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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

Expression of select genes in the A. actinomycetemcomitans strains.The mRNA was isolated, and relative levels of the genes critical in peptidoglycan synthesis (murA, glmS), glycogen synthesis (pgm) and other genes shown were quantified by qPCR as described in the text. Results are means ± standard deviations for triplicate cultures normalized to 16S rRNA. The fold changes in the expression levels of all represented genes are significantly different between the strains as measured by Student’s t-test.
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pone.0134285.g006: Expression of select genes in the A. actinomycetemcomitans strains.The mRNA was isolated, and relative levels of the genes critical in peptidoglycan synthesis (murA, glmS), glycogen synthesis (pgm) and other genes shown were quantified by qPCR as described in the text. Results are means ± standard deviations for triplicate cultures normalized to 16S rRNA. The fold changes in the expression levels of all represented genes are significantly different between the strains as measured by Student’s t-test.

Mentions: For qRT-PCR analysis, mRNA was isolated, transcribed into cDNA, and subjected to qRT-PCR. The results demonstrate that the critical genes for the peptidoglycan synthesis are down-regulated in EA1002 (Fig 6; see Peptidoglycan recycling and cell viability section above). Some genes involved in glycolysis and glycogen synthesis (pgm for example) are significantly up-regulated in EA1002 (lack of production of PGA) except for murA (Fig 6). The fold changes in the mRNA levels between IDH781 and the EA1002 strains in other genes such as aae, apiA, flp-1, which are involved in colonization, have previously shown to be affected as well [4]. Interestingly, the immune evasion genes, ltxA and cdtB were not significantly different [4]. Significance in the fold changes was analyzed using Student’s t-test and the P-values ranged from 0.0008 (mreB) to 0.018 (oppA). In the RNA-seq analysis, the colonization genes were significantly down-regulated (flp-1, D7S_01457, 2.5-fold, P = 8.7E-18; apiA, D7S_00446, 30.6-fold, P = 3E-154; and aae, D7S_2013, 1.5-fold, 5.4E-05) while the toxins were not (ltxA, D7S-00604, 1.2-fold, P = 0.13 and cdtB, D7S_02295, 1.3-fold and P = 0.012). Thus, the phenotypic/physiologic changes we observed in the EA1002 are likely to be reflective of the changes that occur in the pathways as outlined in Fig 3.


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)

Expression of select genes in the A. actinomycetemcomitans strains.The mRNA was isolated, and relative levels of the genes critical in peptidoglycan synthesis (murA, glmS), glycogen synthesis (pgm) and other genes shown were quantified by qPCR as described in the text. Results are means ± standard deviations for triplicate cultures normalized to 16S rRNA. The fold changes in the expression levels of all represented genes are significantly different between the strains as measured by Student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134285.g006: Expression of select genes in the A. actinomycetemcomitans strains.The mRNA was isolated, and relative levels of the genes critical in peptidoglycan synthesis (murA, glmS), glycogen synthesis (pgm) and other genes shown were quantified by qPCR as described in the text. Results are means ± standard deviations for triplicate cultures normalized to 16S rRNA. The fold changes in the expression levels of all represented genes are significantly different between the strains as measured by Student’s t-test.
Mentions: For qRT-PCR analysis, mRNA was isolated, transcribed into cDNA, and subjected to qRT-PCR. The results demonstrate that the critical genes for the peptidoglycan synthesis are down-regulated in EA1002 (Fig 6; see Peptidoglycan recycling and cell viability section above). Some genes involved in glycolysis and glycogen synthesis (pgm for example) are significantly up-regulated in EA1002 (lack of production of PGA) except for murA (Fig 6). The fold changes in the mRNA levels between IDH781 and the EA1002 strains in other genes such as aae, apiA, flp-1, which are involved in colonization, have previously shown to be affected as well [4]. Interestingly, the immune evasion genes, ltxA and cdtB were not significantly different [4]. Significance in the fold changes was analyzed using Student’s t-test and the P-values ranged from 0.0008 (mreB) to 0.018 (oppA). In the RNA-seq analysis, the colonization genes were significantly down-regulated (flp-1, D7S_01457, 2.5-fold, P = 8.7E-18; apiA, D7S_00446, 30.6-fold, P = 3E-154; and aae, D7S_2013, 1.5-fold, 5.4E-05) while the toxins were not (ltxA, D7S-00604, 1.2-fold, P = 0.13 and cdtB, D7S_02295, 1.3-fold and P = 0.012). Thus, the phenotypic/physiologic changes we observed in the EA1002 are likely to be reflective of the changes that occur in the pathways as outlined in Fig 3.

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