Proposed mechanisms of
Chronic Pseudomonas aeruginosa lung infection is the cause of much morbidity and most of the mortality in cystic fibrosis (CF) patients. The high prevalence of P. aeruginosa infections in CF is related to the microbe's large genome and mechanisms of adaptation to the CF lung environment, the host immune system and antibiotic resistance. Among a wide range of P. aeruginosa metabolites involved in infection development in CF, the biosurfactant compounds, rhamnolipids (RLs) and exopolysaccharides (EPSs), have important roles in the early stages of P. aeruginosa infection in CF. RLs and EPSs are involved in bacterial adhesion, biofilm formation, antibiotic resistance, and impairment of host immune system pathways, as well as in processes such as biofilm maintenance and the mucoid phenotype of P. aeruginosa, which lead to development of chronic infection. Due to the proposed roles of RLs and EPSs and the importance of prevention and treatment of P. aeruginosa respiratory infections in CF, these compounds are promising targets for patient therapy. In the future, impairment of P. aeruginosa quorum sensing (QS) pathways and modification of host respiratory mucus epithelial membranes should be considered as potential approaches in preventing respiratory infections caused by this microbe in CF patients.
- cystic fibrosis
- Pseudomonas aeruginosa
Cystic fibrosis is a congenital, recessively inherited disorder. The genetic background of CF development is >1500 mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) on chromosome 7, which lead to malfunction of the chloride channel in CF patients. CF affects a large number of organs and tissues (airways, pancreas, the small intestine, liver, the reproductive tract and sweat glands), resulting in numerous clinical symptoms (viscid mucus, respiratory infections, intestinal malabsorption of fat, diabetes mellitus, meconium ileus, impaired liver function, male infertility and salt loss) .
The malfunction of the chloride channel in CF patients leads to impairment of the noninflammatory defense mechanism of the lower respiratory tract. Therefore, CF patients, from early childhood, suffer recurrent and chronic respiratory tract infections caused by
Recent research indicates that chronic
P. aeruginosainfection in CF
2.2. Pathogenesis of
P. aeruginosainfection in CF
Despite constant exposure to a wide range of microorganisms, CF patients are predisposed to infection by only specific groups of microorganisms . The proximal event in development of CF is mutation of the CFTR gene (see Introduction), but still, it remains unclear how this primary step causes particular infections in CF patients. However, numerous proposed mechanisms are related to CFTR gene mutation, defective CFTR channels and infection development : (1) reduced ion transport; (2) modified salt content in the airway surface liquid; (3) increased levels of acylated glycolipids on the surface of CF airway epithelial cells; (4) defective CFTR exposed on airway epithelial cell membranes become receptors; (5) low levels of antimicrobial compounds (inducible nitric oxide synthase and nitric oxide); and (6) intrinsic hyperinflammation of airways (Table 1) [25–36].
|Decreased ion transport, which results from defective CFTR channels enhances fluid absorption in the airways||Lowered airways surface liquid and impaired ciliary transport of the mucous layer, which results in defects in microbial clearance|
|Altered salt content in the airway surface liquid||Inactivation of immune system defenses pathways; defected neutrophils activity|
|Increased levels of acylated glycolipids on the surface of CF airway epithelial cells due to defective CFTR molecules||Modified glycolipids are receptors for |
|Binding of ||Internalisation of |
|Lowered level of antimicrobial compounds||Propensity of individuals to lung infection|
|Istrinical hyperinflammation of airways||Damage of host cells and disruption of effective microbe clearance|
The first step in infection of CF airways by
Initially, infection of
P. aeruginosaquorum sensing systems and biofilm
One of the most important factors which facilitate
QS is the mechanism by which bacteria engage in cell‐to‐cell communication using diffusible molecules based on a critical cell density . QS molecules manage and regulate diverse physiological processes, some of which are interrelated. In
Biofilms are matrix‐enclosed microbial accretions that adhere to biological or nonbiological surfaces .
2.2.2. Adaptation mechanisms of
P. aeruginosain CF lungs
The CF lungs are an unfriendly and varied environment for invading bacteria due to the presence of stressors such as osmotic stress of viscous mucus, oxidative and nitrosative stresses of the host responses, sublethal concentrations of antibiotics and other microbes presence. Regarding to the environment of CF lungs, it is a great challenge of
It is believed that mechanisms that allow
3. Biosurfactants of
P. aeruginosa—rhamnolipids and exopolysaccharides
Biosurfactants are a group of amphiphilic compounds, comprise a hydrophobic and a hydrophilic moiety and are produced by a range of microorganisms [9, 62].
Rhamnolipids comprise one or two L‐rhamnose units and one or two units of 3‐hydroxy fatty acid. Variations in lipid components contribute to the biodiversity of RLs [9, 67]. Due to their chemical composition, RLs are classified into four homologue groups (Figure 1): RL1—mono‐rhamno‐di‐lipidic, RL2—mono‐rhamno‐mono‐lipidic, RL3—di‐rhamno‐di‐lipidic and RL4–di‐rhamno‐mono‐lipidic structures. RL1 and RL3 are usually classified as principal—common RLs, while RL2 and RL4 are classed as atypical–uncommon RLs . The development of sensitive, high throughput analytical techniques, such as soft ionization mass spectrometry, has led to the further discovery of a wide diversity of RL congeners and homologues (about 60) produced in different concentrations by various
3.1.1. Diversity of rhamnolipid structures
RL biosurfactants are always produced as mixtures of different RL congeners, as observed with various
The presence of different functional groups in RL molecules (the hydrophobic, lipid part and the hydrophilic and carbohydrate part) gives RLs important physicochemical properties. Due to their amphipathic structure, RLs behave as wetting agents, surface active compounds, emulsifiers and detergents. These RL functional groups are, therefore, utilized in enhancing and facilitating bacterial movement, adhesion and contact with surfaces, as well as substrate uptake, or solubilization.
3.1.2. Rhamnolipid biosynthesis and quorum sensing
Biosynthesis of RLs requires three rhamnosyltransferases. The fatty acid dimer moiety in RLs and free 3‐(3‐hydroxyalkanoyloxy) alkanoic acid (HAA) are both synthesized by the rhamnosyltransferase RhlA. Next, dTDP‐L‐rhamnose is transferred to HAA by the rhamnosyltransferase RhlB, or to a previously generated mono‐RL by the rhamnosyltransferase RhlC . HAA precursors are derived from the FASII cycle (bacterial fatty acid synthesis system), while activated L‐rhamnose is derived from the glucose moiety of deoxythymidine di‐phospho (dTDP)‐L‐rhamnose through several reactions catalyzed by four enzymes that, in
In Section 2.2.1, we stated that
In conclusion, in the complex QS network, there is a hierarchy between
Pseudomonads have the potential to produce various types of EPSs such as alginate, levan, marginalan and cellulose, as well as different heteropolysaccharides and protein polysaccharides complexes . Nearly all
3.2.1. Diversity of exopolysaccharide structures
3.2.2. Exopolysaccharide biosynthesis and quorum sensing
EPS biosynthesis requires sugar‐nucleotide precursors and for alginate production, this is GDP‐mannuronate. The enzymes required for GDP‐mannuronate production include: (1) the bifunctional enzyme, AlgA which exhibits phosphomannose isomerase (PMI) and GDP‐mannose pyrophosphorylase (GMP) activity; (2) AlgC, a phosphomannomutase; and (3) AlgD, which is a GDP mannose dehydrogenase [97–99]. AlgD catalyzes the first step in alginate biosynthesis, which is responsible for the mucoid phenotype often observed in clinical
Alginate is first synthesized as a linear homopolymer of D‐mannuronic acid residues. The polymer is then modified in the periplasm through selective O‐acetylation by the concerted action of AlgI, AlgJ and AlgF and epimerized by AlgG [100, 101]. Alginate has a reasonably random structure (Figure 2a). This differentiates alginate from Psl and numerous
AlgC appears to be crucial for general EPS biosynthesis, not just alginate, as it is also required for precursor synthesis of Psl, as well as LPSs and RLs [102, 103]. The LasR from the
4. Physiological role of
P. aeruginosabiosurfactants in CF infection
4.1. Physiological role of rhamnolipids and exopolysaccharides
Among proposed functions of RL biosurfactants, related to their physicochemical properties (surface activity, wetting ability, detergency and other amphipathic‐related properties), are promotion of the uptake and biodegradation of poorly soluble substrates, immune modulators and virulence factors [9, 15]. Additionally, these molecules are involved in the process of swarming, as surface wetting agents and chemotaxis stimuli and in
Physicochemical properties of EPSs, such as surface activity, viscosity, flexibility of molecule, as well as its ability to bind water, protect the microbe from dehydration in the unique CF microenvironment following the switch from nonmucoid to mucoid phenotype . In this regard, the
4.2. Rhamnolipids and exopolysaccharides in
P. aeruginosabiofilm formation
Swarming motility is the rapid and coordinated movement of a bacterial population across a surface, which often results in characteristic flowery, dendritic colony shapes on agar plates . This type of colony movement is related to the production of an extracellular slime layer, mainly composed of EPSs and surface active compounds, which is a pivotal feature of swarming cells, acting as a wetting agent that reduces the surface tension . Several studies suggest that
The importance of swarming motility for biofilm formation indicates that RLs are involved in the process of biofilm formation. Indeed, it was shown that RLs enhance adhesion of planktonic cells in the early stages of biofilm development, when an initial microcolony is formed (Figure 3). Proposed mechanisms for RL effects on cell adhesion include regulation of cell‐surface hydrophobicity and modification of adhesive interactions, especially when nutritional conditions are changed [85, 110–112]. Also, RLs are involved in later differentiation of the biofilm structure, the detachment and dispersion of
EPSs also play an important role in biofilm formation and invasion of pathogenic microorganisms. During biofilm maturation,
In the context of immune system pathways, polymorphonuclear leukocytes (PMNs) are considered as the central line of defense in innate immunity and they are produced as a predominant response to infection, especially in CF lungs . When PMNs phagocytose bacteria, the host cells produce highly reactive oxygen species, which kill
Surfactant protein A (SP‐A) is involved in prevention of alginate‐induced
4.3. Effect of
P. aeruginosarhamnolipids and exopolysaccharides
Respiratory mucosa protects host airways from microbial infection.
RLs concentration of up to 8 μg/ml was found in the sputum of CF patients infected by
RLs produce damage to the bronchial epithelium and inhibit ciliary function [122–124]. Damage to the bronchial epithelia is related to impairment of the protective layer of lung surfactant in CF patients. Phospholipase C and RLs produced by
The effects of
The effect of RLs on immune system pathways with direct impairment and modulation of immune cell activity is well known  (Figure 3). RLs are reported to have hemolytic activity on various erythrocyte species; induce direct neutrophil chemotactic activity ; enhance the oxidative burst response of monocytes; stimulate and release inflammatory mediators from mast cells and platelets; induce lysis of PMNs; stimulate both chemotaxis and chemokinesis of PMNs (depending on concentration); and enhance production of several interleukins produced by granulocyte‐macrophage and nasal epithelial cells (at noncytoxic levels) [131–135]. Furthermore, RLs, especially di‐RLs, are cytolytic for human monocyte‐derived macrophages and at lower concentrations, they inhibit the phagocytic response of macrophages .
The response of
Modification of membrane LPSs in
Figure 3 summarizes the proposed roles, relationships and effects of the biosurfactant RLs and EPSs produced by
5. Rhamnolipids and exopolysaccharides as targets—current and future perspectives
The importance of biofilm formation and maintenance for the establishment and persistence of
|Impairment of biofilm structure and ||Antibiotic cleavage by ||[16, 155 ]|
|Ciprofloxacin||Fluoroquinolones||Mutations by DNA gyrase and topoisomerase IV enzymes and efflux systems||[155, 156]|
|Tobramycin, Gentamicin, Amikacin||Aminoglycosides||Impairment of biofilm structure||Aminoglycoside‐modifying enzymes AMEs and rRNA methylases as well as efflux mechanisms||[16, 155, 157]|
|Patulin, penicillin acid, cis‐2 decanoic acid||Bacterial metabolites||Impairment of biofilm structure and ||No resistance||[16, 158]|
|Solenopisin A||Fire ant venom||Impairment of biofilm structure and ||No resistance||[16, 154]|
|Salicylic acid and 4‐nitro‐pyridine oxide (4‐NPO)||Synthetic compounds||Impairment of biofilm structure and ||No resistance||[16, 152, 154]|
|Garlic extract||Natural mixture||Impairment of biofilm structure and ||No resistance||[16, 152, 159]|
|Halogenated furanones from algae ||Synthetic or modifies natural derived furanones||No resistance||[16, 160]|
Traditional antibiotic therapy is related to the early colonization period, the only possible phase when
A more novel antibiofilm strategy, QS interruption, is a promising approach for treating CF respiratory infections. In this strategy, the QS system is targeted, due to its regulation of the biosynthesis of RLs and EPSs [151–153]. The QS impairment approach involves identification of molecules which can interrupt QS pathways. Generally, these compounds have one of following mechanisms of activity: blocking production of QS signal molecules, degradation of QS signal molecules or prevention of microbe recognition and response to QS stimuli . Various natural compounds inhibited QS or directly impaired biofilm (Table 2) (e.g., garlic extract, metabolites from
In the context of the physiological roles of RLs and EPSs discussed in Section 4, these compounds are also promising targets for future strategies in CF therapy related to specific modulation of respiratory mucus .
RLs and EPSs, biosurfactant molecules, play significant roles in bacterial acquisition, biofilm development and establishment of chronic
This work was supported by projects III43004 and III46010, granted by the Ministry of Education, Science and Technological Development of the Republic of Serbia.