Classifications of CFTR mutations and their impact on the protein.
Abstract
In this chapter, the authors review a major complication associated with cystic fibrosis (CF), problematic bacterial infections of the lungs. Infection by organisms such as Staphylococcus aureus, Burkholderia cepacia complex, and Pseudomonas aeruginosa (a major player in CF related infections) results in complications due to increased inflammation and production of virulence factors produced by the bacteria. In addition to these more canonical organisms associated with CF infection, emergingbacterial species have been found in the CF, including anaerobes that have only within the past 5-10 years have been reported to exist in the lungs. P. aeruginosa has long been a cause of devastating infections, and is often seen as the“hallmark”organism associated with the disease. The authors describe the P. aeruginosa infection, including its conversion to a mucoid phenotype, as well as its ability to utilize the thicker airway surface layer associated with CF to grow in “mode two biofilms.” Finally, the authors discuss treatments for bacterial infections, and some of the new advances that offerhope for treatment of CF symptoms and infections by multi-drug resistant organisms. Among these new treatments is the application of acidified nitrite, a non-antibiotic treatment that has been found to be effective at killing nonmucoid and mucoid variants of P. aeruginosa.
Keywords
- Cystic Fibrosis
- Infections
- Bacteria
- Pseudomonas aeruginosa
- Biofilm
- Acidified Nitrite
1. Introduction
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 picture is becoming clearer.
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 methods of treating bacterial infections.
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,
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].
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. Department 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.
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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.
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 C- and N-terminal regions, the protein is rich in cysteine residues, which can lead to intermolecular 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 membrane 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
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
2.3. Pathology of the CF lung
As might be expected, the buildup of thick mucus in the CF lungs often has severe clinical implications The significant reduction or loss of mucus clearance often results in infection and leads to inflammation due to the dramatic ~1,500-fold increase in airway neutrophils [14]. Coupling the inflammation and buildup of mucus, it is not surprising that the overall lung capacity of CF patients decreases dramatically throughout life. This can be tested using a series of pulmonary function tests (PFTs) [15]. These often involve a spirometry test, which is a measure of the forced efflux volume in one second (FEV1), or how much air the patient can forcefully exhale in one second. This is a hallmark test for overall lung volume and strength. Clinically it has been shown that the FEV1 of a CF patient will be approximately 10% below the expected for a healthy individual of the same age [16].
In older patients, the prolonged effects of the disease often lead to chronic infections. These invading organisms can then be cultured and analyzed to determine the best treatment strategy. While we will be covering the type of infections further in this chapter, it is important to note that clinically, this also affects the patient in other ways, primarily leading to inflammation of the respiratory system and decreased airway capacity [17]. This inflammation is often brought on by increased neutrophil accumulation in the lungs, which not only serve to act as a preliminary means of immune defense, but also to recruit macrophages. These neutrophils and macrophages will phagocytose bacteria and dead immune cells, but also produce pro-inflammatory cytokines that exacerbate the inflammatory process. These cytokines not only attract other immune cells, but also serve as a trigger for the release of proteases and elastases by the immune cells. Normally, these help eradicate the bacteria that triggered this response, but theior over-production in the CF lung can actually damage epithelial cells, leading to the fibrotic nature of cells associated with this disease [18].
3. Bacterial infections
While CF airway disease is based on the genetic mutation of the CFTR gene, this is not usually what leads to the morbidity and mortality associated with the disease. Rather, an infectious agent that grows under the physiological conditions created by this mutation will lead to detrimental symptoms and eventual death. In the lungs, the buildup of thick mucus leads to decreased clearance of bacteria and provides a nutrient rich medium with which they can grow and even thrive. This mucus becomes colonized relatively easily with potentially several different species of bacteria at once [19, 16, 20]. Considering that infection is the major source of morbidity and mortality for CF patients, much research has focused on this aspect of the disease and different means by which to eradicate it. However, time has shown that this is not quite as easy as hoped, but the advancements of alternate treatments are helping this issue.
Bacterial colonization of the lungs of CF patients has been known for many decades. However, our understanding of what bacteria colonize the lungs has evolved dramatically. Early research identified that there were several species of bacteria that could colonize the lungs easily, and were often found associated with CF patients. By far, the most common (and most linked to severe progression of the disease) was
In addition to
The second major strain historically found in addition to
The last of the historically major three bacteria associated with CF,
Several other genera of microbes, including
Although each of these pathogens has been studied as a single organism in the CF pathology, limited efforts have been made into looking at the interaction between the bacteria. Considering that more often than not multiple genera of bacteria are found in the CF lung, this is an important aspect to consider. Research that has been studying this topic has focused generally on the interaction between
While this paradigm of “the big three” bacteria for CF persisted for several decades, along with the recent knowledge that several other species could infect the CF lung, a paradigm shift occurred around 2008, when it was discovered that there were obligate anaerobes present in the lungs of older CF patients [31]. Included in this group of bacteria were the genera
With the discovery of the aforementioned anaerobic bacteria, it became clear that there was most likely a temporal aspect of CF infections as well. Clinical evidence has shown that depending on the age of the patient, certain bacteria are more likely to be cultured with their sputum (Figure 3). Early in the patient’s life, from birth until around ten years of age, it is common for patients to test positive for
As a CF patient progresses through life, the likelihood of culturing positive for a particular microorganism changes. This is due mostly to the changing environment in the CF lungs. This graph depicts the percentage of patients registered in the Cystic Fibrosis Foundation patient registry who tested positive for a particular bacterium, separated by age [4].
Given the presence of anaerobic bacteria, this indicates that current antibiotic regimens for CF patients may have to be revisited. Normal antibiotic treatments include tobramycin, kanamycin, and several other antibiotics of the aminoglycoside class. Those in this class are generally ineffective against anaerobic bacteria due to their mechanism of entry. Aminoglycosides rely on the ability of an organism to respire, using either nitrate or oxygen as the terminal oxygen acceptor [34]. For fermenting bacteria, such as
4. The major contributor: Pseudomonas aeruginosa
Perhaps the most problematic and dangerous of the bacteria associated with CF is
4.1. The role of quorum sensing in PA infections
To control the expression of its virulence factors,
However, the system looping and increasing would mean nothing if there was not an output somewhere. This output happens to be the second major system for quorum sensing, the
Another important output of the
4.2. Alginate production and the conversion to mucoidy
Although these quorum-sensing pathways are important for the virulence associated with
The most important gene involved in the process of alginate production is
Under normal growth conditions in
However, in cases where constitutive mucoidy is found, such as in CF patients chronically infected with
Once the patient acquires mucoid
4.3. PA biofilm formation in CF patients
Herein, we will focus on the biofilm formation of
In CF patients, 65%–80% of all microbial infections are biofilm related [48]. The CF lung provides a suitable environment for
4.4. Genetic alterations during PA infection
In addition to the changes that are phenotypically eminent such as during mucoid conversion, it is also important to note that additional genetic regulation is occurring in this environment. As mentioned earlier, the CF lung is not a hospitable environment for bacteria, due especially to the dessicated mucus layer, yet it is quite nutrient-rich. If the bacteria can benefit from this, then they will be able to survive and flourish in the lung. In order to do this, the bacteria must undergo several layers of genetic regulation in order to activate specific shunt pathways that will allow them to use the nutrients provided. In the case of
Early gene expression analyses showed several important changes in expression levels, but suffered from lack of application. Some of the first analyses were of general biofilm gene expression, which were important as a seminal work. For example, Whiteley et al. performed a transcriptional profiling analysis in 2001 that examined gene expression by
In this regard, several attempts to examine human-associated biofilm infections occurred, but it had to be verified that the gene expression difference was reliable, and not an artifact of the harvesting procedure. Some studies did well with this issue, collecting
Other genes that were found to be changed in the CF lung associated samples seemed to actually be associated with the acquisition of nutrients in the surrounding area. For example, one such study found that there was an increase in genes associated with arginine metabolism and the glyoxylate shunt (a process found often with the β-oxidation of lipids to acetyl-CoA) [59]. This seems to indicate that the CF lung has a higher available amount of arginine and free lipids for degradation, potentially linked to the death of epithelial cells in the lung. This same study also found the cells generally change to a sessile state, where many of the genes associated with motility and chemotaxis are downregulated. This indicates that the cells initially colonizing the CF lung are able to prosper enough that the organisms can rapidly adapt to such conditions. This same down-regulation of motility genes is often seen with the conversion to a biofilm mode of growth. Once the bacteria have shifted to the mucoid state and are able to survive the thick, dessicated mucus, then they will be able to thrive in the lung. This can help explain why
5. Treatment strategies for CF
Considering the relative abundance of CF cases (~70,000 world-wide), it is important that efforts are put forward into treating the symptoms that arise from the disease [60]. These symptoms can be very detrimental, especially in the case of serious bacterial infections and ensuing exacerbations. Initially, the treatments that were available were only focused on chest physical therapy for the buildup of airway mucus, but those efforts have evolved as current research has illuminated more on the pathophysiology of CF lung disease. An example of advanced treatment with continuing research was the initial attempts to provide gene therapy for CF. With the idea that complementing the mutated CFTR with wild-type CFTR would allow for functional ion transport, efforts were made to transfect cells with a functional copy of the gene using either a liposomal or viral vector. However, this eventually ended in failure, as the complementation strategy was unsuccessful and in some cases harmful. Specifically, adenovirus complementation caused a hyper-immune response, and DNA liposome transfection could not properly deliver the DNA to the nucleus [61]. Recent attempts are examining whether transfecting cells with mRNA coding for functional CFTR could be beneficial. However, this still requires much more research. With that in mind, treatment falls primarily into two main camps; the removal of the dessicated mucus, and the treatment of bacterial infections.
5.1. Treatment of mucus buildup
Even without the involvement of pathogens in the mucus plugs that develop in the CF lung, the plugs themselves can cause serious effects on the patients, specifically in their quality of life. As mentioned previously, these plugs develop from a lack of clearance of mucus associated with the pericilliary layer, resulting in a continually developing blockage and a decreased airflow that reaches the respiratory zone of the lungs. With reduced oxygen levels, the patients begin to suffer and can eventually become cyanotic. However, this is one of the more apparent symptoms of CF, and has been a focus since the disease was first identified. Here, the primary mode of treatment has been physical percussion of the patient. This is termed as chest physical therapy (CPT) and has progressed much since it was initially developed. This treatment initially involved pounding on the patient’s back in such a manner as to dislodge mucus that was clinging to the bronchi [62]. After dislodging the mucus, the patient would then cough up any that was loosened, but this process in general was quite painful and not desired by patients, even when it was successful. In an effort to make this a more effective procedure, technology began to be introduced into the process. Initially, a device similar to a back-pack, was created that performed the percussion automatically, rather than rely on the doctor or medical professional. Although this is effective in providing the same amount of force each time, the percussion itself was not eliminated from the process.
Following this, different methods were employed to physically break up the mucus. These have become more advanced in recent decades. Most are reliant on sound waves or vibrations to loosen the mucus that allows for its eventual clearance. These methods include masks and vests that patients don that create vibrations and devices that convert the exhaled air of a patient into vibrations [62]. In all of these, the pressure and pain of physical percussion is eliminated, but the positive effects remain. Even some chemical treatments are prescribed to help loosen the mucus, such as aerosolized sodium bicarbonate that can help return the mucus layer to its normal pH and allow for better clearance. Physicians also routinely prescribe bronchodilators and anti-inflammatory medications to patients to both increase the functional airway passage and to help clear some of the mucus from the airways. In some extreme cases, where this use of medication is ineffective or does not keep up with the overall progression of the disease, the patients may have to endure a lung transplant [62]. While this is occasionally effective, it usually results in the eventual buildup of mucus in the lungs once again, resulting in further treatment or potentially even another transplant.
5.2. Treatment of bacterial infections
While the buildup of mucus within the lungs is quite problematic, it is often not what leads to the overall mortality or morbidity of CF patients. Rather, this is due to secondary infections that arise from this buildup. As stated earlier, this mucus is a rich, largely immobile niche of complex nutrients that enmesh the bacteria. In healthy lungs, the cilia are functioning properly with a thin PCL and clearing most pathogens out of the lungs. However, with the thick mucus, this is not the case, as the cilia do not have enough physical force to push it upwards. As a result, bacteria begin to infect the area. However, the precise location in the lungs where the infection resides is important for the choice of treatment methods. In general, the lungs are divided into two zones. The first is the conductive zone, which includes many of the preliminary branches of the bronchi and does not directly include the alveoli or any of the accompanying areas. The second is the respiratory zone, which includes many of the later branches of the airways and the alveoli. These differences are important due to the necessary changes for treatments. In the conductive zone, the tissue does not have quite as high a vascular exposure, but is much closer to the mouth. As such, infections in this area are often treated with aerosolized medicines. In contrast, the respiratory zone is much deeper into the lungs, but has much higher vascular exposure due to the presence of the alveoli in this zone. Treatments for this zone usually include oral or intravenous medications. While the mode of treatment is known, the actual ability to determine where the infection is occurring is dependent on a series of initial cultures from the patient, and the species present can change once treatment begins.
Once the type of treatment has been determined, the next step is determining what the effective antibiotic(s) is for the particular bacteria being targeted. This is dependent on the class of antibiotic and has to be administered in a patient-specific manner. In general, there are some that work more efficiently in certain patients than in others. For example, tobramycin, a potent aminoglycoside, has been approved by the FDA for clinical treatment of
However, the efficiency of the antibiotics for long-term treatment of
6. Future treatments
While advancements in medicine are not written in stone, there are trends that are observable that can dictate what advancements should be expected. Most of these trends are collected on the website for the CF Foundation [66], which monitors all forms of data related to CF research. In this case, they maintain a log of what is in the pipeline for research, as well as commonly available treatments for patients. In general, the medications available fall into six categories: CFTR modulators, anti-inflammatory drugs, anti-infective drugs, nutrition, mucus alteration, and airway surface restoration. Much of the research on medications has been focused on anti-inflammatory and anti-infective medications. While most of the anti-infective compounds have been modifications of available antibiotics into inhalable forms (such as aerosolized amikacin and vancomycin), the developments in anti-inflammatory medications are slightly more unique. One such example is the development of a drug that is targeting the Type III Secretion System of
Another field that seems to be growing with potential treatments is the area of CFTR modulators. This field of treatment is focused on finding drugs that change the defective behavior of mutant CFTR protein. Since one form of mutant CFTR is produced yet does not make it to the apical membrane of the cells, some thought has gone into making it possible for the protein to make it to the surface, and hopefully in that process, fold properly to allow for a functioning anion channel. All of these are experimental compounds, but have made it to Phase 2 clinical trials at the time of this publication [66]. The first is a “potentiator”, a compound that supposedly will be able to open a defective CFTR once it has reached the surface. While this is not meant for all forms of the disease, it is possible that this will have a great impact. Next is a “corrector” that is meant to move the folded protein to the correct location in the cell. This can help with proteins that have misfolded and are sent for degradation or are sent to a different membrane of the cell (e.g., basolateral). Finally, a synthetic signaling molecule has been developed that is meant to supplement decreased levels of S-nitrosoglutathione (an NO generator) in the cells of CF patients. This signaling molecule has been found to be decreased in these patients, and already evidence from the trial has shown that by supplementing this signal, there is increased and proper folding of the CFTR and function of the channel once it is in the membrane.
7. Conclusions
Bacterial infections of CF patients have been a cornerstone of treatment for decades, and that will not change going forward. Future efforts will be focused on finding alternative treatments that will be able to affect both planktonic and biofilm associated organisms in the lungs. This aspect of bacterial growth is most likely the key to effectively remove the organisms from the CF lung and improve the overall life expectancy for these patients. Already, great advances have been made in the last thirty years, as evidenced by not only the number of new and varying treatments, but also by the increased average life span for CF patients (40.7 years) [4]. While some research will still be focused on finding a way to directly treat the CFTR mutation, short-term research needs to be focused on discovering new and innovative treatment options.
Acknowledgments
The authors wish to thank previous support from the Daniel Tyler Health and Education Foundation (Fort Collins, CO); ARCH Biopartners (Toronto, CA); Cure Finders, Inc. (Sevierville, TN); and Cystic Fibrosis Research (Palo Alto, CA).
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