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

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.

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

“…These findings therefore provide a functional link between natural transformation and biofilm formation. In another report, transcriptome analysis of a pga mutant revealed substantial differential gene expression compared to the wild type (74). As the pga regulon does not encompass tfoX (74), this suggests that tfoX may potentially regulate, or at least act upstream of, pga.…”

“…In another report, transcriptome analysis of a pga mutant revealed substantial differential gene expression compared to the wild type (74). As the pga regulon does not encompass tfoX (74), this suggests that tfoX may potentially regulate, or at least act upstream of, pga. Unfortunately, a tfoX consensus binding site is not available (75), and so we were unable to determine if tfoX binds to the pga promoter.…”

Role of de‐N‐acetylase PgaB from Aggregatibacter actinomycetemcomitans in exopolysaccharide export in biofilm mode of growth

“…For in vitro biofilm growth and cell harvest, inocula obtained from cultures grown as described above were used (3 μL for 45 mL of TSB medium). For biofilm formation experiments, 45 mL of inoculated medium was poured onto 15‐cm polystyrene plates and incubated at 37°C, 10% CO 2 /90% air atmosphere conditions . The spent medium was removed after 16 hours and plates were washed with ice‐cold phosphate‐buffered saline (PBS).…”

“…For assaying the surface attachment of the bacterial cells, 200 μL aliquots of cultures (IDH 781, ∆NpgaB and EA1002) were transferred to the wells of a 96‐well polystyrene microtitre plate (Falcon) and incubated for 24 hours. The biofilm cells were washed with water, air‐dried and stained with 100 μL of 1% Crystal violet (to estimate biofilm) or 100 μL (2 mg mL −1 ) Congo red dye (to estimate exopolysaccharide) for 10 minutes at room temperature . The excess stain was washed thoroughly with water and the bound dye was solubilized in 200 μL of 100% dimethylsulfoxide (Congo red) or 200 μL of 33% acetic acid (Crystal violet).…”

“…Recently, the effect of pga locus on the gene regulation at genome level of A. actinomycetemcomitans was investigated using RNA‐Seq and the differentially expressed genes between mutant strain EA1002 that lacks the pga operon and wild‐type strain IDH781 were analyzed . We demonstrated that the pga locus is involved in bacterial virulent gene regulation in A. actinomycetemcomitans and that the absence of PNAG production resulted in reduced colonization and bone loss in a rat model of periodontitis .…”