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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

Differential gene expression plot.The fold change in the expression of each gene is plotted against mean gene expression. Red points represent genes with significant differential expression in IDH781 and EA1002 (P < 0.05). The blue bars indicate log2 fold change at 1.5.
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pone.0134285.g001: Differential gene expression plot.The fold change in the expression of each gene is plotted against mean gene expression. Red points represent genes with significant differential expression in IDH781 and EA1002 (P < 0.05). The blue bars indicate log2 fold change at 1.5.

Mentions: To investigate the role played by PGA in A. actinomycetemcomitans, transcriptome analysis was conducted with both IDH781 and EA1002 samples taken at 16 h under in vitro biofilm condition. Our main goal was to use RNA-seq to obtain changes in pathways that might be impacted in the absence of pga genes. The A. actinomycetemcomitans genome sequence available for the strain D7S-1 was used for mapping and to identify differentially expressed genes by comparing IDH781 and EA1002 transcriptomes. Gene expression data were normalized among all replicates for differential expression analyses. A total of 1123 genes (out of 2261) were found to be differentially expressed (>1.5-fold change with a P-value less than 0.05). Of these, 579 were down-regulated and 544 genes were up-regulated in IDH781 cells compared to EA1002 (Fig 1; S1 Table). As expected, since we used a knock out strain for comparison, the pga operon genes were higher in IDH781 by as much as 4800-fold (pgaC; P = 4E-162).


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)

Differential gene expression plot.The fold change in the expression of each gene is plotted against mean gene expression. Red points represent genes with significant differential expression in IDH781 and EA1002 (P < 0.05). The blue bars indicate log2 fold change at 1.5.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134285.g001: Differential gene expression plot.The fold change in the expression of each gene is plotted against mean gene expression. Red points represent genes with significant differential expression in IDH781 and EA1002 (P < 0.05). The blue bars indicate log2 fold change at 1.5.
Mentions: To investigate the role played by PGA in A. actinomycetemcomitans, transcriptome analysis was conducted with both IDH781 and EA1002 samples taken at 16 h under in vitro biofilm condition. Our main goal was to use RNA-seq to obtain changes in pathways that might be impacted in the absence of pga genes. The A. actinomycetemcomitans genome sequence available for the strain D7S-1 was used for mapping and to identify differentially expressed genes by comparing IDH781 and EA1002 transcriptomes. Gene expression data were normalized among all replicates for differential expression analyses. A total of 1123 genes (out of 2261) were found to be differentially expressed (>1.5-fold change with a P-value less than 0.05). Of these, 579 were down-regulated and 544 genes were up-regulated in IDH781 cells compared to EA1002 (Fig 1; S1 Table). As expected, since we used a knock out strain for comparison, the pga operon genes were higher in IDH781 by as much as 4800-fold (pgaC; P = 4E-162).

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