IADR Abstract Archives
Study of the Differential Expression of Proteins of Aggregatibacter actinomycetemcomitrans in the Biofilm vs. Planktonic State
Objectives: To study the differential expression of proteins of Aggregatibacter actinomycetemcomitans when the bacteria is forming biofilm when compared to planktonic state using proteomic tools.
Methods: Biofilms of A. actinomycetemcomitans DSM8324 were developed on sterile plasma coated hydroxyapatite slides immersed in the modified BHI medium with bacteria suspension (107 cfu/mL) and the planktonic cell cultures were obtained by inoculating modified BHI medium with bacteria suspension (107 cfu/mL) in 50 mL sterile plastic tubes. Both of them were incubated in anaerobic conditions at 37C during 24 hours (four biological replicates for each condition). The biofilm and planktonic cells were broken by sonication to extract the proteins. Then the samples were cleaned, the proteins were solubilised and were quantified using the Bradford method. The study of differential expression was performed using 2D-DIGE ™ technology. For this, the proteins were labelled with Cy3, Cy5 and Cy2 dyes and these were separated by two-dimensional polyacrylamide gel electrophoresis. For the analysis of scanned images of the gels the DeCyder ™ software was used. In the same software, the statistical analysis (T-Student test) were performed considering 1.5 as threshold value and P<0.05. The differential proteins were identified by mass spectrometry.
Results: 87 differentially expressed proteins were observed, 13 were overexpressed when the bacteria were grown in biofilm and 34 were repressed. Most of overexpressed proteins in biofilm were outer membrane proteins (OMPs) and proteins having an immunogenic character in A. actinomycetemcomitans, such as YaeT (OMP), FtsZ, OMP39, OMP18/16, the chaperone GroEL and OMPA.
Conclusions: Differential protein expression was observed with A. actinomycetemcomitans immunogenic character in biofilm against planktonic state that could be involved in the virulence of the bacteria, and therefore may have a future therapeutic application.
Methods: Biofilms of A. actinomycetemcomitans DSM8324 were developed on sterile plasma coated hydroxyapatite slides immersed in the modified BHI medium with bacteria suspension (107 cfu/mL) and the planktonic cell cultures were obtained by inoculating modified BHI medium with bacteria suspension (107 cfu/mL) in 50 mL sterile plastic tubes. Both of them were incubated in anaerobic conditions at 37C during 24 hours (four biological replicates for each condition). The biofilm and planktonic cells were broken by sonication to extract the proteins. Then the samples were cleaned, the proteins were solubilised and were quantified using the Bradford method. The study of differential expression was performed using 2D-DIGE ™ technology. For this, the proteins were labelled with Cy3, Cy5 and Cy2 dyes and these were separated by two-dimensional polyacrylamide gel electrophoresis. For the analysis of scanned images of the gels the DeCyder ™ software was used. In the same software, the statistical analysis (T-Student test) were performed considering 1.5 as threshold value and P<0.05. The differential proteins were identified by mass spectrometry.
Results: 87 differentially expressed proteins were observed, 13 were overexpressed when the bacteria were grown in biofilm and 34 were repressed. Most of overexpressed proteins in biofilm were outer membrane proteins (OMPs) and proteins having an immunogenic character in A. actinomycetemcomitans, such as YaeT (OMP), FtsZ, OMP39, OMP18/16, the chaperone GroEL and OMPA.
Conclusions: Differential protein expression was observed with A. actinomycetemcomitans immunogenic character in biofilm against planktonic state that could be involved in the virulence of the bacteria, and therefore may have a future therapeutic application.