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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

M. hyorhinis transmission assay.A, Confocal microscopy of HTR8/SVneo trophoblasts infected with M. hyorhinis (red) jointly with the naïve, fluorescently labeled trophoblasts (green). Top panel, HTR8/SVneo trophoblasts were infected for 2 h with M. hyorhinis. Gentamicin (300 μg/ml) was added to the samples for an additional 4 h. Then gentamicin was removed and the naive fluorescently labeled trophoblasts were added to the samples. After 24 h, the secondary infection of the naïve trophoblasts was recorded. The arrows point to the sites of the secondary infection in the fluorescently labeled trophoblasts. B, The non-merged images of the fluorescently labeled trophoblast (Tracker Green) infected by M. hyorhinis (red). DAPI, blue. Bottom panels, uninfected trophoblast cell control.
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pone.0142529.g004: M. hyorhinis transmission assay.A, Confocal microscopy of HTR8/SVneo trophoblasts infected with M. hyorhinis (red) jointly with the naïve, fluorescently labeled trophoblasts (green). Top panel, HTR8/SVneo trophoblasts were infected for 2 h with M. hyorhinis. Gentamicin (300 μg/ml) was added to the samples for an additional 4 h. Then gentamicin was removed and the naive fluorescently labeled trophoblasts were added to the samples. After 24 h, the secondary infection of the naïve trophoblasts was recorded. The arrows point to the sites of the secondary infection in the fluorescently labeled trophoblasts. B, The non-merged images of the fluorescently labeled trophoblast (Tracker Green) infected by M. hyorhinis (red). DAPI, blue. Bottom panels, uninfected trophoblast cell control.

Mentions: To further investigate infectious characteristics of the internalized M. hyorhinis, we studied whether the bacteria can re-infect the naive host cells. For these purposes, we co-incubated HTR8/SVneo cells with M. hyorhinis and then eradicated planktonic mycoplasmas using gentamicin. Naive, fluorescently labeled HTR8/SVneo cells were then added to the infected cultures. The samples were co-incubated further in the gentamicin-free medium to allow re-infection of the newly added cells by the internalized and then released mycoplasmas. We observed that the fluorescently labeled fresh cells readily acquired mycoplasma (Fig 4). These results indicated that the cell internalization did not abolish the ability M. hyorhinis to exit from the infected cells and then to re-infect the additional host cells.


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)

M. hyorhinis transmission assay.A, Confocal microscopy of HTR8/SVneo trophoblasts infected with M. hyorhinis (red) jointly with the naïve, fluorescently labeled trophoblasts (green). Top panel, HTR8/SVneo trophoblasts were infected for 2 h with M. hyorhinis. Gentamicin (300 μg/ml) was added to the samples for an additional 4 h. Then gentamicin was removed and the naive fluorescently labeled trophoblasts were added to the samples. After 24 h, the secondary infection of the naïve trophoblasts was recorded. The arrows point to the sites of the secondary infection in the fluorescently labeled trophoblasts. B, The non-merged images of the fluorescently labeled trophoblast (Tracker Green) infected by M. hyorhinis (red). DAPI, blue. Bottom panels, uninfected trophoblast cell control.
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pone.0142529.g004: M. hyorhinis transmission assay.A, Confocal microscopy of HTR8/SVneo trophoblasts infected with M. hyorhinis (red) jointly with the naïve, fluorescently labeled trophoblasts (green). Top panel, HTR8/SVneo trophoblasts were infected for 2 h with M. hyorhinis. Gentamicin (300 μg/ml) was added to the samples for an additional 4 h. Then gentamicin was removed and the naive fluorescently labeled trophoblasts were added to the samples. After 24 h, the secondary infection of the naïve trophoblasts was recorded. The arrows point to the sites of the secondary infection in the fluorescently labeled trophoblasts. B, The non-merged images of the fluorescently labeled trophoblast (Tracker Green) infected by M. hyorhinis (red). DAPI, blue. Bottom panels, uninfected trophoblast cell control.
Mentions: To further investigate infectious characteristics of the internalized M. hyorhinis, we studied whether the bacteria can re-infect the naive host cells. For these purposes, we co-incubated HTR8/SVneo cells with M. hyorhinis and then eradicated planktonic mycoplasmas using gentamicin. Naive, fluorescently labeled HTR8/SVneo cells were then added to the infected cultures. The samples were co-incubated further in the gentamicin-free medium to allow re-infection of the newly added cells by the internalized and then released mycoplasmas. We observed that the fluorescently labeled fresh cells readily acquired mycoplasma (Fig 4). These results indicated that the cell internalization did not abolish the ability M. hyorhinis to exit from the infected cells and then to re-infect the additional host cells.

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