Structure‐Function Relationships of Rhamnolipid and Exopolysacharide Biosurfactants of Pseudomonas aeruginosa as Therapeutic Targets in Cystic Fibrosis Lung Infections

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 infec‐ tions 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 infec‐ tion. 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.


Introduction
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) [1].
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 P. aeruginosa, Burkholderia cepaci, Achromobacter xylosoxidans, Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, nontuberculous Mycobacteria and Aspergillus fumigatus. In spite of the host inflammatory response and intensive antibiotic therapy, infections persist and lead to respiratory failure requiring lung transplantation or death [1].
Chronic P. aeruginosa lung infection is the cause of much of the morbidity and most of the mortality in CF patients. Chronic infection is considered as growth of P. aeruginosa from multiple respiratory cultures over a 6-month period [2]. About 80% of adults with CF have chronic P. aeruginosa infection [3]. P. aeruginosa is able to survive and persist for several decades in the respiratory tract of CF patients, in spite of the defense mechanisms of the host and intensive antibiotic therapy. However, the microbe has adaptive mechanisms, which explain why it can survive in the hostile CF lung for so long. These include phenotype splitting due to mutations (into nonmucoid or mucoid), their different distributions in respiratory and conductive zones in the lungs and switching to a biofilm mode of growth-mucoid phenotype [4][5][6][7].
Recent research indicates that chronic P. aeruginosa infections are caused by the ability of bacteria to organize themselves into microcolonies regarding to formation of biofilms. In this state, the bacteria are incorporated in a self-produced protective matrix, often with surrounding inflammatory cells, which is very well protected against antibiotics and the host defense [4]. The biosurfactant compounds (RLs and EPSs), due to their structures and physicochemical properties, as well as their interactions and correlation with other metabolites, significantly contribute to colonization, motility and biofilm formation [8][9][10]. Additionally, the mucoid colony morphology of P. aeruginosa is highly correlated with overproduction of alginate (a type of EPS) [8]. Therefore, it is important to consider these biosurfactants and their biosynthetic pathways as possible targets and approaches for CF therapy in order to impair P. aeruginosa mechanisms of pathogenicity. Furthermore, cell-to-cell communication and QS signaling pathways together with their genetic aspects, closely related to RL and EPS biosynthesis, are the most significant targets for new therapy approaches in CF treatment [10][11][12][13].

P. aeruginosa quorum sensing systems and biofilm
One of the most important factors which facilitate P. aeruginosa to colonize and persist in acute and chronic lung infection in CF patients is the ability of this microbe to grow as a biofilm, assembly of which is regulated by a QS system [13,30,40].
QS is the mechanism by which bacteria engage in cell-to-cell communication using diffusible molecules based on a critical cell density [41]. QS molecules manage and regulate diverse physiological processes, some of which are interrelated. In P. aeruginosa, expression, production and/or secretion of many virulence factors, such as EPSs, RLs, enzymes, pigments production, biofilm formation and antibiotic resistance, are controlled by QS [10,13,42]. P. aeruginosa possesses two interrelated QS systems, the las and rhl systems. The las system comprises the transcriptional regulatory protein, LasR and its cognate autoinducer, N-(3-oxododecanoyl) homoserine lactone (3O-C 12 -HSL). The rhl system comprises the RhlR transcription regulator protein (also known as R-protein) and N-butyryl homoserine lactone (C 4 -HSL), its cognate autoinducer [13]. Additionally, these two systems are not independent but are interlinked in a hierarchical manner (the las system directs the rhl system). They are linked by a third signal molecule, 2-heptyl-3-hydroxy-4-quinolone, known as the Pseudomonas quinolone signal (PQS). PQS is produced under the control of the pqs system, which is considered as the third distinct QS system [11,42]. Interestingly, transcriptome analyses have revealed that between 6 and 10% of the P. aeruginosa genome is regulated by the las and/or rhl systems [13].
Biofilms are matrix-enclosed microbial accretions that adhere to biological or nonbiological surfaces [43]. P. aeruginosa biofilms are related to development of different acute and chronic infections, not only in CF patients [16,44,45]. Formation of P. aeruginosa biofilm occurs in stages: bacterial attachment and irreversible adhesion to surface, microcolony development, biofilm maturation and dispersion of bacterial cells from the biofilm [46]. Heterogeneous microenvironments due to oxygen and nutrient diffusion limitations occur in biofilms and

Mechanism Effect
Decreased ion transport, which results from defective CFTR channels enhances fluid absorption in the airways they lead to physiological and phenotype diversity [47,48]. Suggested mechanisms of P. aeruginosa biofilm formation involve QS signaling, which coordinates and protects biofilm assembly and maintenance [44,[49][50][51][52]. The las I, which encodes the biosynthetic pathway for 3O-C 12 -HSL, is critical for biofilm maturation [50]. Heterogeneity of the bacterial population in biofilm is an important characteristic of P. aeruginosa pathogenicity and contributes to the microbe's resistance to antimicrobial therapy. In laboratory conditions, P. aeruginosa can form two types of biofilm, "flat" and "structured", and alginate-producing isolates (the mucoid phenotype) form complex structured type of biofilm which is resistant to tobramycin [53]. Additionally, the QS system is involved in regulation of several genes such as rhlA, rpoS, sad A and genes in the denitrification pathways. These genes are important for all stages of biofilm development, maintenance, or dispersion: (1) biosynthesis of the biofilm matrix (EPSs, extracellular DNA); (2) biosynthesis of RLs; and (3) other metabolic pathways (not discussed here) [13,42].

Adaptation mechanisms of P. aeruginosa in 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 P. aeruginosa populations to overcome these stressors and persist [54].
It is believed that mechanisms that allow P. aeruginosa to cause persistent chronic infection are related to its remarkable potential for adaptation to environmental changes [8,15]. P. aeruginosa adaptations in CF patients' lungs are dynamic and generate subpopulations of bacteria with differing phenotypes [8]. It is thought that primary infection is related to the large P. aeruginosa genome, while development of persistent infection is dependent on spontaneous mutations [55,56]. Mutations are multiple due to different factors such as the presence of hypermutable strains, development of biofilm and downregulation of antioxidant enzymes [57][58][59]. Environmental conditions in CF airways then further favor specific P. aeruginosa phenotypes. This set of adaptations finally leads to development of the subpopulations of bacteria (mentioned above) within the same respiratory tract, which are relatively similar, but which carry unique groups of genes [56,60,61]. Some commonly and intensively studied P. aeruginosa adaptation mechanisms present during respiratory infections in CF involve: transition to mucoid phenotype, antibiotic resistance, alterations in lipopolysacharride (LPS), loss of type III secretion and motility, auxotrophy, small-colony variants, defects in the QS system and hypermutability [8,54].

P. aeruginosa rhamnolipids
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-monorhamno-di-lipidic, RL2-mono-rhamno-mono-lipidic, RL3-di-rhamno-di-lipidic and RL4di-rhamno-mono-lipidic structures. RL1 and RL3 are usually classified as principal-common RLs, while RL2 and RL4 are classed as atypical-uncommon RLs [68]. 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 Pseudomonas spp. and other bacteria [9].
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.

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 [85]. 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 P. aeruginosa, belong to single operon, rmlBDA [11]. dTDP-L-rhamnose has an important role in the regulation of RL biosynthesis, as it is an allosteric regulator for RmlA (which catalyzes transfer of a thymidylmonophosphate nucleotide to glucose-1-phosphate and is a sensor enzyme in this metabolic pathway). Also, this molecule is a precursor for other Lrhamnose containing molecules (LPSs and EPSs). dTDP-L-rhamnose affects the production of mono-RL through coexpression of the operons rmlBDAC and rhlAB, which are responsible for expression of rhamnosyltransferases RhaA and RhaB [86,87]. However, in P. aeruginosa, the QS system has an essential role in regulation of the rhlAB operon and, therefore, in RL biosynthesis.
In Section 2.2.1, we stated that P. aeruginosa QS has two interrelated systems, las and rhl, which are linked by the PQS molecule and that their relationship influences the biosynthesis of various metabolites. Production of RLs is governed by three QS molecules: Pseudomonas autoinducer 1 (PAI-1, also known as 3O-C 12 -HSL), Pseudomonas autoinducer 2 (PAI-2, also known as C 4 -HSL) and PQS. In P. aeruginosa, the las operon consists of two transcriptional activator proteins, LasR and LasI, which direct the synthesis of PAI-1. The production of RLs is regulated by the rhl system. The synthesis of RLs takes place under the coordinated guidance of the rhlAB genes. The rhl system consists of the transcriptional activator proteins, RhlR and RhlI, which regulate the synthesis of PAI-2. The transcriptional activator RhlR activates the transcription of rhlAB operon and gene rhlC (encoding RhlC) [10,11].
The rhlAB operon is clustered on P. aeruginosa DNA together with rhlR and rhlI, which together direct the synthesis of all proteins required for RL production (the rhamnosyltransferases and the transcriptional activators, RhlR and RhlI) [10]. RL synthesis is upregulated and promoted at transcriptional level, related to the QS system, by the Vfr (global virulence regulation) and the pqs systems through activation of RhlR expression and rhlRI operon, respectively [11]. RasL (repressor of las system) and AlgR (biofilm formation) downregulate RL synthesis by repression of LasI and rhlAB/rhlI, respectively [11]. For instance, increasing bacterial cell density induces the las system, resulting in an increased concentration of PAI-1 that binds to the transcriptional activator site LasR and forms the LasR-PAI-1 complex. The LasR-PAI-1 complex induces genes controlled by the rhl system, including the regulator gene rasL, rhlR and pqsH, required for PQS production. PQS acts as a link between the las and rhl systems. The activity of these signals depends on their ability to dissolve in and freely diffuse through aqueous solution [10]. PQS induces the rhlI gene, which directs the production of PAI-2 that binds to and activates RhlR (RhlR-PAI-2 complex). The RhlR-PAI-2 complex induces genes for RL production, which are controlled by the rhl QS system (operons rhlAB, rhlC, rhlI, rhlR and rhlG). The RLs produced enhance the solubility of PQS in aqueous solutions and promote cell-to-cell communication. This is important because of the role PQS plays in the P. aeruginosa stress response, in conditions related to the CF lung environment (oxidative stress and antimicrobial agents) [88].
In conclusion, in the complex QS network, there is a hierarchy between las and rhl systems in RLs biosynthesis. Furthermore, RL biosynthesis is regulated at the transcriptional level according to nutritional and environmental conditions, as well as at the posttranscriptional level [11,42]. However, most of the regulatory mechanisms are not completely understood [11,42].

P. aeruginosa exopolysaccharides
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 [89]. Nearly all Pseudomonas isolates, including P. aeruginosa, Pseudomonas putida and Pseudomonas fluorescens can produce alginate as the main acidic EPS [90][91][92].
Alginate is composed of β-1,4-D-mannuronic and L-guluronic acids linked via β-1,4-glycosidic bonds [93]. Alginates are also produced by Azotobacter isolates and some genera of brown and red algae. In comparison to algal alginates, bacterial alginates are O-acetylated at some of the C-2 and C-3 carbons of the mannuronic acid residues and acetylation occurs during transport through the periplasm. A high degree of O-acetylation increases the viscosity and flexibility of alginate, as well as its ability to bind water [94].

Diversity of exopolysaccharide structures
P. aeruginosa has the genetic ability to produce at least three polysaccharides: alginate, Psl (polysaccharide synthesis locus) and Pel (pellicle formation locus). Alginate and Psl have different chemical structures (Figure 2a) although they have similar biosynthetic mechanisms [89]. In comparison to alginate, a highly O-acetylated linear polymer of 1,4-linked mannuronic acid (M) and guluronic acid (G), Psl is a helicoid polysaccharide composed of a repeating pentamer containing D-mannose, L-rhamnose and D-glucose (Figure 2b). The structure of Pel is not completely characterized and it is supposed that it differs from alginate and Psl   [95]. Pel is proposed to be a glucose-rich polysaccharide, different to cellulose [96]. Each EPS has distinct physiological properties, affecting the cells and the biofilm matrix. While alginate is secreted into the surrounding medium without covalently linking to the cell surface, Psl has helical distribution around the cell surface with a key role in cell-to-cell and cell-to-surface interactions during biofilm formation. Pel forms a connecting matrix allowing it a structured assembly at the air-liquid interface connecting the cells. This matrix could also contain O-antigen-LPS and cyclic glucans [95]. The diversity of EPSs produced by bacterial biofilm subpopulations is one of the proposed P. aeruginosa survival strategies for adaptation to environmental changes, as related to the conditions in CF lungs.
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 E. coli capsule polysaccharides, the structures of which are more regular, with repeating subunits (Figure 2b). The randomness of alginate's structure occurs because during polymerization, AlgG converts D-mannuronic acid residues to L-guluronic acid and critically, either the C-2 and/or C-3 carbons can have acetylated hydroxyl functional groups, which become available for linking the residues.
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 las system might, to some extent, regulate expression of algC and algD, confirming the correlation of QS systems with EPS production [13].

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 P. aeruginosa biofilm structuring, maturation (the formation of water channels in mature biofilms) and dispersion [10]. Probably because they do not present the profile of typical or traditional virulence factors, RLs are sometimes not considered significant members of the virulence arsenal of P. aeruginosa [9]. However, published data strongly demonstrate their importance as virulence determinants and their significant role in infection establishment and persistence [8,9].
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 [94]. In this regard, the P. aeruginosa mucoid phenotype is the most studied adaptation in patients with CF and it is directly proportional to overproduction of EPSs, which is widely considered to be a marker for the transition to chronic infection [8,54]. Alginates are well studied as compounds associated with biofilm formation and invasion of pathogenic microorganisms. The alginate-containing matrix of mucoid P. aeruginosa is thought to allow the formation of protected microcolonies and provide increased resistance to opsonization, phagocytosis and destruction by antibiotics [104]. Alginates also have a protective role in P. aeruginosa infection because they scavenge free radicals released by activated macrophages in vitro, prevent phagocytic clearance and protect the microorganism from the host defense system [13].

Rhamnolipids and exopolysaccharides in P. aeruginosa biofilm 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 [105]. 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 [106]. Several studies suggest that P. aeruginosa expresses swarming motility and that it requires flagella and the production of wetting agents (RLs and its lipidic precursors HAAs) [85,[107][108][109]. Also, HAAs and di-RLs actually modulate the swarming process, as di-RLs and HAAs behave as self-produced chemotactic attractants with opposite activity, while mono-RLs seem to be act solely as wetting agents [107,109]. Additionally, swarming motility is clearly related to biofilm formation [105].
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 cellsurface hydrophobicity and modification of adhesive interactions, especially when nutritional conditions are changed [85,[110][111][112]. Also, RLs are involved in later differentiation of the biofilm structure, the detachment and dispersion of P. aeruginosa cells, where RLs behave as mediators which disturb cell-to-cell and cell-to-substratum interactions and maintenance of open channels inside the biofilm [111,113]. Furthermore, regulation of RL production by P. aeruginosa is regulated not only in temporal terms, but also in quantifiable terms, because overproduction of RLs disrupts biofilm structure or impedes biofilm formation [113].
EPSs also play an important role in biofilm formation and invasion of pathogenic microorganisms. During biofilm maturation, P. aeruginosa begin to excrete EPSs, such that the bacteria in the mature biofilm are encased in a matrix of EPSs that they have produced [114]. Overproduction of alginate is the main indicator of P. aeruginosa converting to the mucoid phenotype and is responsible for the notable microbial resistance to antibiotics as well as defense from the host immune system of CF patients (Figure 3). The mucoid phenotype of P. aeruginosa produces a great amount of alginate as a result of several genes, including algD, which encodes GDP-mannose dehydrogenase, responsible for synthesis of alginate precursor [8,94]. The alginate-containing matrix of the mucoid phenotype allows the formation of protected microcolonies and provides increased resistance to opsonization, phagocytosis and antibiotics, resulting in persistent infection and a worsening prognosis for CF patients [104].
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 [115]. When PMNs phagocytose bacteria, the host cells produce highly reactive oxygen species, which kill P. aeruginosa or induce mutations in the microbial mucA gene. However, the alginate produced by mucoid phenotype P. aeruginosa is also an oxygen radical scavenger, helping to protect this pathogen against host inflammatory defense mechanisms [116]. Airway epithelial cells play a crucial role during establishment of respiratory infection because P. aeruginosa attaches to and enters respiratory epithelia, producing an immune response in the lung by activating lymphocytes at the site of infection [117]. direct role in bacteria opsonization and killing, as well as impairment of bacterial membrane permeabilization [117]. Alginate is surface exposed and levels of SP-A could be crucial in modulating the interaction of P. aeruginosa with the epithelial barrier.

Effect of P. aeruginosa rhamnolipids and exopolysaccharides
Respiratory mucosa protects host airways from microbial infection. P. aeruginosa and other microbial species capable of causing lung infections have developed mechanisms to overcome this barrier, such as alteration of the apical membrane of epithelial cells or alteration and disruption of tight junctions (TJ) [118]. Proposed mechanisms involve alterations of respiratory epithelial ion transport, inhibition of transcellular ion transport and interference with the normal tracheal ciliary function. Bacterial adherence to the basolateral domain of epithelial cells and internalization are suggested as a potential mechanism of P. aeruginosa pathogenicity (Figure 3). The physiological pathways of these processes are not still completely clarified, but reports indicate involvement of virulence factors, production of which is controlled by the type III secretion (cytotoxic proteins) and the las and rhl QS (RLs, elastase) systems [119,120].
RLs concentration of up to 8 μg/ml was found in the sputum of CF patients infected by P. aeruginosa [120], while secretions from a lung removed contained 65 μg/ml RLs [121]. These concentrations of RLs are likely adequate for promotion of P. aeruginosa epithelial cell infiltration. Furthermore, this indicates link between elevated levels of RLs and worsening of patient clinical status.
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 P. aeruginosa can act synergistically to break down lipids and lecithin from lung surfactant [12]. It is believed that RLs, due to their detergency, solubilize the phospholipids in lung surfactant, making them more accessible to cleavage by phospholipase C [12].
The effects of P. aeruginosa RLs on the respiratory epithelia function were studied in several animal models [122]. RLs caused ciliostasis and cell membrane damage to rabbit tissue were a secretagogue in cats and inhibited epithelial ion transport in sheep tissue. Additionally, the authors investigated the effect of RLs on mucociliary transport in the anesthetized guinea pig and guinea pig and human respiratory epithelia in vitro [122]. Reduction of tracheal mucus velocity (TMV) in vivo occurred depending on the applied RL concentration (10 μg of RLs caused cessation of TMV without recovery; 5 μg of RLs reduced TMV by 22.6% over a period of 2 h and 2.5 μg of RLs caused no overall change in TMV). RLs (10 μg) did not disrupt the ultrastructure of guinea pig tracheal epithelium. RL (250 μg/ml) stopped ciliary beating of guinea pig tracheal. Treatment with RL concentration of 100 μg/ml caused immediate slowing of the cilliar beat frequency (CBF) of human nasal brushings, as well as CBF of human nasal turbinate organ culture. Mono-and di-RL had equivalent effects [122]. In addition, RLs stimulate the release of mucus glycoconjugates from feline trachea or human bronchial mucosa [125,126].
In vitro reconstructed respiratory epithelium was exposed to several P. aeruginosa isolates with alterations in genotype: wild type, CF isolates and strains with altered QS system expression [118]. The authors found that only RL-producing P. aeruginosa (those that expressed the rhl QS system) was able to infiltrate the epithelia by modulating the permeability of the tissue. The early stages of infection did not correlate with type III secretion and elastase activity [118], in contrast to previous reports [127,128]. The effect of exogenously applied purified RLs on the epithelial barrier was also studied [118]. The authors used JBR 515, which is commercial mixture of 50% w/v Rha-C 10 -C 10 and 50% w/m Rha-Rha-C 10 -C 10 . RLs produced by bacteria in situ or purified. The applied RLs caused loss of epithelial cell polarity by: incorporation in first, the apical and later, the basolateral epithelial membranes (due to chemical structure); cilia loss; ezrin displacement; and alterations of TJ. The final result was a decrease of transepithelial resistance and higher permeability of respiratory epithelia, without affecting cell viability [118]. After disruption of TJ, paracellular invasion by some P. aeruginosa, involving RL deficient strains, was observed, but they were not internalized [118]. This was in contrast to previous reports [129,130], perhaps due to the in vitro conditions used in the studies as difference. Altogether, the importance of RL biosurfactant and the QS system in P. aeruginosa invasion of respiratory epithelium is acknowledged, but the exact mechanisms of cell polarity and structure alterations remain unclear.
The effect of RLs on immune system pathways with direct impairment and modulation of immune cell activity is well known [9] (Figure 3). RLs are reported to have hemolytic activity on various erythrocyte species; induce direct neutrophil chemotactic activity [130]; 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][132][133][134][135]. Furthermore, RLs, especially di-RLs, are cytolytic for human monocyte-derived macrophages and at lower concentrations, they inhibit the phagocytic response of macrophages [136].
The response of P. aeruginosa mutants (PAO1 and QS, rhlA and pqsA deficient) to the presence of PMNs was studied [115]. Previously reported data showed that in vitro, PMNs performed their immune function and eliminated QS-deficient P. aeruginosa biofilms, although they were incapable of eliminating QS-proficient biofilms [51]. Additionally, purified RLs induced necrosis in PMNs [134]. In biofilm, P. aeruginosa (PAO1 wild type) produced increased levels of various virulence factors in response to PMNs, while P. aeruginosa rhlA mutant was eliminated by PMNs [115]. Additionally, 2000-fold higher levels of RLs from P. aeruginosa PAO1 occurred in biofilm than in surrounding fluid, indicating that RL molecules were grouped around biofilm [115]. Similarly, a P. aeruginosa rhlA mutant was cleared more quickly than the wild strain from two in vivo mouse models of lung infection [137]. Also, microscopic analysis showed that there were no intact PMNs in close contact with outer layers of biofilm. This correlated with microscopic investigations of P. aeruginosa infected ex vivo tissues samples from CF lungs, where PMNs were located peripherally [115]. The RLs isolated in this study were a mixture of mono-and di-RL congeners (Rha-C 10 -C 10 , Rha-C 10 -C 12 , Rha-C 10 -C 12Δ and respective di-RL derivates) [137]. Van et al. [137] proposed that RLs have a role as a protective mechanism in biofilm resistance to phagocytosis and supported a "launch a shield'' model, where RLs surround the biofilm and on contact destroy PMNs. This study [137], in correlation with previous reports about QS regulation of bacterial response to PMNs [50,134] showed that P. aeruginosa pqsA mutant was unable to respond to exposure to PMNs by increasing RL production and that there was impairment of the QS hierarchy. These studies show that RLs probably contribute to the inflammatory-related tissue damage observed in lungs of CF patients, which involves complex and tight regulation by the QS system. RL production, though, is not continued because it affects all host cells, not only immune cells and high levels of RL may create conditions (due to inflammation and host tissue damage) which are not favorable for P. aeruginosa persistence [137]. This study supports a model by which cross-kingdom-based communication contributes significantly to immunomodulation and evasion and which is one reason studying the infective properties of P. aeruginosa is so fascinating.
Modification of membrane LPSs in P. aeruginosa is also an important mechanism in the development of chronic infection in CF patients [138][139][140]. Membrane LPSs in P. aeruginosa are composed of three parts: highly acylated lipid A; a central core oligosaccharide bound to lipid A and O-antigen; and a variable polysaccharide composed of repeated units located out from the core [138,140]. It is not surprising that the structure of LPSs is modified in P. aeruginosa isolated from CF patients because of their direct interface position with the pulmonary environment [8]. Compared to normal lipid A, that from CF patients contains more hexaand hepta-acylated moieties as well as added aminoarabinose, a cationic amino sugar residue which is responsible for resistance to antimicrobials [140]. Acylation levels of lipid A are responsible for LPS recognition by the host and induction of the proinflammatory response, so their modification causes P. aeruginosa to be less visible to the host immune system [141]. Also, in CF isolates, O-antigen is lost, due to mutations in genes responsible for O-antigen production. This loss can facilitate chronic persistence in respiratory tracts of CF patients [138][139][140]. Modification of LPS can directly correlate with overproduction of alginate, which is typical for the mucoid phenotype. Alginate might interact via the carboxylic groups in polyguluronic acid units with modified membrane LPSs in P. aeruginosa, across cationic amino sugar aminoarabinose residues. This likely enhances polymerization and facilitates release of EPSs from the membrane. Thus, study of factors that influence increased production of EPSs and RLs, as well as the structure-function relationships of these compounds would likely be of great importance for improved therapy of CF patients [8].

Rhamnolipids and exopolysaccharides as targets-current and future perspectives
The importance of biofilm formation and maintenance for the establishment and persistence of P. aeruginosa chronic respiratory infection in CF has been discussed in Section 2.2.1. The complex regulation of biofilm development includes the QS network, swarming motility and production of extracellular metabolites and involves significant roles for RLs and EPSs.
Therefore, a logical approach in preventing and treating chronic P. aeruginosa infection in CF patients is focused on antibiofilm strategies. Antibiofilm strategies can take two differing approaches, one common, related to antibiotic therapy and the other novel, related to interruption of QS ( Table 2). Furthermore, vaccination is proposed as a modern approach to prevent P. aeruginosa infection in CF, where virulence factors, such as alginate, have been used as the antigen. However, most vaccines are still in the clinical research phase and have not reached the market [142].
Traditional antibiotic therapy is related to the early colonization period, the only possible phase when P. aeruginosa can be eradicated from CF airways [143,144]. The effectiveness of antibiotics later is significantly reduced due to microbe adaptation mechanisms (membrane changes, efflux system changes, production of various virulence factors and EPS-containing extracellular matrix, mutation and modification of enzymes) [16] ( choice in CF lungs [145]. This is in spite of the fact that alginates produced by the microbe decrease, the movement of aminoglycosides, cationic antimicrobial peptides and quaternary ammonium compounds through P. aeruginosa biofilms [27,146] (Table 2). To overcome obstacles related to antibiotic resistance and increase the antimicrobial effects, an inhaled version of tobramycin, as well as liposomal-encased current antibiotics are available. These antibiotic formulations have improved delivery times and provide higher drug concentrations at the site of infection. Additionally, the importance of biofilm formation as having a crucial role in the antibiotic resistance of P. aeruginosa (as well as other CF pathogens) is now being recognized. Recent research trends include analysis of biofilm formation in terms of P. aeruginosa antibiotic resistance/susceptibility and the potential for antibiotics as efficient therapy agents for biofilm impairment [147][148][149][150].
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][152][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 [16]. Various natural compounds inhibited QS or directly impaired biofilm ( Table 2) (e.g., garlic extract, metabolites from Penicillium spp., salicylic acid, the P. aeruginosa metabolite cis-2-decanoic acid). Furanones are QS blockers and the furanone produced by Delisea pulchra and synthetic furanones, enhanced P. aeruginosa elimination in combination with antibiotic therapy [16]. Furanone C-30 repressed 77% of P. aeruginosa genes induced by exposure to PMNs [50]. The great advantage of using QS inhibitors in CF therapy is that they are not expected to induce bacterial resistance, because their activity is not closely related to bacterial growth [154].
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 [118].

Conclusion
RLs and EPSs, biosurfactant molecules, play significant roles in bacterial acquisition, biofilm development and establishment of chronic P. aeruginosa infections in CF patients. Specifically, RLs and EPSs are, due to their amphipathic structures and physicochemical properties, involved in processes of respiratory mucus alteration, modulation of immune system defense pathways, biofilm development and maintenance and the P. aeruginosa mucoid phenotype. These compounds are responsible for antibiotic resistance and survival and general persistence of P. aeruginosa in the specific, dynamic environmental conditions in CF patients' lungs. Consequently, RLs and EPSs are the direct or indirect cause of bad outcomes and high mortality rates among these patients. Currently, therapy generally based on application of antibiotics fails to prevent and treat chronic P. aeruginosa infection. Therefore, RLs and EPSs are interesting novel targets for dealing with respiratory infection in CF patients. In addition, the P. aeruginosa QS system is an important aspect of CF lung infection, as it regulates synthesis of the biosurfactants and other virulence factors, as well as biofilm formation. Future perspectives to prevent and treat P. aeruginosa respiratory infections in CF certainly should involve impairment of QS pathways. Finally, further study of potential approaches to modify host respiratory mucus epithelial membranes is required.