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Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans.

Shanmugam M, El Abbar F, Ramasubbu N - PLoS ONE (2015)

Bottom Line: Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence.Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth.Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, New Jersey, United States of America.

ABSTRACT
Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus

Confocal imaging analysis.a) Live/Dead staining of cells at 16 h and 48 h. Cell death (propidium iodide stain, red channel) is substantially higher at 48 h than at 16 h. At 48 h, however, there is significantly more death in EA1002 cells and the cell death is apparent throughout the biofilm colonies. In contrast, in IDH781 cells, there are more live cells at the periphery than in the middle. Cell death for the strains were comparable and similar to cell death observed at 16 h (S1 Fig). b) Cell viability for IDH781 and EA1002 strains at different time intervals of growth.
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pone.0134285.g004: Confocal imaging analysis.a) Live/Dead staining of cells at 16 h and 48 h. Cell death (propidium iodide stain, red channel) is substantially higher at 48 h than at 16 h. At 48 h, however, there is significantly more death in EA1002 cells and the cell death is apparent throughout the biofilm colonies. In contrast, in IDH781 cells, there are more live cells at the periphery than in the middle. Cell death for the strains were comparable and similar to cell death observed at 16 h (S1 Fig). b) Cell viability for IDH781 and EA1002 strains at different time intervals of growth.

Mentions: To evaluate whether the apparent attenuated peptidoglycan turnover affects the cell viability, both IDH781 and EA1002 cells grown for 16h and 48 h were analyzed. Syto 9/propidium iodide (Live/Dead) staining was used to compare the amount of cell death using confocal microscopy. The Live/Dead staining showed that while the cell death at 16 h was comparable between the two strains (IDH781 5.7±0.4% vs. EA1002 0.5±0.1%), there was drastic difference at 48 h (IDH781 71±2% vs. EA1002 99±1%; Fig 4). The cell death at 48 h for EA1002 was significantly higher (Student’s t-test, P < 0.001 Fig 4) and was distributed throughout the biofilm colonies. In contrast, in IDH781, cell death was observed in the interior of the biofilm whereas the exterior was enriched with live cells. To confirm the excessive cell death occurring in the EA1002 strain at 48 h and to estimate the amount of viable cells present in comparison to IDH781 strain, we grew the two strains ensuring that equal number of cells were used for growth. After 16 and 48 h, IDH781 and EA1002 cells were enumerated by colony counting (Fig 4b). Clearly, there was no significant difference between the numbers of viable cells at 16 h (P > 0.17) where as there were significantly more viable IDH781 cells than EA1002 at 48 h (P < 0.0001). To test whether or not the cells were at an intermediate time point in their growth, Live/Dead staining for cells grown for 24 h were performed (S1 Fig). The cell death for both IDH781 and EA1002 cells were comparable (IDH781 1.2±0.2% vs. EA1002 0.2±0.02%; P <0.0001; Figure in S1 Fig).


Transcriptome Profiling of Wild-Type and pga-Knockout Mutant Strains Reveal the Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans.

Shanmugam M, El Abbar F, Ramasubbu N - PLoS ONE (2015)

Confocal imaging analysis.a) Live/Dead staining of cells at 16 h and 48 h. Cell death (propidium iodide stain, red channel) is substantially higher at 48 h than at 16 h. At 48 h, however, there is significantly more death in EA1002 cells and the cell death is apparent throughout the biofilm colonies. In contrast, in IDH781 cells, there are more live cells at the periphery than in the middle. Cell death for the strains were comparable and similar to cell death observed at 16 h (S1 Fig). b) Cell viability for IDH781 and EA1002 strains at different time intervals of growth.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4519337&req=5

pone.0134285.g004: Confocal imaging analysis.a) Live/Dead staining of cells at 16 h and 48 h. Cell death (propidium iodide stain, red channel) is substantially higher at 48 h than at 16 h. At 48 h, however, there is significantly more death in EA1002 cells and the cell death is apparent throughout the biofilm colonies. In contrast, in IDH781 cells, there are more live cells at the periphery than in the middle. Cell death for the strains were comparable and similar to cell death observed at 16 h (S1 Fig). b) Cell viability for IDH781 and EA1002 strains at different time intervals of growth.
Mentions: To evaluate whether the apparent attenuated peptidoglycan turnover affects the cell viability, both IDH781 and EA1002 cells grown for 16h and 48 h were analyzed. Syto 9/propidium iodide (Live/Dead) staining was used to compare the amount of cell death using confocal microscopy. The Live/Dead staining showed that while the cell death at 16 h was comparable between the two strains (IDH781 5.7±0.4% vs. EA1002 0.5±0.1%), there was drastic difference at 48 h (IDH781 71±2% vs. EA1002 99±1%; Fig 4). The cell death at 48 h for EA1002 was significantly higher (Student’s t-test, P < 0.001 Fig 4) and was distributed throughout the biofilm colonies. In contrast, in IDH781, cell death was observed in the interior of the biofilm whereas the exterior was enriched with live cells. To confirm the excessive cell death occurring in the EA1002 strain at 48 h and to estimate the amount of viable cells present in comparison to IDH781 strain, we grew the two strains ensuring that equal number of cells were used for growth. After 16 and 48 h, IDH781 and EA1002 cells were enumerated by colony counting (Fig 4b). Clearly, there was no significant difference between the numbers of viable cells at 16 h (P > 0.17) where as there were significantly more viable IDH781 cells than EA1002 at 48 h (P < 0.0001). To test whether or not the cells were at an intermediate time point in their growth, Live/Dead staining for cells grown for 24 h were performed (S1 Fig). The cell death for both IDH781 and EA1002 cells were comparable (IDH781 1.2±0.2% vs. EA1002 0.2±0.02%; P <0.0001; Figure in S1 Fig).

Bottom Line: Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence.Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth.Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, New Jersey, United States of America.

ABSTRACT
Exopolysaccharides have a diverse set of functions in most bacteria including a mechanistic role in protecting bacteria against environmental stresses. Among the many functions attributed to the exopolysaccharides, biofilm formation, antibiotic resistance, immune evasion and colonization have been studied most extensively. The exopolysaccharide produced by many Gram positive as well as Gram negative bacteria including the oral pathogen Aggregatibacter actinomycetemcomitans is the homopolymer of β(1,6)-linked N-acetylglucosamine. Recently, we reported that the PGA-deficient mutant of A. actinomycetemcomitans failed to colonize or induce bone resorption in a rat model of periodontal disease, and the colonization genes, apiA and aae, were significantly down regulated in the mutant strain. To understand the role of exopolysaccharide and the pga locus in the global expression of A. actinomycetemcomitans, we have used comparative transcriptome profiling to identify differentially expressed genes in the wild-type strain in relation to the PGA-deficient strain. Transcriptome analysis revealed that about 50% of the genes are differently expressed (P < 0.05 and fold change >1.5). Our study demonstrated that the absence of the pga locus affects the genes involved in peptidoglycan recycling, glycogen storage, and virulence. Further, using confocal microscopy and plating assays, we show that the viability of pga mutant strain is significantly reduced during biofilm growth. Thus, this study highlights the importance of pga genes and the exopolysaccharide in the virulence of A. actinomycetemcomitans.

No MeSH data available.


Related in: MedlinePlus