Quantification of
Abstract
Pseudomonas aeruginosa is an opportunistic Gram‐negative bacterium that is primarily responsible for infections related to cystic fibrosis (CF) airways, burn wounds, urinary tract infections, surgery‐associated infections, and HIV‐related illness. Pyocyanin and extracellular DNA (eDNA) are the major factors dictating the progression of biofilm formation and infection. Pyocyanin is a potent virulence factor causing cell death in infected CF patients and is associated with high mortality. eDNA is a key player in P. aeruginosa biofilm formation and is also responsible for the high viscosity of CF sputum that blocks the respiratory airway passages. In this chapter, we summarize our recent findings on the role of pyocyanin in facilitating P. aeruginosa biofilm formation. Pyocyanin promotes eDNA release in P. aeruginosa by inducing cell lysis mediated via hydrogen peroxide (H2O2) production. Pyocyanin intercalates with the nitrogenous bases of DNA and creates structural perturbation on the double‐helix structure. Pyocyanin‐eDNA binding significantly influences P. aeruginosa cell surface hydrophobicity and influences the physicochemical interactions facilitating bacterial cell‐to‐cell interaction (aggregation) and ultimately facilitates robust biofilm formation. A pyocyanin knockout (ΔphzA‐G) mutant is shown to have significantly reduced eDNA release and biofilm formation in comparison to its wild‐type. To this end, we discover that antioxidant glutathione directly binds to pyocyanin and modulates pyocyanin structure and function, thus inhibiting pyocyanin‐eDNA binding and consequently hampering biofilm development.
Keywords
- Pseudomonas aeruginosa
- Pyocyanin
- extracellular DNA
- glutathione
- biofilm
1. Introduction
Persistent
In this chapter, we focus our discussion on very recent findings that elucidate the essential role of pyocyanin in promoting eDNA production and interacting with eDNA to facilitate biofilm formation in
2. Pyocyanin production by P. aeruginosa
Figure 1b shows pyocyanin production by various
3. Pyocyanin a virulence factor
Pyocyanin was formerly disregarded as a bacterial secondary metabolite but has recently been ascribed a variety of roles in microbial ecology, and importantly a relationship with the severity of
Depleted GSH levels during the chronic stage of CF infection leads to widespread epithelial cell death and consequently lung damage, respiratory failure, and mortality [21, 22]. Pyocyanin also inhibits catalase activity in the airway epithelial cells and thus aggravates oxidative stress in lung epithelial cells [23].
4. Pyocyanin promotes eDNA release in P. aeruginosa via H2O2 generation
In bacteria, eDNA release is mediated by both lytic and nonlytic mechanisms. The lytic mechanism involves the controlled lysis of a small number of bacterial cells via the production of various QS‐mediated cell lysing agents, such as lytic prophages, autolysin proteins, enzymes, and H2O2. In nonlytic mechanisms, eDNA release is through bacterial outer membrane blebs/vesicles that contain large amounts of DNA [24–26]. QS‐dependent mechanisms involve the AHL and PQS systems, whereas QS‐independent mechanisms operate via release through flagellum and type IV pili [27, 28]. PQS in
A recent work in this field by Das and Manefield has shown that pyocyanin production in
In general, phenazines such as pyocyanin induce H2O2 production and subsequently trigger cell death in host (mammalian) and competing fungal and bacterial cells are well documented. A recent study showed concrete evidence of
By examining pyocyanin production in different strains of
5. Pyocyanin binding to eDNA
Previous studies have demonstrated that eDNA is a key constituent in the construction and structural integrity of the biofilm matrix in many bacterial species, and the cleaving of eDNA by nuclease enzymes such as DNase I disintegrates the biofilm matrix, thereby increasing the susceptibility of bacterial cells within the biofilm matrix to antimicrobial agents such as detergents and antibiotics [33, 34]. eDNA acts as a scaffold for the whole biofilm by binding with other biomolecules such as peptides/enzymes/proteins and polysaccharides. For example, in
5.1. Mechanism of pyocyanin‐DNA binding
The mechanism of pyocyanin‐eDNA binding was elucidated using different types of spectroscopic techniques by Das et al. [10]. In this study, we used
Using fluorescence emission spectroscopy (Varian Cary Eclipse Fluorescence Spectrophotometer, USA), it was found that pyocyanin displaces ethidium bromide (EtBr) bound to dsDNA. All experiments were done in SHE buffer (2 mM HEPES, 10 μM EDTA, and 9.4 mM NaCl in Milli‐Q water adjusted to pH 7 with NaOH). Light emission at 615 nm (ex=480 nm) was quantified at room temperature in 1 ml quartz cuvette. The fluorescence emission spectra suggest that the addition of pyocyanin (70 or 140 μM) reduced the DNA (6 ng/μl)‐EtBr (4 μM) peak maxima to that of an EtBr solution without DNA (Figure 4a). It is well known that EtBr is a classic intercalating agent that strongly binds to DNA via intercalation and the displacement of EtBr by pyocyanin suggests that pyocyanin can bind to DNA. However, the mechanism of pyocyanin‐DNA binding was not immediately clear.
To determine the binding mechanism between pyocyanin and DNA, a Varian Cary 100 Bio UV‐visible (UV‐vis) spectrophotometer technique was used [10]. UV‐vis spectroscopic scans from 200 to 800 nm were performed in 1 ml quartz cuvette on DNA, pyocyanin, and the DNA‐pyocyanin complex in Milli‐Q water. Figure 4b shows the UV‐vis range spectra of DNA (50 ng/μl) in the presence of increasing concentrations of pyocyanin (5.6, 11.2, 16.8, or 28.0 μM). The spectra of DNA with pyocyanin showed a gradual increase in absorbance intensity of the DNA peak and a shift of the DNA peak from 259 to 253 nm with increasing pyocyanin concentrations. The observed hyperchromic (due to the increase in absorbance intensity) and hypochromic (due to the shift in wavelength of DNA peak) effects are indicative of the intercalation of pyocyanin between nitrogenous base pairs of DNA and exposure of nitrogenous base pairs due to the unwinding of the DNA helix [38].
The pyocyanin‐DNA binding mechanism was further confirmed using a Chirascan circular dichroism (CD) spectrophotometer (Applied Photophysics, UK). The experiments were conducted using 1 mm path length quartz cuvette, and mixtures of dsDNA at 135 ng/μl with varying pyocyanin (0, 28, 143, and 286 μM) concentrations in 350 μl Milli‐Q water were scanned from 200 to 320 nm after a 15‐min static incubation at 25°C. The spectra of DNA‐pyocyanin mixtures confirm that DNA binds to pyocyanin with statistically significant changes in peak intensity (
5.2. Pyocyanin‐eDNA binding influences P. aeruginosa cell surface hydrophobicity and physicochemical interactions
Pyocyanin is well known as an electron shuttle [18], and our recent investigation revealed that pyocyanin intercalates with DNA [10]. In line with this, previous studies revealed that eDNA promotes bacterial aggregation through acid‐base interactions involving electron‐donating and electron‐accepting groups [39]. Contact angle measurements on various Gram‐positive and Gram‐negative bacterial cell surfaces revealed that eDNA significantly influences modulation in bacterial cell surface hydrophobicity [8, 39]. For instance, the cell surface of
The hypothesis was tested by measuring contact angles of
The modulation in
5.3. Pyocyanin‐eDNA binding is essential for P. aeruginosa biofilm formation
eDNA was previously acknowledged as a biofilm‐promoting factor, whereas pyocyanin was mainly considered as a secondary metabolite essential for the persistence of
Thickness (μm) | Biomass (μm3/μm2) | |
---|---|---|
Wild‐type | 8.3±0.3 | 1.4±0.4 |
Wild‐type+DNase I (40 U) | ||
Δ |
||
Δ |
||
Δ |
||
Δ |
A previous study by Ramos et al. also showed similar results with phenazine‐deficient mutant but could not elucidate the mechanism behind the influence of phenazine on biofilm formation in
6. Glutathione disrupts P. aeruginosa biofilm formation
With the increased tolerance of bacteria to existing antibiotic therapies [1] and the necessity to use high doses of antibiotics with their related side effects [42, 43], there is an urgent public health priority to identify new methods and targets for the control of
6.1. Glutathione interacts with pyocyanin and inhibits its binding with eDNA
GSH is a thiol tripeptide (γ‐glutamylcysteinylglycine) found in all human cells and in some bacterial species. GSH is considered to be a master antioxidant and its primary functions include defense against ROS and free radicals and maintaining a healthy immune system [13]. In humans, intracellular GSH levels vary from 2 to 10 mM, whereas, in the extracellular lung lining fluid (ELF), levels range from 250 to 800 μM [44]. In contrast, intracellular GSH concentration in bacterial cell differs significantly from species to species [45–47]. For instance, in
Pyocyanin undergoes oxidation by donating electron to molecular oxygen to form superoxides and H2O2 [49]. GSH, being a thiol antioxidant, will donate electron/protons to pyocyanin directly through the ‐SH group from cysteine [50], thereby interfering in the pyocyanin oxidation process by inhibiting H2O2 generation [50]. The antioxidant property of GSH makes it a potential inhibitor of pyocyanin toxicity.
Our recent investigation using CD and UV‐vis suggests that pyocyanin‐GSH complex interferes with pyocyanin binding to DNA (Figure 7a and b) [10], whereas nuclear magnetic resonance (NMR; Bruker Avance 400 spectrometer) of the pyocyanin-GSH complex at various pyocyanin‐GSH ratios clearly indicates that GSH (with at least fivefold higher concentration than pyocyanin) is required to modulate pyocyanin aromatic structure (Figure 7c; unpublished data). As discussed earlier, pyocyanin is a planar molecule that intercalates into the nitrogenous base of DNA. By instead conjugating with GSH, intercalation with DNA is restricted. However, it is interesting to observe that with the increases in GSH concentration, the inhibition of pyocyanin binding to DNA is increased; almost complete inhibition was observed at a molar ratio of pyocyanin/GSH of ∼1:6. A similar observation was reported recently by Muller and Merrett [50] demonstrating that thiol concentration needs to be available in the millimolar range to neutralize pyocyanin toxicity. Their work also suggests that GSH forms a cell‐impermanent conjugate with pyocyanin. This suggests that thiol antioxidants could be a potential clinical target against
6.2. Glutathione disrupts clinical strains of P. aeruginosa biofilms and facilitates bactericidal activity
Biophysical techniques confirmed that GSH inhibits pyocyanin‐eDNA intercalation. This made us to hypothesize that GSH could disrupt biofilms, as pyocyanin and eDNA are the crucial factors that initiate biofilm formation. Our recent investigation confirmed that interrupting the pyocyanin‐eDNA intercalation using GSH results in a significant disruption of the biofilms of pathogenic Australian epidemic strain (AES‐1R and AES‐1M isolated from a CF patient; unpublished data). Figure 8 shows the effect of GSH, DNase I treatment (12 h, 37°C, 150 rpm) on preestablished biofilms grown in LB medium (24 h, 37°C, 150 rpm) imaged using CLSM.
An analysis of biofilm image using ImageJ software revealed that GSH or DNase I‐treated AES‐1R and AES‐1M biofilm showed significant difference (Student's
Treatment | Thickness (μm) | Biomass (μm3/μm2) | Average Live/Dead (%) | |
---|---|---|---|---|
AES‐1R | — | 8.6±1.1 | 1.8±0.4 | 83/17 |
AES‐1R | GSH (2 mM) | 37/ |
||
AES‐1R | DNase I (40 U) | 84/16 | ||
AES‐1M | — | 7.4±1.4 | 1.1±0.3 | 79/21 |
AES‐1M | GSH (2 mM) | 0.7±0.4 | 12/ |
|
AES‐1M | DNase I (40 U) | 0.2±0.1 | 93/7 |
Biofilm image (Figure 8) and quantification of live (green) and dead (red) biofilm biomass (Table 2) clearly show significant increase in dead biomass when biofilm exposed to GSH treatment. GSH‐mediated bactericidal activity in
7. Conclusion and perspective
Bacterial biofilms form a highly protective biological matrix that enables persisting populations of bacteria to survive in otherwise highly hostile environments. These matrices vary highly between species; however, they share a common structural element (eDNA). Within
Biofilm formation is associated with increased resistance to antibiotic therapies and persistence of bacterial colonization within the CF lung. Novel treatment strategies seek to act on molecules that are essential for bacterial persistence such as biofilm constituents. Biofilm disruption is associated with increased antibiotic susceptibility and the clearance of bacteria. We have shown that, by disrupting the biofilm association with thiol‐based antioxidants, namely GSH, which directly binds and clears freely available pyocyanin, intercellular aggregation and overall biofilm structure are perturbed. This is enhanced by the activity of nucleases such as DNase I, which remove the underlying eDNA scaffold, resulting in a complete disruption of the biofilm structure by decreasing the water contact angle of bacterial cells and increasing the Gibbs free energy between cells.
Thus, the intercalation between pyocyanin and eDNA is both a unique and highly exploitable interaction in
Acknowledgments
We thank Professor Dianne K. Newman (Department of Biology, California Institute of Technology, Pasadena, CA, USA) for providing us with the
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