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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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Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene.(A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods. Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp1–37; 7, A549 cells transfected with plasmid constructs of Tnp1–224; 8, A549 cells transfected with plasmid constructs of Tnp1–230; 9, A549 cells transfected with plasmid constructs of Tnp1–362. (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods. Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p<0.05).
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pone-0038974-g003: Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene.(A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods. Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp1–37; 7, A549 cells transfected with plasmid constructs of Tnp1–224; 8, A549 cells transfected with plasmid constructs of Tnp1–230; 9, A549 cells transfected with plasmid constructs of Tnp1–362. (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods. Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p<0.05).

Mentions: To determine whether nuclear targeting of A. baumannii Tnp induced cellular damage, cells were transfected with plasmid constructs containing the full-length A. baumannii Tnp gene cloned in the pcDNA™6.2/N-EmGFP-DEST and incubated for 48 h. The viability of COS-7 cells transfected with the full-length A. baumannii Tnp gene was slightly increased (126±2.8%) as compared to that of COS-7 cells transfected with the empty destination vector. Expression of A. baumannii Tnp fused with GFP in the nuclei of A549 cells did not induce any morphological change relative to control cells transfected with the destination vector (Fig. 1). To determine whether A. baumannii Tnp induced epigenetic changes in host cells, A549 cells were transfected with plasmid constructs of the full-length A. baumannii Tnp gene cloned in pcDNA™6.2/N-EmGFP-DEST and incubated for 48 h. A549 cells that originated from human lung carcinoma were used because the respiratory tract is the most common infection site of A. baumannii[2]. Genomic DNA was extracted from A549 cells and methylation-specific polymerase chain reaction (MSP) was performed using primers specific for the CpG regions of p16INK4A, hMLH1, and E-cadherin genes, which are involved in inhibiting cell cycle progression, DNA mismatch repair, and adhesion of epithelial cells to one another, respectively [35]–[39]. A. baumannii Tnp specifically induced DNA methylation of CpG regions in the promoters of E-cadherin gene (Fig. 3A), but not in CpG regions of p16INK4A and hMLH1 (data not shown). To determine whether DNA methylation of CpG regions in the promoter of E-cadherin gene was dependent on nuclear targeting of A. baumannii Tnp, A549 cells were transfected with three mutant clones, Tnp1–37, Tnp1–224, and Tnp1–230, fused with GFP and then MSP specific for the CpG regions of E-cadherin gene was performed. The truncated Tnp1–230 with NLSs induced DNA methylation, whereas the two mutant clones without NLSs, Tnp1–37 and Tnp1–224, did not induce DNA methylation (Fig. 3A). An aberrant DNA methylation in the promoters of genes can down-regulate transcription level. We determined mRNA expression of E-cadherin gene in A549 cells transfected with plasmid constructs of the full-length of A. baumannii Tnp fused with GFP. When transfection efficiency reached to 60–70%, total RNA of cells was harvested and quantitative reverse transcriptase-PCR (qRT-PCR) was performed. As a control, A549 cells were transfected with pcDNA™6.2/N-EmGFP-DEST vector. A. baumannii Tnp down-regulated mRNA expression of E-cadherin gene (0.82±0.16) as compared to the empty destination vector (1.0±0.06) (p<0.05) (Fig. 3B). These results suggest that nuclear targeting of A. baumannii Tnp specifically induces DNA methylation of CpG regions in the promoters of E-cadherin gene and then down-regulates gene expression.


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)

Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene.(A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods. Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp1–37; 7, A549 cells transfected with plasmid constructs of Tnp1–224; 8, A549 cells transfected with plasmid constructs of Tnp1–230; 9, A549 cells transfected with plasmid constructs of Tnp1–362. (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods. Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p<0.05).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369853&req=5

pone-0038974-g003: Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene.(A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods. Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp1–37; 7, A549 cells transfected with plasmid constructs of Tnp1–224; 8, A549 cells transfected with plasmid constructs of Tnp1–230; 9, A549 cells transfected with plasmid constructs of Tnp1–362. (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods. Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p<0.05).
Mentions: To determine whether nuclear targeting of A. baumannii Tnp induced cellular damage, cells were transfected with plasmid constructs containing the full-length A. baumannii Tnp gene cloned in the pcDNA™6.2/N-EmGFP-DEST and incubated for 48 h. The viability of COS-7 cells transfected with the full-length A. baumannii Tnp gene was slightly increased (126±2.8%) as compared to that of COS-7 cells transfected with the empty destination vector. Expression of A. baumannii Tnp fused with GFP in the nuclei of A549 cells did not induce any morphological change relative to control cells transfected with the destination vector (Fig. 1). To determine whether A. baumannii Tnp induced epigenetic changes in host cells, A549 cells were transfected with plasmid constructs of the full-length A. baumannii Tnp gene cloned in pcDNA™6.2/N-EmGFP-DEST and incubated for 48 h. A549 cells that originated from human lung carcinoma were used because the respiratory tract is the most common infection site of A. baumannii[2]. Genomic DNA was extracted from A549 cells and methylation-specific polymerase chain reaction (MSP) was performed using primers specific for the CpG regions of p16INK4A, hMLH1, and E-cadherin genes, which are involved in inhibiting cell cycle progression, DNA mismatch repair, and adhesion of epithelial cells to one another, respectively [35]–[39]. A. baumannii Tnp specifically induced DNA methylation of CpG regions in the promoters of E-cadherin gene (Fig. 3A), but not in CpG regions of p16INK4A and hMLH1 (data not shown). To determine whether DNA methylation of CpG regions in the promoter of E-cadherin gene was dependent on nuclear targeting of A. baumannii Tnp, A549 cells were transfected with three mutant clones, Tnp1–37, Tnp1–224, and Tnp1–230, fused with GFP and then MSP specific for the CpG regions of E-cadherin gene was performed. The truncated Tnp1–230 with NLSs induced DNA methylation, whereas the two mutant clones without NLSs, Tnp1–37 and Tnp1–224, did not induce DNA methylation (Fig. 3A). An aberrant DNA methylation in the promoters of genes can down-regulate transcription level. We determined mRNA expression of E-cadherin gene in A549 cells transfected with plasmid constructs of the full-length of A. baumannii Tnp fused with GFP. When transfection efficiency reached to 60–70%, total RNA of cells was harvested and quantitative reverse transcriptase-PCR (qRT-PCR) was performed. As a control, A549 cells were transfected with pcDNA™6.2/N-EmGFP-DEST vector. A. baumannii Tnp down-regulated mRNA expression of E-cadherin gene (0.82±0.16) as compared to the empty destination vector (1.0±0.06) (p<0.05) (Fig. 3B). These results suggest that nuclear targeting of A. baumannii Tnp specifically induces DNA methylation of CpG regions in the promoters of E-cadherin gene and then down-regulates gene expression.

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