumannii Tnp with a molecular mass of 39.8 kDa was detected in both the bacterial cell lysates and OMVs (Fig. 2C). Next, to determine whether OMVs could deliver A. baumannii Tnp to host cells, COS-7 cells were treated with A. baumannii OMVs and the subcellular distribution of A. baumannii Tnp was analyzed by confocal laser microscopy. A. baumannii Tnp was mainly distributed in the nuclei of COS-7 cells (Fig. 2D). These results suggest that A. baumannii OMVs deliver their component Tnp to the cytoplasm of host cells.

Bottom Line: A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells.Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation.In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Kyungpook National University School of Medicine, Daegu, Korea.

ABSTRACT
Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

Show MeSH
Related in: MedlinePlus