**2. Overview of CF and the role of CFTR in the lung**

### **2.1. CFTR: Role and function**

The function of CFTR in the lungs has been established previously, supporting the fact that its absence or modification (depending on the mutation) leads to the dangerous symptoms that are diagnostic hallmarks in CF patients. Although some canonically think of CFTR as only being influential in the development of the lung problems, CFTR is actually expressed in many areas of the body, including the liver, intestines, pancreas, skin, and reproductive organs (Figure 1). In all these cases, a defect in CFTR can cause many problems for the affected organ. The reason is due to the large role the transporter plays in the maintenance of osmotically balanced fluids in these tissues. CFTR is an anion channel found in the apical membrane of epithelial cells, primarily responsible for pumping chloride ions into the fluids surrounding the epithelial cells, and allowing for the passage of water from the epithelial cells into the fluid layers lining the cells (e.g, the pericilliary layer (PCL) of the airways) [5, 6, 7]. This allows the fluids to maintain their function, which, in the lung, is usually to facilitate clearance of opportunistic pathogens and cellular debris from the area or to transport the fluid to a different area (as is the case in the reproductive system). The transporter itself is ATP-driven, and is activated due to rising cAMP levels in the cells [7].

**Figure 1.** Organs affected by CF and its effect on airways.

**1. Introduction**

172 Cystic Fibrosis in the Light of New Research

picture is becoming clearer.

infected individuals.

**2.1. CFTR: Role and function**

methods of treating bacterial infections.

**2. Overview of CF and the role of CFTR in the lung**

Cystic fibrosis (CF) is the second most common genetic disease in the United States, second only to sickle cell anemia. A mutation in the associated gene, cystic fibrosis transmembrane regulator (CFTR), results in the clinical symptoms seen for the disease. While the disease itself is devastating, it does not usually result in the immediate death of the patient. Rather, the bodily conditions that the CFTR mutation creates, especially in the lungs, results in the acquisition of problematic bacterial biofilm infections that remain in the thick, inspissated mucus layer for the remainder of the patient's life. This unique niche provides many complex nutrients that the bacteria utilize and offers an environment that is protected against the host's innate immune system. While only a few bacterial species have generally been thought of as dominating the CF lung, it has recently been revealed that there are many species inhabiting the lung, and that these species vary from normal flora to even obligate anaerobes [1]. While the exact role of all these bacterial species in the lung has not yet been determined, the clinical

When the genetic cause of CF was first identified in 1989 [2], the average lifespan of a CF patient was approximately 15 years of age. Since then, it has increased to roughly 40 [3, 4]. This is due to the tremendous efforts that have been made in determining new and improved means of treating patients suffering from CF, including addressing physiological parameters and

This chapter will focus on the bacterial infections that develop during CF and the conditions of the lung that make it so favorable for bacterial infection. We will discuss some of the major bacterial species that contribute to the morbidity of the disease, and focus on perhaps the largest contributor to airway infection, *Pseudomonas aeruginosa* (PA). In addition, we will define some of the niches that the bacteria inhabit within the lung, and discuss treatment options for

The function of CFTR in the lungs has been established previously, supporting the fact that its absence or modification (depending on the mutation) leads to the dangerous symptoms that are diagnostic hallmarks in CF patients. Although some canonically think of CFTR as only being influential in the development of the lung problems, CFTR is actually expressed in many areas of the body, including the liver, intestines, pancreas, skin, and reproductive organs (Figure 1). In all these cases, a defect in CFTR can cause many problems for the affected organ. The reason is due to the large role the transporter plays in the maintenance of osmotically balanced fluids in these tissues. CFTR is an anion channel found in the apical membrane of epithelial cells, primarily responsible for pumping chloride ions into the fluids surrounding the epithelial cells, and allowing for the passage of water from the epithelial cells into the fluid A) An overview of the organs affected by CF, with a brief description of the complications associated with it. B) A normal airway depicting open passages. C) A CF airway depicting the buildup of mucus, inflammation, and bacterial infection that will lead to further complications. (Source: National Heart, Lung, and Blood Institute; National Institutes of Health; U.S. Depart‐ ment of Health and Human Services. [8])

In cases where the CFTR is not active or only partially active, the consequences can be quite severe. This is due to a myriad of potential mutations in the CFTR gene. These mutations have been categorized into classes, as previously reviewed by Rowntree and Harris [9] and summated by the Cystic Fibrosis Foundation. As shown in Table 1, these classes focus on the means by which the CFTR is rendered dysfunctional, such as mutations affecting protein maturation (Class II) or those leading to dysregulation of Cl- conductance (Class IV) [9, 4]. The most common mutation is a deletion of a phenylalanine residue at position 508 of the protein, referred to as homozygous recessive ΔF508 [7]. Although the exact reasoning of why this mutation was clinically deleterious was at first a mystery, it has come to be discovered that this leads to a misfolding of the channel, causing it to never reach the cell membrane and instead be destroyed in the Golgi apparatus. Although one could surmise that the loss of CFTR alone is not enough to cause harm and the body could compensate for this loss by redundant channels, it is also suspected that CFTR can help mediate the activation and use of other channels in the membrane. Thus, its loss may be far more reaching than simply its anion channeling properties. With this loss of function, the surrounding fluid begins to become osmotically imbalanced and often viscous and impervious to other ions [6]. With no new water coming into the fluids from the epithelial cells, the fluid layer begins to thicken, eventually forming mucus plugs in the respective organ. As such, the associated ducts are no longer able to perform their proper functions.


**Table 1.** Classifications of CFTR mutations and their impact on the protein.

Although there are numerous mutations that have been associated with CF, they can generally be broken down into five classes based on the way this mutation affects CFTR. This table briefly summarizes these classes and provides an example of each mutation [4].

### **2.2. The CF lung**

Within the CF lung, the loss of functional CFTR is quite dramatic, and usually leads to the canonical respiratory symptoms associated with CF lung disease. A healthy functioning lung will have a thin, hydrated pericilliary mucus layer lining the airway. This mucus rests above the cilia of the epithelial cells in a biphasic layer [7]. The top layer is slightly more viscous than the bottom layer and serves to trap bacteria and particles that enter the lung. The bottom layer, referred to as the PCL, is much more fluid, and allows for the cilia to beat within it, pushing the entire mucus layer up the lung for expectoration [7, 10]. Through this mechanism, the lungs can clear bacteria and debris that has been inhaled or otherwise entered the airway passages. The PCL is kept hydrated by the action of the CFTR and other ion channels present in the epithelial cells lining the airway that also maintain the osmotic balance.

**Figure 2.** A model of the CFTR in the apical membrane of lung epithelia.

maturation (Class II) or those leading to dysregulation of Cl- conductance (Class IV) [9, 4]. The most common mutation is a deletion of a phenylalanine residue at position 508 of the protein, referred to as homozygous recessive ΔF508 [7]. Although the exact reasoning of why this mutation was clinically deleterious was at first a mystery, it has come to be discovered that this leads to a misfolding of the channel, causing it to never reach the cell membrane and instead be destroyed in the Golgi apparatus. Although one could surmise that the loss of CFTR alone is not enough to cause harm and the body could compensate for this loss by redundant channels, it is also suspected that CFTR can help mediate the activation and use of other channels in the membrane. Thus, its loss may be far more reaching than simply its anion channeling properties. With this loss of function, the surrounding fluid begins to become osmotically imbalanced and often viscous and impervious to other ions [6]. With no new water coming into the fluids from the epithelial cells, the fluid layer begins to thicken, eventually forming mucus plugs in the respective organ. As such, the associated ducts are no longer able

to perform their proper functions.

174 Cystic Fibrosis in the Light of New Research

**2.2. The CF lung**

**Table 1.** Classifications of CFTR mutations and their impact on the protein.

summarizes these classes and provides an example of each mutation [4].

Although there are numerous mutations that have been associated with CF, they can generally be broken down into five classes based on the way this mutation affects CFTR. This table briefly

Within the CF lung, the loss of functional CFTR is quite dramatic, and usually leads to the canonical respiratory symptoms associated with CF lung disease. A healthy functioning lung will have a thin, hydrated pericilliary mucus layer lining the airway. This mucus rests above

The CFTR, embedded in the apical membrane of epithelial cells, serves to transport anions, specifically chloride and bicarbonate, into the lumen of the associated organ. The CFTR is composed of several domains, including two transmembrane domains (red ovals), two nucleotide binding domains (blue, squares), and a regulatory domain that controls the opening of the transporter. The nucleotide binding domains use ATP to provide energy for the transport of the anions into the lumen bordering the cell.

However, in a case such as CF, where the CFTR is functionally absent and proper ion transport is lacking, the mucus layer begins to thicken. This is believed to be due to the primary transport of Cl- [11, 6, 7] and the secondary transport of HCO3 by CFTR (Figure 2). Recent studies have shown that the ability of CFTR to transport HCO3 is very important, as it seems to play a large role in the regulation of the mucin folding [7]. Mucin, the primary protein component of airway mucus, is a long chain-like, repetitive peptide that is heavily O- and N-glycosylated. At the Cand N-terminal regions, the protein is rich in cysteine residues, which can lead to intermolec‐ ular disulfide bridges. These disulfide bridges will link the chains together, creating a larger oligomer. It is suspected that in a more acidic environment, the mucin molecules contract, causing the overall density of the mucus to increase. This causes impermeability issues [6] that can be devastating to the patient. In addition to the effects that decreased HCO3 levels have on the density of the mucus, the general inability to transport anions across the apical mem‐ brane also affects the airway mucus layer. The PCL that lines the cilia of the epithelial cells is very sensitive to changes in water concentration. When the cells are not exporting significant amounts of ions, the PCL will then lose water as a sequela, resulting in it becoming denser and reducing the effectiveness of ciliary beating. This leads to an overall larger amount of material that cannot be cleared from the airway, causing a buildup or mucus plug.

In addition to the buildup of mucus as the disease progresses, the patient will experience several other symptoms as well. Commonly, the bronchi become inflamed, caused by an overreactive response from the immune system due to both mucus buildup (containing a plethora of bacterial components such as virulence factors, DNA, and cell debris from lysed bacteria or airway epithelial cells) and a potential infection. While the infections will be covered in more detail later in this chapter, bacteria such as *Pseudomonas aeruginosa* (*PA), H. influen‐ zae,* and *S. aureus* have been found to infect CF patients early in life (roughly 1 year of age) [1], and even if eradicated once, will often arise from a re-infection later in life. *PA* is the organism most commonly associated with a decline in the clinical course of CF patients, as its ability to form biofilms and convert to a mucoid phenotype often provides a large level of resistance to antibiotics and other disease treatments.

Interestingly, the CF lung will also develop an oxygen gradient in its luminal mucus [12, 13]. As mentioned earlier, the increased density of the mucus makes it more difficult for oxygen to diffuse across it freely and into the blood. While this has consequences for the overall health of the individual, it also has implications for growth of bacteria enmeshed within it. The oxygen gradient is severe enough that the basal layer of the mucus could be termed microaerobic, or in more severe cases, anaerobic. This leads to the growth and development of bacteria that would normally not be found in the lung, and eventually to the growth of the mucoid form of *PA*. This will be covered in more depth later in this chapter.
