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Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Fujiwara N, Ohara N, Ogawa M, Maeda S, Naka T, Taniguchi H, Yamamoto S, Ayata M - PLoS ONE (2015)

Bottom Line: Mycobacterium smegmatis has been widely used as a mycobacterial infection model.The mps1-complemented J15cs mutant restored the expression of GPLs.The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Nutrition, Faculty of Contemporary Human Life Science, Tezukayama University, Nara City, Nara, Japan; Department of Bacteriology, Osaka City University Graduate School of Medicine, Osaka City, Osaka, Japan.

ABSTRACT
Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc(2)155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc(2)155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc(2)155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

No MeSH data available.


Related in: MedlinePlus

Survival of mps1-complemented J15cs mutant in J774.1 cells.The J774.1 cells were grown on 24-well flat-bottom tissue culture plates. A bacterial single-cell suspension of approximately 1×107 CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed, and the infected J774.1 cells were further incubated for 2 h with the medium plus 200 μg/ml gentamicin. The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells cultured for 8 days. At various time intervals after inoculation, the adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The bacterial burden was evaluated by counting CFU. The separate experiments were done in triplicate, and statistical analysis was performed using Dunnett’s test. * and ** indicate a p value of <0.05, compared to the J15cs strain inserted with a pNN2 vector only respectively.
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pone.0126813.g006: Survival of mps1-complemented J15cs mutant in J774.1 cells.The J774.1 cells were grown on 24-well flat-bottom tissue culture plates. A bacterial single-cell suspension of approximately 1×107 CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed, and the infected J774.1 cells were further incubated for 2 h with the medium plus 200 μg/ml gentamicin. The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells cultured for 8 days. At various time intervals after inoculation, the adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The bacterial burden was evaluated by counting CFU. The separate experiments were done in triplicate, and statistical analysis was performed using Dunnett’s test. * and ** indicate a p value of <0.05, compared to the J15cs strain inserted with a pNN2 vector only respectively.

Mentions: To determine the effect of GPLs on bacterial survival in host cells, J774.1 cells were infected with the mps1-complemented J15cs mutant. Surprisingly, this GPL-restored J15cs mutant did not survive as long as the parent J15cs strain. On day 2 after infection, the survival rate of the GPL-restored J15cs mutant was significantly lower than that of the parent strain, and the survival curve was almost the same as that of the mc2155 strain that expressed GPLs (Fig 6).


Glycopeptidolipid of Mycobacterium smegmatis J15cs Affects Morphology and Survival in Host Cells.

Fujiwara N, Ohara N, Ogawa M, Maeda S, Naka T, Taniguchi H, Yamamoto S, Ayata M - PLoS ONE (2015)

Survival of mps1-complemented J15cs mutant in J774.1 cells.The J774.1 cells were grown on 24-well flat-bottom tissue culture plates. A bacterial single-cell suspension of approximately 1×107 CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed, and the infected J774.1 cells were further incubated for 2 h with the medium plus 200 μg/ml gentamicin. The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells cultured for 8 days. At various time intervals after inoculation, the adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The bacterial burden was evaluated by counting CFU. The separate experiments were done in triplicate, and statistical analysis was performed using Dunnett’s test. * and ** indicate a p value of <0.05, compared to the J15cs strain inserted with a pNN2 vector only respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4430512&req=5

pone.0126813.g006: Survival of mps1-complemented J15cs mutant in J774.1 cells.The J774.1 cells were grown on 24-well flat-bottom tissue culture plates. A bacterial single-cell suspension of approximately 1×107 CFU/well was inoculated into the J774.1 cells at an approximate multiplicity of infection 10. After cocultivation for 3 h, the infected J774.1 cells were washed, and the infected J774.1 cells were further incubated for 2 h with the medium plus 200 μg/ml gentamicin. The medium was replaced with fresh medium containing 2 μg/ml of gentamicin (this time point was day 0), and the infected cells cultured for 8 days. At various time intervals after inoculation, the adherent cells were treated with 1% Triton-X100/PBS, and were sonicated. The bacterial burden was evaluated by counting CFU. The separate experiments were done in triplicate, and statistical analysis was performed using Dunnett’s test. * and ** indicate a p value of <0.05, compared to the J15cs strain inserted with a pNN2 vector only respectively.
Mentions: To determine the effect of GPLs on bacterial survival in host cells, J774.1 cells were infected with the mps1-complemented J15cs mutant. Surprisingly, this GPL-restored J15cs mutant did not survive as long as the parent J15cs strain. On day 2 after infection, the survival rate of the GPL-restored J15cs mutant was significantly lower than that of the parent strain, and the survival curve was almost the same as that of the mc2155 strain that expressed GPLs (Fig 6).

Bottom Line: Mycobacterium smegmatis has been widely used as a mycobacterial infection model.The mps1-complemented J15cs mutant restored the expression of GPLs.The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Nutrition, Faculty of Contemporary Human Life Science, Tezukayama University, Nara City, Nara, Japan; Department of Bacteriology, Osaka City University Graduate School of Medicine, Osaka City, Osaka, Japan.

ABSTRACT
Mycobacterium smegmatis has been widely used as a mycobacterial infection model. Unlike the M. smegmatis mc(2)155 strain, M. smegmatis J15cs strain has the advantage of surviving for one week in murine macrophages. In our previous report, we clarified that the J15cs strain has deleted apolar glycopeptidolipids (GPLs) in the cell wall, which may affect its morphology and survival in host cells. In this study, the gene causing the GPL deletion in the J15cs strain was identified. The mps1-2 gene (MSMEG_0400-0402) correlated with GPL biosynthesis. The J15cs strain had 18 bps deleted in the mps1 gene compared to that of the mc(2)155 strain. The mps1-complemented J15cs mutant restored the expression of GPLs. Although the J15cs strain produces a rough and dry colony, the colony morphology of this mps1-complement was smooth like the mc(2)155 strain. The length in the mps1-complemented J15cs mutant was shortened by the expression of GPLs. In addition, the GPL-restored J15cs mutant did not survive as long as the parent J15cs strain in the murine macrophage cell line J774.1 cells. The results are direct evidence that the deletion of GPLs in the J15cs strain affects bacterial size, morphology, and survival in host cells.

No MeSH data available.


Related in: MedlinePlus