IADR Abstract Archives
The Role of Butyrate in Fusobacterium Nucleatum Biofilms
Objectives: To study the implication of the butyrate pathway in Fusobacterium nucleatum biofilmS, since, out of the eight enzymes that regulate the synthesis of butyrate, six of them were differentially expressed (over- and down-) when the bacteria were forming biofilms.
Methods: Monospecies biofilms of F. nucleatum DSMZ 20482 [107 colony forming units (cfu)/mL] were developed on sterile hydroxyapatite slides, while planktonic cell cultures were obtained by inoculating the medium with bacterial suspensions in sterile plastic tubes. Both were incubated in anaerobic conditions at 37 C during 24 hours. The bacterial cells were recovered and disrupted by sonication. The supernatants were concentrated and the protein concentration was determined using the Bradford assay. These protein extracts were used to measure the enzymatic activity of thiolase (acetyl-CoA acetyl transferase) and 3-hydroxybutyryl-CoA dehydrogenase. All enzyme measurements were carried out in 1-cm-path-length quartz cuvettes at room temperature using a final volume of 1 mL. The thiolase activity was determined in the thiolytic direction with acetoacetyl-CoA as substrate by monitoring the decrease in absorbance at 303 nm (DA303nm) and with 500 µg protein extract from biofilm or planktonic. In the case of the 3-hydroxybutyryl-CoA dehydrogenase activity, it was determined by measuring the decrease of β-nicotinamide adenine dinucleotide reduced (NADH) absorption at 340 nm (DA340nm) and with 350 µg protein extract from biofilm or planktonic. The enzymatic assays were repeated three times on different days. For comparing the differences in DA303nm and DA340nm between both conditions, unpaired Student’s t tests were performed.
Results: Statistically significant differences were found in the amount of both enzymes activity (thiolase, p=0.0078; 3-hydroxybutyryl-CoA dehydrogenase, p=0.0027) between biofilm and planktonic states. These results confirm the differential expression of the same proteins previously studied with proteomics tools.
Conclusions: Butyrate pathway may play a critical role in the development of F. nucleatum biofilms..
Methods: Monospecies biofilms of F. nucleatum DSMZ 20482 [107 colony forming units (cfu)/mL] were developed on sterile hydroxyapatite slides, while planktonic cell cultures were obtained by inoculating the medium with bacterial suspensions in sterile plastic tubes. Both were incubated in anaerobic conditions at 37 C during 24 hours. The bacterial cells were recovered and disrupted by sonication. The supernatants were concentrated and the protein concentration was determined using the Bradford assay. These protein extracts were used to measure the enzymatic activity of thiolase (acetyl-CoA acetyl transferase) and 3-hydroxybutyryl-CoA dehydrogenase. All enzyme measurements were carried out in 1-cm-path-length quartz cuvettes at room temperature using a final volume of 1 mL. The thiolase activity was determined in the thiolytic direction with acetoacetyl-CoA as substrate by monitoring the decrease in absorbance at 303 nm (DA303nm) and with 500 µg protein extract from biofilm or planktonic. In the case of the 3-hydroxybutyryl-CoA dehydrogenase activity, it was determined by measuring the decrease of β-nicotinamide adenine dinucleotide reduced (NADH) absorption at 340 nm (DA340nm) and with 350 µg protein extract from biofilm or planktonic. The enzymatic assays were repeated three times on different days. For comparing the differences in DA303nm and DA340nm between both conditions, unpaired Student’s t tests were performed.
Results: Statistically significant differences were found in the amount of both enzymes activity (thiolase, p=0.0078; 3-hydroxybutyryl-CoA dehydrogenase, p=0.0027) between biofilm and planktonic states. These results confirm the differential expression of the same proteins previously studied with proteomics tools.
Conclusions: Butyrate pathway may play a critical role in the development of F. nucleatum biofilms..