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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 transposase targets in the nucleus of host cells via NLSs.COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp1–362 and Tnp1–230, were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp1–37 and Tnp1–224, were located in the cytoplasm.
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pone-0038974-g001: A. baumannii transposase targets in the nucleus of host cells via NLSs.COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp1–362 and Tnp1–230, were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp1–37 and Tnp1–224, were located in the cytoplasm.

Mentions: Tnp of A. baumannii ATCC 17978 (NCBI accession no. gi/126640304) was composed of 362 amino acids and was predicted to carry the putative NLSs, 225RKRKRK230[31]. To determine whether A. baumannii Tnp targeted the nuclei of host cells, the full-length A. baumannii Tnp gene was cloned into pcDNA™6.2/N-EmGFP-DEST using the Gateway recombinational cloning system (Invitrogen) and the constructed plasmids were transfected into COS-7 cells. As a control, COS-7 cells were transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST. The green fluorescent protein (GFP) composed of a molecular mass of 27 kDa, was observed in both the cytoplasm and the nucleus of COS-7 cells transfected with the destination vector. GFP behaves within the exclusion limit of NPC and passively diffuses into the nuclei of host cells, whereas GFP-tagged A. baumannii Tnp fusion proteins composed of a molecular mass of 66.8 kDa, are exclusively present in the nuclei (Fig. 1). To determine whether nuclear targeting of A. baumannii Tnp was dependent on NLSs, three mutant clones, Tnp1–37, Tnp1–224, and Tnp1–230, fused with GFP were constructed and their subcellular localization was determined by confocal laser microscopy. Two A. baumannii Tnp mutant clones without NLSs, Tnp1–37 and Tnp1–224, were present in the cytoplasm of host cells, whereas the mutant clone with NLSs, Tnp1–230, appeared in the nuclei (Fig. 1). These results suggest that A. baumannii Tnp targets the nuclei of host cells via functional NLSs.


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 transposase targets in the nucleus of host cells via NLSs.COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp1–362 and Tnp1–230, were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp1–37 and Tnp1–224, were located in the cytoplasm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369853&req=5

pone-0038974-g001: A. baumannii transposase targets in the nucleus of host cells via NLSs.COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp1–362 and Tnp1–230, were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp1–37 and Tnp1–224, were located in the cytoplasm.
Mentions: Tnp of A. baumannii ATCC 17978 (NCBI accession no. gi/126640304) was composed of 362 amino acids and was predicted to carry the putative NLSs, 225RKRKRK230[31]. To determine whether A. baumannii Tnp targeted the nuclei of host cells, the full-length A. baumannii Tnp gene was cloned into pcDNA™6.2/N-EmGFP-DEST using the Gateway recombinational cloning system (Invitrogen) and the constructed plasmids were transfected into COS-7 cells. As a control, COS-7 cells were transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST. The green fluorescent protein (GFP) composed of a molecular mass of 27 kDa, was observed in both the cytoplasm and the nucleus of COS-7 cells transfected with the destination vector. GFP behaves within the exclusion limit of NPC and passively diffuses into the nuclei of host cells, whereas GFP-tagged A. baumannii Tnp fusion proteins composed of a molecular mass of 66.8 kDa, are exclusively present in the nuclei (Fig. 1). To determine whether nuclear targeting of A. baumannii Tnp was dependent on NLSs, three mutant clones, Tnp1–37, Tnp1–224, and Tnp1–230, fused with GFP were constructed and their subcellular localization was determined by confocal laser microscopy. Two A. baumannii Tnp mutant clones without NLSs, Tnp1–37 and Tnp1–224, were present in the cytoplasm of host cells, whereas the mutant clone with NLSs, Tnp1–230, appeared in the nuclei (Fig. 1). These results suggest that A. baumannii Tnp targets the nuclei of host cells via functional NLSs.

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