Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation

Abstract

Calcium (Ca(2+)) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca(2+) and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca(2+) binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca(2+) had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca(2+) at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (ΔG) values in iTC data confirmed that the interaction between DNA and Ca(2+) is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca(2+) alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca(2+) to eDNA thereby mediating bacterial aggregation and biofilm formation.

Figure 1. Quantification of eDNA in planktonic growth culture.

Concentration of eDNA in supernatants of planktonic cultures of Gram-negative and Gram-positive bacterial strains grown for 24 h. Error bars represent standard deviations from multiple cultures (n = 4). Asterisks indicate statistically significant differences (p<0.05) in eDNA concentration in comparison to S. aureus, S. epidermidis and E. faecalis whereas, hash indicates P. aeruginosa supernatants has statistically significantly higher concentration of eDNA in comparison to all other bacterial strains.

Figure 2. Influence of naturally occurring eDNA and added Ca²⁺ on bacterial aggregation.

Percentage reduction in optical density of Gram-negative and Gram-positive bacteria in PBS, showing patterns of aggregation. The black, white and grey bars represent bacterial aggregation in presence of naturally occurring eDNA, absence of naturally occurring eDNA (DNase I treated) and heat inactivated DNase I treated respectively. Error bars represents standard deviations from multiple cultures (n = 5). Asterisks and hash indicate statistically significant differences (p<0.05) between data obtained in the presence (including heat inactivated DNase I) of eDNA in comparison to absence of eDNA and in presence and absence of Ca²⁺ respectively (A). Fluorescence microscopy imaging showing patterns of S. aureus and E. faecalis adhesion and aggregation in presence and absence of eDNA and added Ca²⁺ (1000 μM) on 6 polystyrene well plates surfaces after incubation for 90 min in room temperature under static condition (scale bar 50 μm) (B and C).

Figure 3. Influence of exogenous addition of DNA and Ca²⁺ on bacterial aggregation.

Percentage reduction in optical density after 90²⁺ (1000 μM). Error bars represent standard deviations from multiple culture (n = 4). Asterisks and hash indicate statistically significant differences (P<0.05) between data obtained in the presence or absence of exogenous DNA and in presence and absence of Ca²⁺ respectively.

Figure 4. Interfacial free energy of aggregation of P. aeruginosa wildtype.

The interfacial free energy of aggregation in the presence and absence of eDNA and Ca²⁺ (1000 μM) includes components: Lifshitz-Van der Waals (LW ΔG) (A) and acid-base (AB ΔG) (B) and total interfacial free energy (Total ΔG) (C) of aggregation of PA14. Error bars represents standard deviations from the mean (n = 4). Asterisks indicate statistically significant (p<0.05) differences in the free energy of aggregation in comparison to absence of naturally occurring eDNA, regardless of presence of Ca²⁺.

Figure 5. Thermodynamic of binding of Ca²⁺ with DNA.

Isothermal titration calorimetry (iTC) studies to evaluate the interaction between DNA and Ca²⁺. Upper panel: Raw data for the titration of total 200 μl DNA (50 ng/μl) with total 1200 μM Ca²⁺. Lower panel: Integrated, dilution-corrected and concentration normalized titration data of the DNA with Ca²⁺. Data were fitted with the “One binding site model” of the Origin 7.0 data analysis software (MicroCal) with derived thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) and showing number of moles of Ca²⁺ binding to per mole of DNA at 25°C.

Figure 6. Influence of Ca²⁺ in biofilm formation before and after DNase I treatment and eDNA concentration during biofilm growth.

Biofilm biomass quantification over 24(black) or absence (white) of naturally occurring eDNA (A). Error bars represents standard deviations from multiple cultures (n = 5). Asterisks and hash indicate statistically significant (P<0.05) differences between data obtained in the presence or absence of eDNA and in presence or absence of Ca²⁺ respectively. Concentration of eDNA at different growth time of biofilm formation for Gram-negative and Gram-positive bacterial strains (B). Error bars represent standard deviations from multiple cultures (n = 4). Asterisks indicate statistically significant (p<0.05) differences in eDNA concentration in comparison to both S. aureus and E. faecalis. Hash indicates the difference is statistically significant only in comparison to E. faecalis.

Figure 7. eDNA mediated bacterial aggregation via acid-base interactions and Ca²⁺ assisted cationic bridging.

Schematic representation showing removal of eDNA influence acid-base interactions, and Ca²⁺ mediated cationic bridging between bacterial cells (B, D) and consequently bacterial aggregation (A, C).