Theerthankar Das

Role of Extracellular DNA in Initial Bacterial Adhesion and Surface Aggregation

ABSTRACT Extracellular DNA (eDNA) is an important component of the biofilm matrix. We show that removal of eDNA from Gram-positive bacteria reduces initial adhesion to and aggregation of bacteria on surfaces. Thermodynamic analyses indicated that eDNA introduces favorable acid-base interactions, explaining the effect of eDNA on aggregation and adhesion to the surface.

Pyocyanin Promotes Extracellular DNA Release in Pseudomonas aeruginosa

Bacterial adhesion and biofilm formation are both dependent on the production of extracellular polymeric substances (EPS) mainly composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA). eDNA promotes biofilm establishment in a wide range of bacterial species. In Pseudomonas aeruginosa eDNA is major component of biofilms and is essential for biofilm formation and stability. In this study we report that production of pyocyanin in P. aeruginosa PAO1 and PA14 batch cultures is responsible for promotion of eDNA release. A phzSH mutant of P. aeruginosa PAO1 that overproduces pyocyanin displayed enhanced hydrogen peroxide (H2O2) generation, cell lysis, and eDNA release in comparison to its wildtype strain. A ΔphzA-G mutant of P. aeruginosa PA14 deficient in pyocyanin production generated negligible amounts of H2O2 and released less eDNA in comparison to its wildtype counterpart. Exogenous addition of pyocyanin or incubation with H2O2 was also shown to promote eDNA release in low pyocyanin producing (PAO1) and pyocynain deficient (PA14) strains. Based on these data and recent findings in the biofilm literature, we propose that the impact of pyocyanin on biofilm formation in P. aeruginosa occurs via eDNA release through H2O2 mediated cell lysis.

The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development

Bacteria adhere to natural and engineered surfaces and develop into mature biofilms encased in self-produced extracellular polymeric substances (EPSs). EPS consists of polysaccharides, proteins, metabolites and extracellular DNA (eDNA). Extracellular DNA release by bacteria is mediated by both quorum-sensing (QS)-dependent and -independent mechanisms. Quorum-sensing-independent mechanisms are responsible for basal levels of eDNA release, whereas QS-dependent mechanisms control the production of prophages, phenazines and proteins involved in cell lysis and subsequent release of elevated amounts of eDNA. Extracellular DNA binds with other biopolymers such as polysaccharides, proteins or metabolites like phenazines, thereby providing structural integrity to EPS. Extracellular DNA promotes attractive acid-base interactions between bacterial cells and between bacteria and surfaces. It therefore plays an essential structural role in stabilising biofilms and protecting bacterial cells from physical and chemical challenges. Accordingly, with current knowledge, it becomes clear that targeting and destroying eDNA in bacterial EPS is a promising strategy for treatment of bacterial-associated infections in a medical context and biofilm control on surfaces to prevent biocorrison in an engineering context. In contrast, the addition of DNA can be applied to engineering of biofilms for beneficial purposes such as remediation of environmental pollutants and electricity or fuel production in bioelectrochemical systems or bioreactors.

Pyocyanin Facilitates Extracellular DNA Binding to Pseudomonas aeruginosa Influencing Cell Surface Properties and Aggregation

Pyocyanin is an electrochemically active metabolite produced by the human pathogen Pseudomonas aeruginosa. It is a recognized virulence factor and is involved in a variety of significant biological activities including gene expression, maintaining fitness of bacterial cells and biofilm formation. It is also recognized as an electron shuttle for bacterial respiration and as an antibacterial and antifungal agent. eDNA has also been demonstrated to be a major component in establishing P. aeruginosa biofilms. In this study we discovered that production of pyocyanin influences the binding of eDNA to P. aeruginosa PA14 cells, mediated through intercalation of pyocyanin with eDNA. P. aeruginosa cell surface properties including cell size (hydrodynamic diameter), hydrophobicity and attractive surface energies were influenced by eDNA in the presence of pyocyanin, affecting physico-chemical interactions and promoting aggregation. A ΔphzA-G PA14 mutant, deficient in pyocynain production, could not bind with eDNA resulting in a reduction in hydrodynamic diameter, a decrease in hydrophobicity, repulsive physico-chemical interactions and reduction in aggregation in comparison to the wildtype strain. Removal of eDNA by DNase I treatment on the PA14 wildtype strain resulted in significant reduction in aggregation, cell surface hydrophobicity and size and an increase in repulsive physico-chemical interactions, similar to the level of the ΔphzA-G mutant. The cell surface properties of the ΔphzA-G mutant were not affected by DNase I treatment. Based on these findings we propose that pyocyanin intercalation with eDNA promotes cell-to-cell interactions in P. aeruginosa cells by influencing their cell surface properties and physico-chemical interactions.

Role of eDNA on the adhesion forces between Streptococcus mutans and substratum surfaces: influence of ionic strength and substratum hydrophobicity.

The aim of this study was to investigate the role of extracellular DNA (eDNA) on the adhesion strength of Streptococcus mutans LT11 on substrata with different hydrophobicities at high and low ionic strengths. AFM adhesion forces to a hydrophilic and hydrophobic substratum increased with increasing surface-delay times and ionic strength and were stronger on a hydrophobic than on a hydrophilic substratum. The presence of eDNA on the streptococcal cell surface enhanced its adhesion force to a hydrophobic substratum significantly more than to a hydrophilic substratum, especially after bond maturation. Bond maturation on a hydrophilic substratum was accompanied by an increasing number of minor adhesion peaks, indicating the involvement of acid-base interactions, whereas on the hydrophobic substratum surface the number of minor adhesion peaks remained low. More minor adhesion peaks developed on the hydrophilic substratum at low ionic strength than at high ionic strength. The final rupture distance in retraction force-distance curves was independent of ionic strength on a hydrophilic substratum and increased with increasing surface delay time. On the hydrophobic surface, the final rupture distance did not increase with surface delay time but was significantly smaller at low than at high ionic strength. Final rupture distances were different in presence and absence of eDNA, and the lower values of this difference coincided with the decrease in hydrodynamic radius of the streptococci upon increasing ionic strength, measured using dynamic light scattering. AFM also yielded higher values for the ionic strength induced difference in final rupture distance because in AFM rupture is forced, while in dynamic light scattering differences in radius are only induced by ionic strength differences.

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

Calcium (Ca2+) 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 Ca2+ 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 Ca2+ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca2+ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ to eDNA thereby mediating bacterial aggregation and biofilm formation.

Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation

Bacterial resistance to conventional antibiotics necessitates the identification of novel leads for infection control. Interference with extracellular phenomena, such as quorum sensing, extracellular DNA integrity and redox active metabolite release, represents a new frontier to control human pathogens such as Pseudomonas aeruginosa and hence reduce mortality. Here we reveal that the extracellular redox active virulence factor pyocyanin produced by P. aeruginosa binds directly to the deoxyribose-phosphate backbone of DNA and intercalates with DNA nitrogenous base pair regions. Binding results in local perturbations of the DNA double helix structure and enhanced electron transfer along the nucleic acid polymer. Pyocyanin binding to DNA also increases DNA solution viscosity. In contrast, antioxidants interacting with DNA and pyocyanin decrease DNA solution viscosity. Biofilms deficient in pyocyanin production and biofilms lacking extracellular DNA show similar architecture indicating the interaction is important in P. aeruginosa biofilm formation.

Phenazine production enhances extracellular DNA release via hydrogen peroxide generation in Pseudomonas aeruginosa

In Pseudomonas aeruginosa eDNA is a crucial component essential for biofilm formation and stability. In this study we report that release of eDNA is influenced by the production of phenazine in P. aeruginosa. A ∆phzA-G mutant of P. aeruginosa PA14 deficient in phenazine production generated significantly less eDNA in comparison with the phenazine producing strains. The relationship between eDNA release and phenazine production is bridged via hydrogen peroxide (H2O2) generation and subsequent H2O2 mediated cell lysis and ultimately release of chromosomal DNA into the extracellular environment as eDNA.

Interface Thermodynamic State-Induced High-Performance Memristors

A new class of memristors based on long-range-ordered CeO2 nanocubes with a controlled degree of self-assembly is presented, in which the regularity and range of the nanocubes can be greatly improved with a highly concentrated dispersed surfactant. The magnitudes of the hydrophobicity and surface energy components as functions of surfactant concentration were also investigated. The self-assembled nanostructure was found to demonstrate excellent degradation in device threshold voltage with excellent uniformity in resistive switching parameters, particularly a set voltage distribution of ∼ 0.2 V over 30 successive cycles and a fast response time for writing (0.2 μs) and erasing (1 μs) operations, thus offering great potential for nonvolatile memory applications with high performance at low cost.

Serratia Secondary Metabolite Prodigiosin Inhibits Pseudomonas aeruginosa Biofilm Development by Producing Reactive Oxygen Species that Damage Biological Molecules

Prodigiosin is a heterocyclic bacterial secondary metabolite belonging to the class of tripyrrole compounds, synthesized by various types of bacteria including Serratia species. Prodigiosin has been the subject of intense research over the last decade for its ability to induce apoptosis in several cancer cell lines. Reports suggest that prodigiosin promotes oxidative damage to double-stranded DNA (dsDNA) in the presence of copper ions and consequently leads to inhibition of cell-cycle progression and cell death. However, prodigiosin has not been previously implicated in biofilm inhibition. In this study, the link between prodigiosin and biofilm inhibition through the production of redox active metabolites is presented. Our study showed that prodigiosin (500 μM) (extracted from Serratia marcescens culture) and a prodigiosin/copper(II) (100 μM each) complex have strong RNA and dsDNA cleaving properties while they have no pronounced effect on protein. Results support a role for oxidative damage to biomolecules by H2O2 and hydroxyl radical generation. Further, it was demonstrated that reactive oxygen species scavengers significantly reduced the DNA and RNA cleaving property of prodigiosin. P. aeruginosa cell surface hydrophobicity and biofilm integrity were significantly altered due to the cleavage of nucleic acids by prodigiosin or the prodigiosin/copper(II) complex. In addition, prodigiosin also facilitated the bactericidal activity. The ability of prodigiosinto cause nucleic acid degradation offers novel opportunities to interfere with extracellular DNA dependent bacterial biofilms.