Cystic fibrosis (CF) is a genetic disorder that predominantly affects Caucasian populations. Pseudomonas aeruginosa is the most important Gram‐negative pathogen that persists in CF patients’ lungs. By evading host defence mechanisms and persisting, it is ultimately responsible for the morbidity and mortality of about 80% of CF patients worldwide. P. aeruginosa is also responsible for infections in burns, wounds, eyes, nosocomial patients and HIV patients. Prevalence and progression of infection by P. aeruginosa in the host is dependent on secretion of numerous extracellular molecules such as polysaccharides, proteases, eDNA, pyocyanin and pyoverdine. These molecules have multiple roles in facilitating P. aeruginosa colonisation and virulence. Pyocyanin is one of the major factors dictating progression of infection and biofilm formation. Pyocyanin is a potent virulence factor causing host cell death in CF patients. In this chapter, we have outlined the roles of various extracellular molecules secreted by P. aeruginosa and specifically focused on the role of pyocyanin in inducing eDNA production, binding to eDNA via intercalation and facilitating biofilm promoting factors, whilst inducing oxidative stress to host cells via production of reactive oxygen species. In line with this, we have described the current challenges in treatment of CF infections and the development of new strategies to control P. aeruginosa infections.
- Pseudomonas aeruginosa
Cystic fibrosis (CF) is a genetic disorder whose effects are felt from birth. It predominantly affects Caucasian populations; however, it is also present in non‐Caucasians . The prevalence of CF varies around the globe; however, extensive evidence suggests that in the USA, Canada, Australia, New Zealand and European countries the ratio of newborns with CF is 1:2000–3000 . CF is induced by mutations (amino acid deletions/substitutions) in the cystic fibrosis transmembrane conductance regulator (CFTR), with a loss of the phenylalanine at position 508 (∆F508) leading to the most severe outcome. The dysfunctional CFTR leads to greatly reduced transport of ions across epithelial cells and membranes, resulting in dehydration of the mucus in the host respiratory tract/lungs and the digestive pathway, reduced mucus clearance and severe breathing problems [1, 2]. The slow‐moving mucous facilitates the growth of microbes, including potentially life‐threatening bacteria such as
Persistence of bacterial infections in the host is due to the bacterium's ability to form biofilms via secretion of numerous extracellular biopolymers, collectively known as extracellular polymeric substances (EPS) and small molecules [7, 8]. Different extracellular biopolymers and small molecules conjugate with each other through physico‐chemical interactions to form a highly complexed and structurally integrated matrix . This matrix represents a critical interface between bacterial cells and the host or its environment. Extracellular biopolymers (EPSs) play a primary role in immobilising planktonic cells (cell adhesion) and cell‐cell communication (aggregation), leading to colonisation and biofilm formation on both biotic and abiotic surfaces. It also provides bacterial cells/biofilms with inherent protection against physical stress, traditional antibiotic therapy and host immune defences, thus making eradication extremely difficult [7, 9]. Potentially all biopolymers (e.g. proteins, polysaccharides, eDNA) in EPS serve as an excellent source of nutrients and specifically eDNA promotes horizontal gene transfer between cells within the biofilm .
Of the many extracellular molecules secreted by
2. Role of
P. aeruginosa secreted extracellular molecules in development of biofilm and pathogenesis
Alginate (capsular polysaccharide) is acknowledged as a virulence factor responsible for mucoidal
Other polysaccharides that are essential and partly associated with biofilm formation include Psl and Pel (coded by the
Iron is an important cofactor required for bacterial metabolism, growth and survival and also essential for induction of infection in host by various pathogenic bacteria including
Various factors influence the bioavailability of iron for
Interestingly, mammalian biological systems have an innate defence strategy against siderophores, a neutrophil‐gelatinase‐associated lipocalin (NGAL). NGAL functions as a scavenger by directly binding with siderophores, blocking
2.4. Role of eDNA
eDNA is currently recognised as an essential constituent of EPS and plays a pivotal role in the various processes of biofilm formation in numerous medicallyrelevant Gram‐negative and Gram‐positive bacteria [8, 9]. In
eDNA also serves as a nutrient source (an excellent source of carbon,phosphate and nitrogen), facilitates horizontal gene transfer through Type IV pili and competence stimulating peptides and helps maintain the structural integrity of the biofilm by binding to various extracellular molecules (proteins, polysaccharides, metabolites) in the biofilm matrix [7, 8]. Recent investigations have revealed that eDNA protects bacterial cells in biofilm from physical challenges such as shear stress by increasing biofilm viscosity, and from chemical challenges by antibiotics and detergents. For example, eDNA binds to various positively charged antibiotics (aminoglycosides) thus shielding
While eDNA is well‐recognised as one of the prime factors in the establishment of
2.5. Role of pyocyanin
2.5.1. Pyocyanin production in P. aeruginosa
Pyocyanin, a member of the phenazine class, is a molecule only known to be expressed by
In chronic CF lung infection, up to 85 µM of pyocyanin has been recorded in
2.5.2. Pyocyanin facilitates eDNA release
Pyocyanin is a redox molecule and electrochemically active (has potential to accept and donate electrons as a shuttle) with a multitude of biological activities . Recent investigations have demonstrated that pyocyanin facilitates eDNA release in
2.5.3. Pyocyanin and eDNA intercalate in biofilms
Pyocyanin's intercalation with DNA has been demonstrated using various bio‐physical techniques (circular dichroism, Fourier transform infrared spectroscopy, fluorescence and UV‐Vis spectroscopy) . In a preliminary study using fluorescence emission spectroscopy, it was shown that pyocyanin displaces ethidium bromide bound to dsDNA, indicating pyocyanin is an intercalating agent. Fluorescence emission spectroscopy data were further complemented using the UV‐Vis spectra of the DNA‐pyocyanin complex. Results indicated a significant shift (from 259 to 253 nm) and increase in absorbance intensity in the DNA peak. This marked change in the DNA peak from 259 nm indicates effective intercalation of pyocyanin molecules between the nitrogenous base‐pairs of DNA . Meanwhile, the circular dichroism spectra of the DNA‐pyocyanin mixtures confirmed that pyocyanin binds to the sugar‐phosphate backbone of DNA and strongly intercalates with the nitrogenous bases of DNA, consequently creating local perturbations in the DNA double helix structure . This type of interaction is a typical characteristic feature of all intercalating molecules. In the same study, Das et al. also discovered that pyocyanin significantly increased the viscosity of DNA solutions, and that by intercalating with DNA pyocyanin‐facilitated electron transfer . These results are in line with previous studies concluding that in order to remain viable in biofilms,
2.5.4. Pyocyanin‐eDNA binding influences biofilm formation via physico‐chemical interactions
Molecules that bind to both biological and non‐biological surfaces are known to influence hydrophobicity, charge and the physico‐chemical properties that assist or resist interactions. Previous studies have demonstrated that in both bacteria and fungi, the presence of such bio‐molecules (eDNA or proteins) plays a significant role in dictating cell surface hydrophobicity and physico‐chemical interactions . In
Analysis of bacteria‐to‐bacteria and bacteria‐to‐substratum physico‐chemical interactions (Lifshitz‐Van der Waals interactions forces, acid‐base interactions forces) has revealed that the presence of pyocyanin and eDNA facilitates attractive physico‐chemical interactions . Removal of eDNA from the
It should be noted, however, that physico‐chemical interactions do not explain bacterial interaction in all cases, since bacterial cell structures (pili, fimbriae) and bio‐polymers (polysaccharides, proteins, eDNA) extend up to hundreds of nanometres from the bacterial cell surface and can affect other interaction types . These cell structures and bio‐polymers initiate hydrogen bonding and ionic interactions by colliding with bio‐molecules anchored on the bacterial cell surface to stabilise the biofilm matrix and also to its adjacent cells and thereby help bacterial cells to overcome the physico‐chemical energy barrier and promote bacterial cell‐to‐cell interactions and biofilm formation [7, 64]. Confocal laser scanning microscopy (CLSM) analysis revealed that the intercalation of pyocyanin with eDNA facilitates
2.5.5. Pyocyanin as a virulence factor
Pyocyanin was formerly recognised only as a bacterial secondary metabolite, but has recently gained significant attention for its involvement in a variety of crucial roles in microbial ecology, specifically correlated with the severity of
In the host, pyocyanin appears to participate in a reduction mechanism, which is capable of reducing and releasing the iron from transferrin in host cells to stimulate the growth of
Pyocyanin has also been extensively studied due to its electrochemical and redox activity. The diffusible nature and small size of pyocyanin means it can easily pass through the host cell membrane and undergo redox reactions with other molecules . For example, it accepts electrons from NADH and subsequently donates electrons to molecular oxygen to form reactive oxygen species (ROS) such as H2O2  (Figure 3). Pyocyanin‐mediated ROS cause oxidative stress and affect calcium homeostasis while also obstructing cellular respiration and depleting intracellular cAMP and ATP levels . Pyocyanin significantly alters human protease activity, inhibits nitric oxide production and consequently influences blood flow, blood pressure and immune functions. It also modulates the host immune response to support evasion of the host immune system and establish chronic infection . In CF, pyocyanin‐mediated ROS oxidise host intracellular and extracellular reduced glutathione (GSH) to form glutathione disulphide or oxidised glutathione (GSSG) . Depleted GSH levels during the chronic stage of CF infection lead to widespread epithelial cell death and consequent lung damage and leading to respiratory failure and death [75, 76]. Pyocyanin also inhibits catalase activity in airway epithelial cells, thus increasing oxidative stress in these cells and initiating pulmonary tissue damage . In a recent study, Rada et al. showed that pyocyanin promotes neutrophil extracellular trap (NET) formation . NET formation is an important innate immune mechanism initiated by neutrophils to trap and kill pathogens, however, the aberrant NET release triggered by pyocyanin‐mediated intracellular ROS production directly damages host tissues and has been linked to the severity of many diseases including CF .
P. aeruginosa infections
Substantial research over many decades has led to a good degree of understanding of the mechanisms
3.1. Current antibiotic treatment and challenges against
P. aeruginosa infections in CF patients
Many antibiotics developed in recent decades such as aminoglycosides, ticarcillin, ureidopenicillins, ceftazidime, cefepime, aztreonam, the carbapenems, ciprofloxacin and levofloxacin display anti‐pseudomonal activity. However, the choice of best antibiotic to use in a particular case remains a major challenge as
Antibiotics commonly used to treat
Other serious challenges with nebuliser treatment (in comparison to dry powder inhalation) strategies are that the antibiotic particles do not reach infection sites at a faster rate, but even with dry powder inhalation does not provide immediate relief to CF patients . For example, studies with CF patients demonstrated that inhaled tobramycin is effective in reducing
3.2. Current non‐antibiotic strategies against CF lung infection
Non‐antibiotic treatment strategies that have shown potential to reduce the severity of respiratory symptoms in CF patients and bacterial associated infections have largely centred on the use of aerosolised recombinant human DNase I (rhDNase I (Pulmozyme)) in a nebuliser . Earlier studies showed DNase I reduced the viscosity of CF sputum by cleaving DNA present in sputum and thus leading to increased pulmonary function . As noted above, eDNA is an essential biofilm promoting factor in many pathogenic bacterial species, is the backbone of the
3.3. New non‐antibiotic treatments
A new potential treatment strategy involves the use of reduced GSH to bind to pyocyanin and prevent its intercalation with eDNA. Intracellular GSH levels in mammalian cells are in the millimolar (mM) range, and lower concentrations are found in some bacterial cells. However, in CF patients, GSH levels in whole blood, blood neutrophils lymphocytes and epithelial lung fluid are markedly decreased . Replenishment of GSH levels in CF has thus been investigated in a number of human studies using either inhaled GSH [90, 91] or oral N‐acetylcysteine, a GSH precursor . These studies demonstrated the feasibility of successfully delivering GSH to human lung, with a significant improvement in lung function (FEV1), especially in patients with moderate lung disease. The GSH therapy was well tolerated by CF patients with no noticeable side effects .
GSH, being a thiol antioxidant, will donate electrons/protons to pyocyanin directly through the –SH group from cysteine [53, 76], thereby interfering in the pyocyanin oxidation process by inhibiting H2O2 generation . The antioxidant properties of GSH make it a potential inhibitor of pyocyanin toxicity. GSH binding to pyocyanin tends to modulate pyocyanin's structure, and this has been confirmed using nuclear magnetic resonance (NMR) spectroscopyand mass spectrometry [53, 93]. This structural change consequently inhibits the intercalation of pyocyanin with DNA, confirmed using circular dichroism . In line with this, Muller and Merrett concluded that GSH forms a cell‐impermanent conjugate with pyocyanin and consequently inhibits pyocyanin entry into host cells, thus preventing pyocyanin‐mediated lung epithelial cell lysis .
Recent studies in the Manos laboratory by Klare et al. have demonstrated the excellent utility of GSH in disrupting
In comparison to other techniques, GSH treatment has a distinct advantage, being an intrinsic and essential antioxidant for host cells that not only has antibiofilm properties but has also been proven to enhance lung epithelial growth and increase pulmonary function in CF patients .
3.4. Development of new antibacterial agents
Several new antibacterial agents are being developed and undergoing stringent testing both in vitro and in vivo (animal models) against
Other antibiofilm agents under investigation include nitric oxide (NO) which has recently been discovered to induce dispersal of
Extracellular molecules released by bacteria form a scaffold for biofilm formation. In
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