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Depletion of CG-Specific Methylation in Mycoplasma hyorhinis Genomic DNA after Host Cell Invasion.

Chernov AV, Reyes L, Peterson S, Strongin AY - PLoS ONE (2015)

Bottom Line: We determined that genomic GATC sites were consistently highly methylated in the bacterial chromosome suggesting that the bacterial GATC-specific 5-methylcytosine DNA methyltransferase was fully functional both pre- and post-infection.In turn, two distinct populations, with either high or low CG methylation, were detected in the M. hyorhinis cultures continually grown in the rich medium independently of host cells.We also identified that M. hyorhinis efficiently evaded endosomal degradation and uses exocytosis to exit infected human cells enabling re-infection of additional cells.

View Article: PubMed Central - PubMed

Affiliation: Infectious & Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America.

ABSTRACT
Adaptation to the environment requires pathogenic bacteria to alter their gene expression in order to increase long-term survival in the host. Here, we present the first experimental evidence that bacterial DNA methylation affects the intracellular survival of pathogenic Mycoplasma hyorhinis. Using bisulfite sequencing, we identified that the M. hyorhinis DNA methylation landscape was distinct in free-living M. hyorhinis relative to the internalized bacteria surviving in the infected human cells. We determined that genomic GATC sites were consistently highly methylated in the bacterial chromosome suggesting that the bacterial GATC-specific 5-methylcytosine DNA methyltransferase was fully functional both pre- and post-infection. In contrast, only the low CG methylation pattern was observed in the mycoplasma genome in the infective bacteria that invaded and then survived in the host cells. In turn, two distinct populations, with either high or low CG methylation, were detected in the M. hyorhinis cultures continually grown in the rich medium independently of host cells. We also identified that M. hyorhinis efficiently evaded endosomal degradation and uses exocytosis to exit infected human cells enabling re-infection of additional cells. The well-orchestrated changes in the chromosome methylation landscape play a major regulatory role in the mycoplasma life cycle.

No MeSH data available.


Related in: MedlinePlus

Methylation of M. hyorhinis genomic DNA.A and B, schematic diagrams show the presence of two GATC and 27 CG methylation sites in the 368 bp CG-rich region of the M. hyorhinis genome in free-living (A) and intracellular (B) mycoplasmas. Circles indicate methylated CGs and GATC sites (red and blue, respectively), and unmethylated (empty) sites. Each row corresponds to a methylation pattern in the individual mycoplasma cell. Bottom, a fragment of the sequencing chromatogram of the bisulfate-converted DNA confirms the presence of the methylated GATC and CG sites in the 368 bp CG-rich region of the M. hyorhinis genome. Stars, 5mC. C, Circular histogram shows the distribution of the CG and GATC sites in the M. hyorhinis genome. Histogram bar heights correspond to a number of the individual CG and GATC marks (light red and light blue, respectively) within a 1 kb window. Regions with >10 sites are shown in dark red (CG) and dark blue (GATC). Genes are named and their positions are shown along the chromosome as orange bars. In the genomic DNA, positions of mycoplasma CPGI-like regions (mCPGIs) are indicated by thin red lines on the white background. The histogram was generated using Circos [26]. D, Dinucleotide analysis of M. hyorhinis genome.
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pone.0142529.g005: Methylation of M. hyorhinis genomic DNA.A and B, schematic diagrams show the presence of two GATC and 27 CG methylation sites in the 368 bp CG-rich region of the M. hyorhinis genome in free-living (A) and intracellular (B) mycoplasmas. Circles indicate methylated CGs and GATC sites (red and blue, respectively), and unmethylated (empty) sites. Each row corresponds to a methylation pattern in the individual mycoplasma cell. Bottom, a fragment of the sequencing chromatogram of the bisulfate-converted DNA confirms the presence of the methylated GATC and CG sites in the 368 bp CG-rich region of the M. hyorhinis genome. Stars, 5mC. C, Circular histogram shows the distribution of the CG and GATC sites in the M. hyorhinis genome. Histogram bar heights correspond to a number of the individual CG and GATC marks (light red and light blue, respectively) within a 1 kb window. Regions with >10 sites are shown in dark red (CG) and dark blue (GATC). Genes are named and their positions are shown along the chromosome as orange bars. In the genomic DNA, positions of mycoplasma CPGI-like regions (mCPGIs) are indicated by thin red lines on the white background. The histogram was generated using Circos [26]. D, Dinucleotide analysis of M. hyorhinis genome.

Mentions: To reveal the functionality of mycoplasma MTases in the course of host cell infection, we determined the methylation pattern in the genomic DNA of planktonic versus intracellular M. hyorhinis. For these purposes, we analyzed a 368 bp CG-rich region of the M. hyorhinis genome by bisulfite sequencing that included two GATC and 27 CG sites. In all cultured bacteria, GATC sites were fully methylated (Fig 5A). As based on sequencing of individual colonies, half of the in vitro grown bacteria exhibited complete methylation of 27 CG sites. Remarkably, in approximately half of the cultured bacteria, CG methylation was limited.


Depletion of CG-Specific Methylation in Mycoplasma hyorhinis Genomic DNA after Host Cell Invasion.

Chernov AV, Reyes L, Peterson S, Strongin AY - PLoS ONE (2015)

Methylation of M. hyorhinis genomic DNA.A and B, schematic diagrams show the presence of two GATC and 27 CG methylation sites in the 368 bp CG-rich region of the M. hyorhinis genome in free-living (A) and intracellular (B) mycoplasmas. Circles indicate methylated CGs and GATC sites (red and blue, respectively), and unmethylated (empty) sites. Each row corresponds to a methylation pattern in the individual mycoplasma cell. Bottom, a fragment of the sequencing chromatogram of the bisulfate-converted DNA confirms the presence of the methylated GATC and CG sites in the 368 bp CG-rich region of the M. hyorhinis genome. Stars, 5mC. C, Circular histogram shows the distribution of the CG and GATC sites in the M. hyorhinis genome. Histogram bar heights correspond to a number of the individual CG and GATC marks (light red and light blue, respectively) within a 1 kb window. Regions with >10 sites are shown in dark red (CG) and dark blue (GATC). Genes are named and their positions are shown along the chromosome as orange bars. In the genomic DNA, positions of mycoplasma CPGI-like regions (mCPGIs) are indicated by thin red lines on the white background. The histogram was generated using Circos [26]. D, Dinucleotide analysis of M. hyorhinis genome.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636357&req=5

pone.0142529.g005: Methylation of M. hyorhinis genomic DNA.A and B, schematic diagrams show the presence of two GATC and 27 CG methylation sites in the 368 bp CG-rich region of the M. hyorhinis genome in free-living (A) and intracellular (B) mycoplasmas. Circles indicate methylated CGs and GATC sites (red and blue, respectively), and unmethylated (empty) sites. Each row corresponds to a methylation pattern in the individual mycoplasma cell. Bottom, a fragment of the sequencing chromatogram of the bisulfate-converted DNA confirms the presence of the methylated GATC and CG sites in the 368 bp CG-rich region of the M. hyorhinis genome. Stars, 5mC. C, Circular histogram shows the distribution of the CG and GATC sites in the M. hyorhinis genome. Histogram bar heights correspond to a number of the individual CG and GATC marks (light red and light blue, respectively) within a 1 kb window. Regions with >10 sites are shown in dark red (CG) and dark blue (GATC). Genes are named and their positions are shown along the chromosome as orange bars. In the genomic DNA, positions of mycoplasma CPGI-like regions (mCPGIs) are indicated by thin red lines on the white background. The histogram was generated using Circos [26]. D, Dinucleotide analysis of M. hyorhinis genome.
Mentions: To reveal the functionality of mycoplasma MTases in the course of host cell infection, we determined the methylation pattern in the genomic DNA of planktonic versus intracellular M. hyorhinis. For these purposes, we analyzed a 368 bp CG-rich region of the M. hyorhinis genome by bisulfite sequencing that included two GATC and 27 CG sites. In all cultured bacteria, GATC sites were fully methylated (Fig 5A). As based on sequencing of individual colonies, half of the in vitro grown bacteria exhibited complete methylation of 27 CG sites. Remarkably, in approximately half of the cultured bacteria, CG methylation was limited.

Bottom Line: We determined that genomic GATC sites were consistently highly methylated in the bacterial chromosome suggesting that the bacterial GATC-specific 5-methylcytosine DNA methyltransferase was fully functional both pre- and post-infection.In turn, two distinct populations, with either high or low CG methylation, were detected in the M. hyorhinis cultures continually grown in the rich medium independently of host cells.We also identified that M. hyorhinis efficiently evaded endosomal degradation and uses exocytosis to exit infected human cells enabling re-infection of additional cells.

View Article: PubMed Central - PubMed

Affiliation: Infectious & Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California, United States of America.

ABSTRACT
Adaptation to the environment requires pathogenic bacteria to alter their gene expression in order to increase long-term survival in the host. Here, we present the first experimental evidence that bacterial DNA methylation affects the intracellular survival of pathogenic Mycoplasma hyorhinis. Using bisulfite sequencing, we identified that the M. hyorhinis DNA methylation landscape was distinct in free-living M. hyorhinis relative to the internalized bacteria surviving in the infected human cells. We determined that genomic GATC sites were consistently highly methylated in the bacterial chromosome suggesting that the bacterial GATC-specific 5-methylcytosine DNA methyltransferase was fully functional both pre- and post-infection. In contrast, only the low CG methylation pattern was observed in the mycoplasma genome in the infective bacteria that invaded and then survived in the host cells. In turn, two distinct populations, with either high or low CG methylation, were detected in the M. hyorhinis cultures continually grown in the rich medium independently of host cells. We also identified that M. hyorhinis efficiently evaded endosomal degradation and uses exocytosis to exit infected human cells enabling re-infection of additional cells. The well-orchestrated changes in the chromosome methylation landscape play a major regulatory role in the mycoplasma life cycle.

No MeSH data available.


Related in: MedlinePlus