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

Milena G. Rikalovic, Natasa S. Avramovic and

Ivanka M. Karadzic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66687

#### **Abstract**

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.

**Keywords:** cystic fibrosis, *Pseudomonas aeruginosa*, biosurfactant, rhamnolipid, exopolysaccharide, biofilm

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

Cystic fibrosis is a congenital, recessively inherited disorder. The genetic background of CF development is >1500 mutations in the cystic fibrosis transmembrane conductance regula‐ tor 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 intes‐ tine, liver, the reproductive tract and sweat glands), resulting in numerous clinical symptoms (viscid mucus, respiratory infections, intestinal malabsorption of fat, diabetes mellitus, meco‐ nium ileus, impaired liver function, male infertility and salt loss) [1].

The malfunction of the chloride channel in CF patients leads to impairment of the non‐ inflammatory 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 split‐ ting due to mutations (into nonmucoid or mucoid), their different distributions in respira‐ tory and conductive zones in the lungs and switching to a biofilm mode of growth—mucoid phenotype [4–7].

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 sur‐ rounding 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 physi‐ cochemical properties, as well as their interactions and correlation with other metabolites, significantly contribute to colonization, motility and biofilm formation [8–10]. 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 [10–13].

## **2.** *P. aeruginosa* **infection in CF**

#### **2.1.** *P. aeruginosa*

**1. Introduction**

128 Progress in Understanding Cystic Fibrosis

or death [1].

phenotype [4–7].

[10–13].

Cystic fibrosis is a congenital, recessively inherited disorder. The genetic background of CF development is >1500 mutations in the cystic fibrosis transmembrane conductance regula‐ tor 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 intes‐ tine, liver, the reproductive tract and sweat glands), resulting in numerous clinical symptoms (viscid mucus, respiratory infections, intestinal malabsorption of fat, diabetes mellitus, meco‐

The malfunction of the chloride channel in CF patients leads to impairment of the non‐ inflammatory 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

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 split‐ ting due to mutations (into nonmucoid or mucoid), their different distributions in respira‐ tory and conductive zones in the lungs and switching to a biofilm mode of growth—mucoid

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 sur‐ rounding 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 physi‐ cochemical properties, as well as their interactions and correlation with other metabolites, significantly contribute to colonization, motility and biofilm formation [8–10]. 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

nium ileus, impaired liver function, male infertility and salt loss) [1].

*Pseudomonas* is ubiquitously present worldwide, being an extremely diverse bacterial genus. Pseudomonads are frequently closely associated with animals and plants; they are common and numerous in a wide range of environmental habitats. Their ability to adapt genetically, producing varying physiological advantages as a response to their pervasiveness, is the sub‐ ject of much scientific speculation and study. *P. aeruginosa*, as all species that belong to the genus *Pseudomonas*, due to its metabolic diversity, has potential for adaptation, survival and growth in a wide range of environmental conditions [14, 15].

*P. aeruginosa* produces an arsenal of secondary metabolites, including EPSs, RLs, enzymes (elastase, alkalne protease, exoenzyme S, phospholipase C and hemolysins), pigments and toxins (exotoxin A), using these virulence factors for infecting and colonizing a wide range of hosts (animals, plats, insect and nematodes) and surfaces [12–24]. The major biosurfactant compounds produced by *P. aeruginosa*, RLs and EPSs, are involved in bacterial adherence, biofilm formation and maintenance, which all are necessary for respiratory infection estab‐ lishment, development and progression in CF patients [4, 8, 12, 13, 16].

#### **2.2. Pathogenesis of** *P. aeruginosa* **infection in CF**

Despite constant exposure to a wide range of microorganisms, CF patients are predisposed to infection by only specific groups of microorganisms [8]. The proximal event in develop‐ ment 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 devel‐ opment [8]: (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) intrin‐ sic hyperinflammation of airways (**Table 1**) [25–36].

The first step in infection of CF airways by *P. aeruginosa* is microbe acquisition [8]. Due to the abundance of *P. aeruginosa* in many natural environments, most individuals acquire *P. aeru‐ ginosa* through casual contact with natural bacterial sources, while some individuals acquire *P. aeruginosa* directly or indirectly from other CF patients. Transmission data and genotype/ phenotype properties of clinical and environmental *P. aeruginosa* isolates indicate that char‐ acterizing the ecology of *P. aeruginosa* originating from natural environments would lead to a better understanding CF epidemiology [8].

Initially, infection of *P. aeruginosa* in CF is usually the result of an alternating series of acquisitions of different isolates and in the first stage of infection, most of the isolates are nonmucoid and highly antibiotic sensitive [8, 37–39]. Eventually, one or two isolates establish themselves and, due to their genetic, phenotypic and physiological changes, develop chronic infection [13, 16, 40].


**Table 1.** Proposed mechanisms of *P. aeruginosa* in development of respiratory infection in CF airways.

#### *2.2.1. 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 dif‐ fusible molecules based on a critical cell density [41]. QS molecules manage and regulate diverse physiological processes, some of which are interrelated. In *P. aeruginosa*, expres‐ sion, 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‐C12‐HSL). The *rhl* system comprises the RhlR transcription regulator protein (also known as R‐protein) and N‐butyryl homoserine lactone (C<sup>4</sup> ‐HSL), its cognate autoinducer [13]. Additionally, these two systems are not indepen‐ dent 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 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, 49–52]. The *las I*, which encodes the biosynthetic pathway for 3O‐C12‐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 bio‐ film 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].

#### *2.2.2. Adaptation mechanisms of P. aeruginosa in CF lungs*

*2.2.1. P. aeruginosa quorum sensing systems and biofilm*

**Mechanism Effect** Decreased ion transport, which results from defective CFTR channels enhances fluid absorption in the airways

Increased levels of acylated glycolipids on the surface of CF airway epithelial cells due to defective CFTR molecules

Binding of *P. aeruginosa* to defective CFTR molecule exposed

on airway epithelial cells membranes

130 Progress in Understanding Cystic Fibrosis

(C<sup>4</sup>

the *las* and/or *rhl* systems [13].

assembly of which is regulated by a QS system [13, 30, 40].

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,

Istrinical hyperinflammation of airways Damage of host cells and disruption of effective microbe

Altered salt content in the airway surface liquid Inactivation of immune system defenses pathways;

Lowered level of antimicrobial compounds Propensity of individuals to lung infection

**Table 1.** Proposed mechanisms of *P. aeruginosa* in development of respiratory infection in CF airways.

Lowered airways surface liquid and impaired ciliary transport of the mucous layer, which results in defects

Modified glycolipids are receptors for *P. aeruginosa*

in microbial clearance

adherence

clearance

defected neutrophils activity

Internalisation of *P. aeruginosa*

QS is the mechanism by which bacteria engage in cell‐to‐cell communication using dif‐ fusible molecules based on a critical cell density [41]. QS molecules manage and regulate diverse physiological processes, some of which are interrelated. In *P. aeruginosa*, expres‐ sion, 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‐C12‐HSL). The *rhl* system comprises the RhlR transcription regulator protein (also known as R‐protein) and N‐butyryl homoserine lactone

‐HSL), its cognate autoinducer [13]. Additionally, these two systems are not indepen‐ dent 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

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 The CF lungs are an unfriendly and varied environment for invading bacteria due to the pres‐ ence 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. aerugi‐ nosa* 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 [57–59]. Environmental conditions in CF airways then further favor specific *P. aeruginosa* phe‐ notypes. 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. aerugi‐ nosa* 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].

## **3. Biosurfactants of** *P. aeruginosa***—rhamnolipids and exopolysaccharides**

Biosurfactants are a group of amphiphilic compounds, comprise a hydrophobic and a hydro‐ philic moiety and are produced by a range of microorganisms [9, 62]. *Pseudomonas* spp. are the most common producers of biosurfactants [63], with *P. aeruginosa* being the preeminent RL and EPS biosurfactant‐producing species [9, 63]. Up to date, a variety of biosurfactants have been studied, but RLs (glycolipid biosurfactants) and EPSs (polymeric biosurfactants) are currently attracting the most attention, as they are relevant in medicine, environmental protection, food and the pharmaceutical industry [15, 24, 64–66].

#### **3.1.** *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—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 [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].

#### *3.1.1. Diversity of rhamnolipid structures*

RL biosurfactants are always produced as mixtures of different RL congeners, as observed with various *P. aeruginosa* isolates [15, 69–74]. The complexity of the RL mixtures produced depends on various factors such as bacterial isolate origin, type of carbon substrate, culture conditions and isolation procedure and age of the culture and of course, the *P. aeruginosa* iso‐ late itself [15, 23, 63, 72, 75–80]. Despite the number of such factors reported, some particular RL congeners are predominant in all *P. aeruginosa* producer isolates. These are classified as the major RL structures (Rha‐C10‐C<sup>8</sup> , Rha‐C10‐C10, Rha‐C10‐C12, Rha‐C10‐C12:1, Rha‐Rha‐C10‐C<sup>8</sup> , Rha‐Rha‐C10‐C10, Rha‐Rha‐C10‐C12 and Rha‐C10‐C12:1) [23, 72, 81–84]. Other RLs, produced only sometimes or in low abundance, are the minor RL structures [23, 72, 81–84]. Both the major and the minor RL congeners contribute to the complete profile of RLs. In all studies of RL mixtures produced by various *P. aeruginosa* isolates, mono‐rhamnolipid Rha–C10–C10 and di‐ rhamnolipid Rha–Rha–C10–C10 were the predominant congeners, in spite of the varying com‐ positions produced [23, 72, 81–84].

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 rham‐ nosyltransferase 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 Structure‐Function Relationships of Rhamnolipid and Exopolysacharide... http://dx.doi.org/10.5772/66687 133

have been studied, but RLs (glycolipid biosurfactants) and EPSs (polymeric biosurfactants) are currently attracting the most attention, as they are relevant in medicine, environmental

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 [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].

RL biosurfactants are always produced as mixtures of different RL congeners, as observed with various *P. aeruginosa* isolates [15, 69–74]. The complexity of the RL mixtures produced depends on various factors such as bacterial isolate origin, type of carbon substrate, culture conditions and isolation procedure and age of the culture and of course, the *P. aeruginosa* iso‐ late itself [15, 23, 63, 72, 75–80]. Despite the number of such factors reported, some particular RL congeners are predominant in all *P. aeruginosa* producer isolates. These are classified as

Rha‐Rha‐C10‐C10, Rha‐Rha‐C10‐C12 and Rha‐C10‐C12:1) [23, 72, 81–84]. Other RLs, produced only sometimes or in low abundance, are the minor RL structures [23, 72, 81–84]. Both the major and the minor RL congeners contribute to the complete profile of RLs. In all studies of RL mixtures produced by various *P. aeruginosa* isolates, mono‐rhamnolipid Rha–C10–C10 and di‐ rhamnolipid Rha–Rha–C10–C10 were the predominant congeners, in spite of the varying com‐

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

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 rham‐ nosyltransferase 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

, Rha‐C10‐C10, Rha‐C10‐C12, Rha‐C10‐C12:1, Rha‐Rha‐C10‐C<sup>8</sup>

,

protection, food and the pharmaceutical industry [15, 24, 64–66].

**3.1.** *P. aeruginosa* **rhamnolipids**

132 Progress in Understanding Cystic Fibrosis

*3.1.1. Diversity of rhamnolipid structures*

the major RL structures (Rha‐C10‐C<sup>8</sup>

positions produced [23, 72, 81–84].

*3.1.2. Rhamnolipid biosynthesis and quorum sensing*

uptake, or solubilization.

**Figure 1.** Structure of rhamnolipid congeners: RL1 (mono‐rhamno‐di‐lipidic), RL2 (mono‐rhamno‐mono‐lipidic), RL3 (di‐rhamno‐di‐lipidic) and RL4 (di‐rhamno‐di‐lipidic).

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 L‐ rhamnose 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‐C12‐HSL), *Pseudomonas* autoinducer 2 (PAI‐2, also known as C<sup>4</sup> ‐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 guid‐ ance 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 den‐ sity 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. aerugi‐ nosa* 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].

#### **3.2.** *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 polysaccha‐ rides complexes [89]. Nearly all *Pseudomonas* isolates, including *P. aeruginosa*, *Pseudomonas putida* and *Pseudomonas fluorescens* can produce alginate as the main acidic EPS [90–92]. Alginate is composed of β‐1,4‐D‐mannuronic and L‐guluronic acids linked via β‐1,4‐gly‐ cosidic 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 viscos‐ ity and flexibility of alginate, as well as its ability to bind water [94].

#### *3.2.1. Diversity of exopolysaccharide structures*

of various metabolites. Production of RLs is governed by three *QS* molecules: *Pseudomonas* autoinducer 1 (PAI‐1, also known as 3O‐C12‐HSL), *Pseudomonas* autoinducer 2 (PAI‐2, also

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 guid‐ ance 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 *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 den‐ sity 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. aerugi‐ nosa* stress response, in conditions related to the CF lung environment (oxidative stress and

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

Pseudomonads have the potential to produce various types of EPSs such as alginate, levan, marginalan and cellulose, as well as different heteropolysaccharides and protein polysaccha‐ rides complexes [89]. Nearly all *Pseudomonas* isolates, including *P. aeruginosa*, *Pseudomonas putida* and *Pseudomonas fluorescens* can produce alginate as the main acidic EPS [90–92]. Alginate is composed of β‐1,4‐D‐mannuronic and L‐guluronic acids linked via β‐1,4‐gly‐ cosidic 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

the transcription of *rhlAB* operon and gene *rhlC* (encoding RhlC) [10, 11].

‐HSL) and PQS. In *P. aeruginosa*, the *las* operon consists of two transcriptional

known as C<sup>4</sup>

134 Progress in Understanding Cystic Fibrosis

antimicrobial agents) [88].

**3.2.** *P. aeruginosa* **exopolysaccharides**

[11, 42].

*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 dif‐ ferent 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 mannu‐ ronic acid (M) and guluronic acid (G), Psl is a helicoid polysaccharide composed of a repeat‐ ing 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

**Figure 2.** Structures of extracellular polysaccharides produced by *P. aeruginosa*: (a) alginate and (b) exopolysaccharide 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‐sur‐ face interactions during biofilm formation. Pel forms a connecting matrix allowing it a struc‐ tured 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 envi‐ ronmental changes, as related to the conditions in CF lungs.

#### *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‐man‐ nose 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 *P. aeruginosa* from chronically infected CF patients [13].

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].

## **4. Physiological role of** *P. aeruginosa* **biosurfactants 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 modula‐ tors 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 fac‐ tors, 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].

[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‐sur‐ face interactions during biofilm formation. Pel forms a connecting matrix allowing it a struc‐ tured 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 envi‐

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‐man‐ nose 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 *P.* 

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

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

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 modula‐ tors 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

**4. Physiological role of** *P. aeruginosa* **biosurfactants in CF infection**

**4.1. Physiological role of rhamnolipids and exopolysaccharides**

ronmental changes, as related to the conditions in CF lungs.

*3.2.2. Exopolysaccharide biosynthesis and quorum sensing*

*aeruginosa* from chronically infected CF patients [13].

for linking the residues.

136 Progress in Understanding Cystic Fibrosis

systems with EPS production [13].

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* infec‐ tion because they scavenge free radicals released by activated macrophages *in vitro*, prevent phagocytic clearance and protect the microorganism from the host defense system [13].

#### **4.2. 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 stud‐ ies 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, 107–109]. 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 bio‐ film 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 plank‐ tonic 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 nutri‐ tional conditions are changed [85, 110–112]. 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 micro‐ organisms. 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 precur‐ sor [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 con‐ sidered as the central line of defense in innate immunity and they are produced as a predomi‐ nant 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 muta‐ tions 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].

Surfactant protein A (SP‐A) is involved in prevention of alginate‐induced *P. aeruginosa* inva‐ sion of lung epithelial cells. SP‐A plays a part in the innate immunity in the lung, with a

**Figure 3.** Proposed roles, relations and effects of *P. aeruginosa* biosurfactants RLs and EXPs in development and persistence of chronic respiratory infection in CF patients.

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.

#### **4.3. Effect of** *P. aeruginosa* **rhamnolipids and exopolysaccharides**

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 precur‐ sor [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 con‐ sidered as the central line of defense in innate immunity and they are produced as a predomi‐ nant 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 muta‐ tions 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

Surfactant protein A (SP‐A) is involved in prevention of alginate‐induced *P. aeruginosa* inva‐ sion of lung epithelial cells. SP‐A plays a part in the innate immunity in the lung, with a

**Figure 3.** Proposed roles, relations and effects of *P. aeruginosa* biosurfactants RLs and EXPs in development and

persistence of chronic respiratory infection in CF patients.

infection [117].

138 Progress in Understanding Cystic Fibrosis

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 respira‐ tory 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* pathogenic‐ ity (**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 infiltra‐ tion. Furthermore, this indicates link between elevated levels of RLs and worsening of patient clinical status.

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 *P. aeruginosa* can act syner‐ gistically 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 ultra‐ structure 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‐C10‐C10 and 50% w/m Rha‐Rha‐C10‐C10. 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 tran‐ sepithelial 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 chemo‐ kinesis of PMNs (depending on concentration); and enhance production of several interleu‐ kins 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 macro‐ phages [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 elimi‐ nated 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 cor‐ related 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‐C10‐C10, Rha‐C10‐C12, Rha‐C10‐C12Δ and respective di‐RL derivates) [137]. Van et al. [137] proposed that RLs have a role as a protective mecha‐ nism 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 produc‐ tion and that there was impairment of the QS hierarchy. These studies show that RLs prob‐ ably 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.

[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‐C10‐C10 and 50% w/m Rha‐Rha‐C10‐C10. 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 tran‐ sepithelial 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

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 chemo‐ kinesis of PMNs (depending on concentration); and enhance production of several interleu‐ kins 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 macro‐

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 elimi‐ nated 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 cor‐ related 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‐C10‐C10, Rha‐C10‐C12, Rha‐C10‐C12Δ and respective di‐RL derivates) [137]. Van et al. [137] proposed that RLs have a role as a protective mecha‐ nism in biofilm resistance to phagocytosis and supported a "launch a shield'' model, where

and structure alterations remain unclear.

140 Progress in Understanding Cystic Fibrosis

phages [136].

Modification of membrane LPSs in *P. aeruginosa* is also an important mechanism in the devel‐ opment of chronic infection in CF patients [138–140]. 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. aerugi‐ nosa* 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 hexa‐ and hepta‐acylated moieties as well as added aminoarabinose, a cationic amino sugar resi‐ due 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 [138–140]. 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 poly‐ guluronic 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].

**Figure 3** summarizes the proposed roles, relationships and effects of the biosurfactant RLs and EPSs produced by *P. aeruginosa* in the development of chronic respiratory CF infection.

## **5. 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**.**


**Table 2.** Antibiofilm approaches in therapy of P. *auruginosa* infection of CF patients.

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 differ‐ ing 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] (**Table 2**). Furthermore, tobramycin (an aminoglycoside) is the most common antibiotic for *P. aeruginosa* therapy 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 obsta‐ cles 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 recog‐ nized. 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 [147–150].

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 [151–153]. The QS impairment approach involves identifica‐ tion of molecules which can interrupt QS pathways. Generally, these compounds have one of following mechanisms of activity: blocking production of QS signal molecules, degrada‐ tion 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 com‐ pounds are also promising targets for future strategies in CF therapy related to specific modu‐ lation of respiratory mucus [118].

## **6. Conclusion**

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 differ‐ ing 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

**Agents Type Strategy Resistance References**

Antibiotic cleavage by *β*‐ lactamase enzymes, antibiotic expulsion by encoded efflux mechanisms and reduced drug uptake due to loss of outer membrane porin proteins

and topoisomerase IV enzymes and efflux

Aminoglycoside‐modifying enzymes AMEs and rRNA methylases as well as efflux

No resistance [16, 158]

No resistance [16, 154]

No resistance [16, 152, 154]

No resistance [16, 152, 159]

No resistance [16, 160]

systems

mechanisms

[16, 155 ]

[155, 156]

[16, 155, 157]

structure and *QS* inhibitors

Ciprofloxacin Fluoroquinolones *QS* inhibitors Mutations by DNA gyrase

structure

structure and *QS* inhibitors

structure and *QS* inhibitors

structure and *QS* inhibitors

structure and *QS* inhibitors

*QS*‐inhibitor and *P. aeruginosa* elimination in combination with antibiotics

*β*‐Lactams Impairment of biofilm

Aminoglycosides Impairment of biofilm

Bacterial metabolites Impairment of biofilm

Synthetic compounds Impairment of biofilm

**Table 2.** Antibiofilm approaches in therapy of P. *auruginosa* infection of CF patients.

Solenopisin A Fire ant venom Impairment of biofilm

Garlic extract Natural mixture Impairment of biofilm

Synthetic or modifies natural derived furanones

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] (**Table 2**). Furthermore, tobramycin (an aminoglycoside) is the most common antibiotic for *P. aeruginosa* therapy

not reached the market [142].

Ticarcillin, Piperacillin Cefrazidime, Cefepime Imipenem, Meropenem Aztreonam

142 Progress in Understanding Cystic Fibrosis

Tobramycin, Gentamicin, Amikacin

Patulin, penicillin acid, cis‐2 decanoic acid

Salicylic acid and 4‐nitro‐pyridine oxide (4‐NPO)

Halogenated furanones from algae *D. pulchra*, Furanone C‐30

> 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 persis‐ tence 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 mor‐ tality rates among these patients. Currently, therapy generally based on application of anti‐ biotics 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 perspec‐ tives 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.

## **Acknowledgment**

This work was supported by projects III43004 and III46010, granted by the Ministry of Education, Science and Technological Development of the Republic of Serbia.

## **Author details**

Milena G. Rikalovic1,\*, Natasa S. Avramovic<sup>2</sup> and Ivanka M. Karadzic<sup>2</sup>


## **References**


[7] Jelsbak L, Johansen HK, Frost AL, Thøgersen R, Thomsen LE, Ciofu O, Yang L, Haagensen JA, Høiby N, Molin S: Molecular epidemiology and dynamics of *Pseudomonas aeruginosa* populations in lungs of cystic fibrosis patients. Infect. Immun. 2007;**75**:2214‐2224. DOI:10.1128/IAI.01282‐06

*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 perspec‐ tives 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

This work was supported by projects III43004 and III46010, granted by the Ministry of

[1] Høiby N: Mini review recent advances in the treatment of *Pseudomonas aeruginosa* infec‐ tions in cystic fibrosis. BMC Medicine. 2011;**9**:32. DOI: 10.1186/1741‐7015‐9‐32

[2] Jansen HK, Høiby N: Seasonal onset of initial colonisation and chronic infection with *Pseudomonas aeruginosa* in patients with cystic fibrosisi in Denmark. Thorax. 1992;**47**:109‐

[3] Hansen CR, Pressler T, Høiby N: Early aggressive eradication therapy for intermittent *Pseudomonas aeruginosa* airway colonization in cystic fibrosis patients: 15 years experi‐

[4] Bjarnsholt T, Jensen PØ, Fiandaca MJ, Pedersen J, Hansen CR, Andersen CB, Pressler T, Givskov M, Høiby N: *Pseudomonas aeruginosa* biofilms in the respiratory tract of cystic

[5] Hoffmann N, Rasmussen TB, Jensen PØ, Stub C, Hentzer M, Molin S, Ciofu O, Givskov M, Johansen HK, Høiby N: Novel mouse model of chronic *Pseudomonas aeruginosa* lung infection mimicking cystic fibrosis. Infect. Immun. 2005;**73**:2504‐2514. DOI: 10.1128/

[6] Høiby N, Ciofu O, Bjarnsholt T: *Pseudomonas aeruginosa* biofilms in cystic fibrosis. Future

fibrosis patients. Pediatr. Pulmonol. 2009;**44**:547‐558. DOI: 10.1002/ppul.21011

and Ivanka M. Karadzic<sup>2</sup>

Education, Science and Technological Development of the Republic of Serbia.

1 Sport Academy Belgrade College of Higher Vocational Studies, Belgrade, Serbia

2 University School of Medicine, University of Belgrade, Belgrade, Serbia

ence. J. Cyst. Fibros. 2008;**7**:523‐530. DOI: 10.1016/**j**.jcf.2008.06.009

Microbiol. 2010;**5**:1663‐1674. DOI: 10.2217/fmb.10.125

respiratory mucus epithelial membranes is required.

Milena G. Rikalovic1,\*, Natasa S. Avramovic<sup>2</sup>

111. DOI: 10.1136/thx.47.2.109

IAI.73.4.2504‐2514.2005

\*Address all correspondence to: mrikalovic@gmail.com

**Acknowledgment**

144 Progress in Understanding Cystic Fibrosis

**Author details**

**References**


immobilization and purification. Carbohydr. Polym. 2011;**83**:1397‐1401. DOI:10.1016/j. carbpol.2010.10.005


[31] Machen TE: Innate immune response in CF airway epithelia: Hyperinflammatory? Am. J. Physiol. Cell. Physiol. 2006;**291**:C218‐C230. DOI: 10.1152/ajpcell.00605.2005

immobilization and purification. Carbohydr. Polym. 2011;**83**:1397‐1401. DOI:10.1016/j.

[19] Izrael‐Zivkovic L, Gojgic‐Cvijovic G, Gopcevic K, Vrvic M, Karadzic I: Enzymatic charac‐ terization of 30 kDa lipase from *Pseudomonas aeruginosa* ATCC 27853. J. Basic. Microbiol.

[20] Jakovetić SM, Knezević‐Jugović ZD, Grbavčić SŽ, Bezbradica DI, Avramović NS, Karadžić IM: Rhamnolipid and lipase production by *Pseudomonas aeruginosa san‐ai*: The process comparison analysis by statistical approach. Chem. Ind. 2013;**67**:677‐685. DOI:

[21] Karadzic I, Masui A, Izrael‐Zivkovic L, Fujiwara N: Purification and characterization of an alkaline lipase from *Pseudomonas aeruginosa* isolated from putrid mineral cutting oil

[22] Karadzic I, Masui A, Fujiwara N: Purification and characterization of a protease from *Pseudomonas aeruginosa* grown in cutting oil. J. Biosci. Bioeng. 2004;**98**:145‐152. DOI:

[23] Rikalovic MG, Abdel‐Mawgoud AM, Déziel E, Gojgic‐Cvijovic G Dj, Nestorovic Z, Vrvic MM, Karadzic IM: Comparative analysis of rhamnolipids from novel environmental isolates of *Pseudomonas aeruginosa*. J. Surfact.. Deterg. 2013;**16**:673‐682. DOI: 10.1007/

[24] Rikalovic MG, Vrvic MM, Karadžić IM: Rhamnolipid biosurfactant from *Pseudomonas aeruginosa*—From discovery to application in contemporary technology. J. Serb. Chem.

[25] Davies JC: *Pseudomonas aeruginosa* in cystic fibrosis: Pathogenesis and persistence. Paediatr. Respir. Rev. 2002;**3**:128‐134. DOI: http://dx.doi.org/10.1016/S1526‐0550(02)00003‐3

[26] Doring G, Gulbins E: Cystic fibrosis and innate immunity: How chloride channel mutations provoke lung disease. Cell. Microbiol. 2009;**11**:208‐216. DOI: 10.1111/j.1462‐5822.2008.01271.x

[27] Gibson RL, Burns JL, Ramsey BW: Pathophysiology and management of pulmonary infections in cystic fibrosis. Am. J. Respir. Crit. Care. Med. 2003;**168**:918‐951. DOI:

[28] Kelley TJ, Drumm ML: Inducible nitric oxide synthase expression is reduced in cystic fibrosis murine and human airway epithelial cells. J. Clin. Invest. 1998;102:1200‐1207.

[29] Konstan MW: Therapies aimed at airway inflammation in cystic fibrosis. Clin. Chest.

[30] Lyczak JB, Cannon CL, Pier GB: Lung infections associated with cystic fibrosis. Clin.

as metal working fluid. J. Biosc. Bioeng. 2006;**102**:82‐89. DOI: 10.1263/jbb.102.82

carbpol.2010.10.005

146 Progress in Understanding Cystic Fibrosis

10.2298/HEMIND121008114J

10.1263/jbb.98.145

10.1164/rccm.200304‐505SO

2009;**49**:452‐462. DOI: 10.1002/jobm.200800229

s11743‐013‐1462‐4 DOI:10.1007/s11743‐013‐1462‐4

Soc. 2015;**80**:279‐304. DOI: 10.2298/JSC140627096R

DOI: 10.1172/JCI2357 DOI:10.1172%2FJCI2357#pmc\_ext

Med. 1998;**19**:505‐513. DOI: 10.1016/S0272‐5231(05)70096‐4

Microbiol. Rev. 2002;**15**:194‐222. DOI: 10.1128/CMR.15.2.194‐222.2002


[55] Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FSL, Hufnagle WO, Kowalk DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, Westbrock‐ Wadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK‐S, Wu Z, Paulsen IT, Reizer J, Saler MH, Hancock REW, Lory S, Olson MV: Complete genome sequence of *Pseudomonas aeruginosa* PAO1, an opportunis‐ tic pathogen. Nature. 2000;**406**:959‐964. DOI: 10.1038/35023079

[43] Hall‐Stoodley L, Costerton JW, Stoodley P: Bacterial biofilms: from the natural environ‐ ment to infectious diseases. Nat. Rev. Microbiol. 2004;**2**:95‐108. DOI:10.1038/nrmicro821

[44] Parsek MR, Greenberg EP: Sociomicrobiology: The connections between quorum sens‐ ing and biofilms. Trends. Microbiol. 2005;**13**:27‐33. DOI:10.1016/j.tim.2004.11.007 [45] Costerton JW: Anaerobic biofilm infections in cystic fibrosis. Mol. Cell. 2002;**10**: 699‐700. DOI: http://dx.doi.org/10.1016/S1097‐2765(02)00698‐6 DOI:10.1016/S1097‐2765(02)

[46] Sauer K, Camper AK, Ehrlich GD, Costerton JW, Davies DG: *Pseudomonas aeruginosa* displays multiple phenotypes during development as a biofilm. J. Bacteriol. 2002;**184**:1140‐1154. DOI: 10.1128/jb.184.4.1140‐1154.2002 DOI:10.1128%2Fjb.184.4.1140‐1154.2002#pmc\_ext

[47] Hentzer M, Eberl L, Givskov M:Transcriptome analysis of *Pseudomonas aeruginosa* bio‐ film development: Anaerobic respiration and iron limitation. Biofilms. 2005;**2**:37‐61.

[48] Xu KD, Stewart PS, Xia F, Huang CT, McFeters GA: Spatial physiological heterogeneity in *Pseudomonas aeruginosa* biofilm is determined by oxygen availability. Appl. Environ.

[49] Davies DG, Parsek MR, Pearson JP, Iglewski BH, Costerton JW, Greenberg EP: The involvement of cell‐to‐cell signals in the development of a bacterial biofilm. Science.

[50] Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, Manefield M, Costerton JW, Molin S,Eberl L, Steinberg P, Kjelleberg S, Høiby N, Givskov M: Attenuation of *Pseudomonas aeruginosa* virulence by quorum sensing inhibitors. EMBO J. 2003;**22**:3803‐3815. DOI: 10.1093/emboj/cdg366

[51] Bjarnsholt T, Jensen PØ, Burmølle M, Hentzer M, Haagensen JA, Hougen HP, Calum H, Madsen KG, Moser C, Molin S, Høiby N, Givskov M: *Pseudomonas aeruginosa* tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum‐sens‐

[52] Hassett DJ, Ma JF, Elkins JG, Iglewski BH: Quorum sensing in *Pseudomonas aerugi‐ nosa* controls expression of catalase and superoxide dismutase genes and mediates biofilm susceptibility to hydrogen peroxide. Mol. Microbiol. 1999;**34**:1082‐1093. DOI:

[53] Hentzer M, Teitzel GM, Balzer GJ, Heydorn A, Molin S, Givskov M, Parsek MR: Alginate overproduction affect *Pseudmonas aeruginosa* biofilm structure and function. J. Bacetriol.

[54] Winstanley C, O'Brien S, Brockhurst AM, *Pseudomonas aeruginosa* evolutionary adap‐ tation and diversification in cystic fibrosis chronic lung infections. Trends. Microbiol. 2016;**24**:327‐337 (in Special Issue Microbial Endurance). DOI: http://dx.doi.org/10.1016/j.

ing dependent. Microbiology. 2005;**151**:373‐383. DOI: 10.1099/mic.0.27463‐0

2001; 183:5395‐5401. DOI: 10.1128/JB.183.18.5395‐5401.2001

DOI: http://dx.doi.org/10.1017/S1479050505001699

1998;**280**:295‐298. DOI: 10.1126/science.280.5361.295

10.1046/j.1365‐2958.1999.01672.x

tim.2016.01.008

Microbiol. 1998;**64**:4035‐4039. DOI: 0099‐2240/98/\$04.0010

00698‐6#doilink

148 Progress in Understanding Cystic Fibrosis


[77] Rahman KSM, Rahman TJ, McClean S, Marchant R, Banat IM: Rhamnolipid biosur‐ factants production by strains of *Pseudomonas aeruginosa* using low cost raw materials. Biotechnol. Prog. 2002;**18**:1277‐1281. DOI: 10.1021/bp020071x

[66] Muthusamy K, Gopalakrishnan S, Ravi TK, Sivachidambaram P: Biosurfactants: Properties, commercial production and application. Curr. Sci. 2008;**94**:736‐747. DOI:

[67] Van Hamme JD, Singh A, WardOP: Surfactants in microbiology and biotechnology: Part 1. Physiological aspects. Biotechnol. Adv. 2006;**24**:604‐620. DOI: doi:10.1016/j.

[68] Tahzibi A, Kamal F, Assadi MM: Improved production of rhamnolipids by a *Pseudomonas aeruginosa* mutant. Iran. Biomed. J. 2004;**8**:25‐31. http://ibj.pasteur.ac.ir/browse.

[69] Abalos A, Pinazo A, Infante MR, Casals M, Garcia F, Manresa A: Physicochemical and antimicrobial properties of new rhamnolipids produced by *Pseudomonas aeruginosa* AT10 from soybean oil refinery wastes. Langmuir. 2001*;***17**:1367‐1371. DOI: 10.1021/

[70] Abdel‐Mawgoud AM, Aboulwafa MM, Hassouna NAH: Characterizations of rham‐ nolipid produced by *Pseudomonas aeruginosa* isolate Bs 20. Appl. Biochem. Biotechnol.

[71] Benincasa M, Abalos A, Oliveira I, Manresa A: Chemical structure, surface prop‐ erties and biological activities of the biosurfactant produced by *Pseudomonas aeru‐ ginosa* LBI from soapstock. Antonie. Van. Leeuwen. 2004*;***85**:1‐8. DOI: 10.1023/B:A

[72] Haba E, Abalos A, Jauregui O, Espuny MJ, Manresa A: Use of liquid chromatography‐ mass spectroscopy for studying the composition and properties of rhamnolipids pro‐ duced by different strains of *Pseudomonas aeruginosa.* J. Surfact. Deterg. 2003;**6**:155‐161.

[73] Mata‐Sandoval J, Karns J, Torrens A: Effect of nutritional and environmental conditions on the production and composition of rhamnolipids by *P. aeruginosa* UG2. Microbiol.

[74] Pornsunthorntawee O, Wongpanit P, Chavadej S, Abe M, Rujiravanit R: Structural and physiochemical characterization of crude biosurfactant produced by *Pseudomonas aeru‐ ginoa* SP4 isolated from petroleum‐contaminated soil. Bioresour. Technol. 2008;**99**:1589‐

[75] Benincasa M, Contiero J, Manresa MA, Moraes IO: Rhamnolipid production by *Pseudomonas aeruginosa* LBI growing on soapstock as the sole carbon source. J. Food. Eng. 2002;**54**:283‐288. DOI: http://dx.doi.org/10.1016/S0260‐8774(01)00214‐X DOI:10.1016/

[76] Benincasa M, Accorsini FR: *Pseudomonas aeruginosa* LBI production as an integrated process using the wastes from sunflower‐oil refining as substrate. Bioresour. Technol.

10.1016/j.chemosphere.2008.01.003

php?a\_id=508&slc\_lang=en&sid=1&ftxt=1

2009;**157**:329‐345. DOI: 10.1007/s12010‐008‐8285‐1

Res. 2001;**155**: 249‐256. DOI: 10.1016/S0944‐5013(01)80001‐X

2008;99:3843‐3849. DOI: 10.1016/j.biortech.2007.06.048

biotechadv.2006.08.001

150 Progress in Understanding Cystic Fibrosis

NTO.0000020148.45523.41

DOI: 10.1007/s11743‐003‐0260‐7

1595. DOI: 10.1016/j.biortech.2007.04.020

S0260‐8774(01)00214‐X#doilink

la0011735


[100] Franklin MJ, Ohman DE: Identification of algF in the alginate biosynthetic gene clus‐ ter of *Pseudomonas aeruginosa* which is required for alginate acetylation. J Bacteriol. 1993;**175**:5057‐5065. DOI: 0021‐9193/93/165057‐09\$02.00/0

[88] Haussler S, Becker T: The pseudomonas quinolone signal (PQS) balances life and death in *Pseudomonas aeruginosa* populations. PLoS Pathog. 2008;4(3): e1000166. DOI: http://

[89] Franklin MT., Nivens DE., Weadge JT., Lynne‐Howell L, Biosynthesis of *P. aeruginosa* extracellular polysacharides alginate, Pel and Psl. Front. Microbiol. 2011;**2**:1‐16. DOI:

[90] Halverson LJ. Role of alginate in bacterial biofilms. In Rehm BHA editor. Alginates: Biology and Applications. Springer‐Verlag, Berlin Heidelberg; 2009. pp. 135‐151. DOI:

[91] Gulez G, Altıntas A, Fazli, M, Dechesne A, Workman CT, Tolker‐Nielsen T, Smets BF: Colony morphology and transcriptome profiling of *Pseudomonas putida* KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials.

[92] Fett WF, Wells JM, Cescutti P, Wijey C: Identification of exopolysaccharides produced by fluorescent Pseudomonads associated with commercial mushroom (*Agaricus bisporus*) production. Appl. Environ. Microbiol. 1995;**61**:513‐517. DOI: 0099‐2240/95/\$04.0010 [93] Remminghorst U, Rehm BHA: Bacterial alginates: From biosynthesis to applications.

[94] Hay ID, Rehman ZU, Moradali MF, Wang Y, Rehm BHA: Microbial alginate pro‐ duction, modification and its applications. Microb. Biotechnol. 2013;**6**:637‐650. DOI:

[95] Coulon C, Vinogradov E, Filloux A, Sadovskaya I: Chemical analysis of cellular and extracellular carbohydrates of a biofilm forming strain *Pseudomonas aeruginosa* PA14.

[96] Friedman L, Kolter R: Two genetic loci produce distinct carbohydrate‐rich structural com‐ ponents of the *Pseudomonas aeruginosa* biofilm matrix. J. Bacteriol. 2004;**186**:4457‐4465. DOI: 10.1128/JB.186.14.4457‐4465.2004 DOI:10.1128%2FJB.186.14.4457‐4465.2004#pmc\_ext

[97] Darzins A, Frantz B, Vanags RI, Chakrabarty AM: Nucleotide sequence analysis of the phosphomannose isomerase gene (pmi) of *Pseudomonas aeruginosa* and compari‐ son with the corresponding *Escherichia coli* gene manA. Gene. 1986; **42**:293‐302. DOI:

[98] Deretic V, Gill JF, Chakrabarty AM: Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid *Pseudomonas aeruginosa*. J. Bacteriol. 1987;**169**:351‐

[99] Zielinski NA*,* Chakrabarty, AM, Berry A: Characterization and regulation of the *Pseudomonas aeruginosa* algC gene encoding phosphomannomutase. J. Biol. Chem.

1991;**266**:9754*‐*9763. http://www.jbc.org/content/266/15/9754.full.pdf

Microbiol. Open. 2014;**3**:457‐469. DOI: 10.1002/mbo3.180

Biotechnol. Lett. 2006;**28**:1701‐1712. DOI: 10.1007/s10529‐006‐9156‐x

10.1111/1751‐7915.12076 DOI:10.1111%2F1751‐7915.12076#pmc\_ext

PLoS One. 2010;**5**:e14220. DOI: 10.1371/journal.pone.001422

dx.doi.org/10.1371/journal.ppat.1000166

10.3389/fmicb.2011.00167

152 Progress in Understanding Cystic Fibrosis

10.1007/978‐3‐540‐92679‐5\_2

10.1016/0378‐1119(86)90233‐7

358. DOI: 0021‐9193/87/010351‐08\$02.00/0


[123] Graham A, Steel DM, Wilson R, Cole PJ, Alton E, Geddes DM: Effects of purified *Pseudomonas rhamnolipids* on bioelectric properties of sheep tracheal epithelium. Exp. Lung. Res. 1993;**19**:77‐89. DOI: http://dx.doi.org/10.3109/01902149309071082

[112] Zhang YM, Miller RM: Effect of a *Pseudomonas* rhamnolipid biosurfactant on cell hydro‐ phobicity and biodegradation of octadecane. Appl. Environ. Microbiol. 1994;**60**:2101*‐*

[113] Davey ME, Caiazza NC, O'Toole GA: Rhamnolipid surfactant production affects bio‐ film architecture in *Pseudomonas aeruginosa* PAOI. J. Bacetriol. 2003;**185**:1027‐1036. DOI:

[114] Byrd MS, Sadovskaya I, Vinogradov E, Lu H, Sprinkle AB, Richardson SH, Ma L, Ralston B, Parsek MR, Anderson EM, Lam JS, Wozniak DJ: Genetic and biochemical analyses of the *Pseudomonas aeruginosa* Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol. Microbiol. 2009;**73**:622‐638. DOI: 10.1111/j.1365‐2958.2009.06795.x DOI:10.1111%2Fj.

[115] Alhede M, Bjarnsholt T, Jensen PO, Phipps RK, Moser C, Christophersen L, Christensen LD, van Gennip M, Parsek M, Hoiby N, Rasmussen TB, Givskov M: *Pseudomonas aeru‐ ginosa* recognizes and responds aggressively to the presence of polymorphonuclear

[116] Høiby N: *P. aeruginosa* in cystic fibrosis patients resists host defenses, antibiotics.

[117] Barbier M, Martínez‐Ramos I, Townsend P, Albertí S: Surfactant protein A blocks rec‐ ognition of *Pseudomonas aeruginosa* by CKAP4/P63 on airway epithelial cells. J. Infect.

[118] Zulianello L, Canard C, Kӧhler T, Caille D, Lacroix JS, Meda P: Rhamnolipids are vir‐ ulence factors that promote early infiltration of primary human airway epithelia by *Pseudomonas aeruginosa*. Infect. Immun. 2006;**74**:3134‐3147. DOI: 10.1128/IAI.01772‐05

[119] Moss J, Ehrmantraut ME, Banwart BD, Frank DW, Barieri JT: Sera from adult patients with cystic fibrosis contain antibodies to *Pseudomonas aeruginosa* type III apparatus.

[120] Winzer K, Williams P: Quorum sensing and the regulation of virulence gene expression in pathogenic bacteria. Int. J. Med. Microbiol. 2001;**291:**131‐143. DOI:

[121] Kownatzki R, Tummler B, Doring G: Rhamnolipid of *Pseudomonas aeruginosa* in sputum of cystic fibrosis patients. Lancet. 1987;**329**:1026‐1027. DOI: http://dx.doi.org/10.1016/

[122] Read RC, Roberts P, Munro N, Rutman A, Hastie A, Shryock T, Hall R, McDonald‐ Gibson W, Lund V, Taylor G: Effect of *Pseudomonas aeruginosa* rhamnolipids on muco‐ ciliary transport and ciliary beating. J. Appl. Physiol. (1985). 1992;**72**:2271‐2277. http://

Infect. Immun. 2001;**69**:1185‐1188. DOI: 10.1128/IAI.69.2.1185‐1188.2001

leukocytes. Microbiol. Sgm. 2009;**155**:3500‐3508. DOI: 10.1099/mic.0.031443‐0

2106. DOI: 0099‐2240/94/\$04.00+0

154 Progress in Understanding Cystic Fibrosis

10.1128/JB.185.3.1027‐1036.2003

1365‐2958.2009.06795.x#pmc\_ext

Microbe. 2006;**1**:571‐577.

10.1078/1438‐4221‐00110

S0140‐6736(87)92286‐0

jap.physiology.org/content/72/6/2271

Dis. 2012;**206**:1753‐1762. DOI: 10.1093/infdis/jis587


[147] Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A: The Calgary Biofilm Device: New technology for rapid determination of antibiotic susceptibilities of bacterial bio‐ films. J. Clin. Microbiol. 1999;**37**:1771‐1776. DOI: 0095‐1137/99/\$04.0010

[135] Bédard M, McClure CD, Schiller NL, Francoeur C, Cantin A, Denis M: Release of interleukin‐8, interleukin‐6, and colony stimulating factors by upper airway epithelial cells: implication for cystic fibrosis. Am. J. Respir. Cell. Mol. Biol. 1993;**9**:455‐462. DOI:

[136] McClure CD, Schiller NL: Inhibition of macrophage phagocytosis by *Pseudomonas aeruginosa* rhamnolipids in vitro and in vivo. Curr. Microbiol. 1996;**33**:109‐117. DOI:

[137] Van Gennip M, Christensen LD, Alhede M, Phipps R, Jensen PO, Christophersen L, Pamp SJ, Moser C, Mikkelsen PJ, Koh AY, Tolker‐Nielsen T, Pier GB, Hoiby N, Givskov M, Bjarnsholt T: Inactivation of the rhlA gene in *Pseudomonas aeruginosa* prevents rham‐ nolipid production, disabling the protection against polymorphonuclear leukocytes.

[138] Ernst RK, Hajjar AM, Tsai JH, Moskowitz SM, Wilson CB, Miller SI: *Pseudomonas aeru‐ ginosa* lipid A diversity and its recognition by Toll‐like receptor 4. J. Endotoxin. Res.

[139] Ernst RK, Moskowitz SM, Emerson JC, Kraig GM, Adams KN, Harvey MD, Ramsey B, Speert DP, Burns JL, Miller SI: Unique lipid A modifications in *Pseudomonas aeruginosa* isolated from the airways of patients with cystic fibrosis. J. Infect. Dis. 2007;**196**:1088‐

[140] Ernst RK, Yi EC, Guo L, Lim KB, Burns JL, Hackett M, Miller SI: Specific lipopolysac‐ charide found in cystic fibrosis airway *Pseudomonas aeruginosa*. Science. 1999;**286**:1561.

[141] Hajjar AM, Ernst RK, Tsai JH, Wilson CB, Miller SI: Human Toll‐like receptor 4 recog‐ nizes host‐specific LPS modifications. Nat. Immunol. 2002;**3**:354‐359. DOI: 10.1038/ni777

[142] Doring G, Pier GB: Vaccines and immunotherapy against *Pseudomonas aeruginosa*.

[143] Stuart B, Lin JH, Mogayzel PJ Jr: Early eradication of *Pseudomonas aeruginosa* in patients with cystic fibrosis. Paediatr. Respir. Rev. 2010;**11**:177‐184. DOI: 10.1016/j.

[144] Hayes D, Feola JrDJ, Murphy BS, Kuhn RJ, Davis GA: Eradication of *Pseudomonas aeru‐ ginosa* in an adult patient with cystic fibrosis. Am. J. Health. Syst. Pharm. 2011;**68**:319‐

[145] Chan C, Burrows LL, Deber CM: Alginate as an auxiliary bacterial membrane: binding of membrane‐active peptides by polysaccharides. J. Pept. Res. 2005;**65**:343‐351. DOI:

[146] Ryan G, Singh M, Dwan K: Inhaled antibiotics for long‐term therapy in cystic fibrosis. Cochrane Database Syst. Rev. 2011;**3**:CD001021. DOI: 10.1002/14651858.CD001021.pub2

Vaccine. 2008;**26(8):**1011‐1124. DOI:10.1016/j.vaccine.2007.12.007

Apmis. 2009;**117**:537‐546. DOI: 10.1111/j.1600‐0463.2009.02466.x

2003;**9**:395‐400. DOI:10.1179/096805103225002764

10.1165/ajrcmb/9.4.455

156 Progress in Understanding Cystic Fibrosis

10.1007/s002849900084

1092. DOI: 10.1086/521367

prrv.2010.05.003

322. DOI: 10.2146/ajhp10010

10.1111/j.1399‐3011.2005.00217.x

DOI: 10.1126/science.286.5444.1561

