Cystic Fibrosis

Bielecki et al. Research Paper Pseudomonas aeruginosa is a Gram-negative bacteria, particularly known for causing nosocomial infections. As a pathogen, it effectively causes disease by acquiring resistance to antibiotics that would otherwise inhibit growth. Reported rates of infection range from 0. 6 to 32% across various clinical environments because Pseudomonas aeruginosa has gained multi-drug resistance. Certain strains of Pseudomonas aeruginosa treated with gamma rays can break down the hydrocarbons in crude oil and are thus useful in cleaning up oil spills.

The genome of Pseudomonas aeruginosa is 6. 3 million base pairs long, which is the largest bacterial genome to be sequenced. It contains about 5,570 open reading frames. Argyrin is a naturally synthesized antibiotic peptide extracted from myxobacteria. It has cytotoxic properties, suppresses the immune system, and is a highly active antibiotic used against Pseudomonas strains. Figure 1. Argyrin A structure. Bielecki et al chose to isolate these resistant clones in order to observe the mechanisms by which the P. eruoginosa acquires resistance to Argyrin A within the fusA1 gene. They isolated these clones by growing Pseudomonas aeruginosa strains on agar that contained Argyrin A. After incubation, the colonies that formed were able to grow in the presence of argyrin; these colonies were then streaked onto plates with Argyrin A again to ensure accuracy of obtaining resistant strains. A point mutation is an alteration of one base pair within a DNA sequence. The point mutations, which caused changes in the amino acid sequence within the fusA1 gene, were different among the six isolates.

They might have conferred resistance because the mutations caused the same impact on the resulting protein. The gene was identified by sequencing the whole genome of Pseudomonas aeruginosa strains with the bacterial target of Argyrin A, which showed mutations within fusA1 that encode for the elongation factor EF-G in resistant strains of Pseudomonas aeruginosa. The diagram below illustrates the process of elongation during the translational phase in EF-G along with EF-Tu. Figure 2. Elongation during ribosome-catalyzed translation. Bielecki et al confirmed the identity of the gene by using genetic maps. This required sequencing the resistant strain a second time to make a reference strain to compare the genes at a specific loci. Adding a mutation into the sensitive Pseudomonas aeruginosa strain demonstrated a resistance phenotype. Surface plasmon resonance is a lab technique that involves aiming a beam of light at a thin metal sheet, which catalyzes a reaction by causing movement in the molecules behind the metal sheet.

SPR was useful in this experiment because it confirmed that fusA1 is the target gene for Argyrin A, rather than fusidic acid, the antibiotic previously recognized. A heterologous protein, or a heterologue, is a protein that differs in structure and function relative to a given protein; not all proteins with different amino acid sequence necessarily differ in function. N-terminal His6-tags were fused to the fusA1 genes before undergoing the SPR experiments, causing the production of heterologous proteins in relation to the original fusA1.

According to Bielecki et al, the SPR procedures supported that Argyrin A binds to fusA1 by the resulting KD value. This shows that Argyrin A has a target on the heterologous protein. It is important to compare the variations made in the mutations because the other bacteria may have a different sequence that can still achieve resistance. It cannot be assumed that all bacterial strains will be identically resistant or sensitive because they all contain differences in their genomes. By mapping the mutated genes, the authors found the locations of the mutations in different domains.

They deduced that the mutations exhibiting resistance to Argyrin A in Pseudomonas aeruginosa are found on opposite sides of the domain, despite the fact that most mutations involving fusidic acid and Argyrin A are located on the same side of the domain. This shows that the binding sites for fusidic acid and Argyrin A must be independent of each other. Both fusA1 and the second gene, fusA2, encode for the elongation factor EF-G. The fusA2 gene was expressed 30 times less in the strains of Pseudomonas aeruginosa than in the fusA1 gene, as shown by RNA sequencing.

Homology modeling uses the model of a target protein to produce an estimated structure of a homologous template protein. After creating a homology model of Argyrin A’s protein structure, Bielecki et al concluded that it “most likely binds to a site distinct from that of fusidic acid, indicating a new mode of protein biosynthesis inhibition by Argyrin A”. Multi-drug resistant pathogens pose a very big risk on the world because they can easily mutate their genomes to adopt resistance to a given antibiotic and persist in causing harmful diseases.

The authors used MDR clinic isolates in order to observe the mechanisms by which these pathogens mutate to build resistance to Argyrin. The fact that eleven of the twelve isolates showed sensitivity to Argyrin suggests that Argyrin is a useful antibiotic in preventing infections by Pseudomonas aeruginosa. There are other factors besides the uptake and export of Argyrin that affect Pseudomonas aeruginosa’s sensitivity to Argyrin, such as efflux pumps; however, the uptake and export of Argyrin in other bacteria does play a role in its sensitivity.

A proteasome is a hollow protein complex with active sites that break down proteins by proteolysis. The degraded peptides that are produced can be used for other functions in the cell. Argyrin A is a factor used to inhibit proteasome function, yet there is no distinct evidence that Argyrin A binds to the site on the proteasome. This paper is important because it analyzes the resistance and sensitivity to Argyrin A in various strains of Pseudomonas aeruginosa.

This bacteria has been a leading cause in nosocomial infections, so it is important to determine which antibiotics best work to stop the spread of disease. About ten percent of patients in hospitals across the United States obtain a significant nosocomial infection. Although there are effective methods to prevent the spread of pathogens in clinical environments, it is important to study how bacteria acquire resistance, so that scientists can develop ways to inhibit the spread of nosocomial infections by multi-drug resistant pathogens.

Bibliography

1. Bielecki, P. , Lukat, P. , Husecken, K. , Dotsch, A. , Steinmetz, H. , Hartmann, R. W. , Muller, R. , and Houssler, S. (2012)
2. Mutation in elongation factor G confers resistance to the antibiotic Argyrin in the opportunistic pathogen Pseudomonas aeruginosa. Chembiochem. 13, 2339-2345. Obritsch, M. D. , Fish, D. N. , MacLaren, R. , and Jung, R. (2005)
3. Nosocomial infections due to multidrug-resistant Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy. 25, 1353-1364. Iqbal, S. , Khalid, Z. M. and Malik, K.
4. A. (1995) Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas aeruginosa. Lett. Appl. Microbiol. 21, 176–179.
5. C. K. Stover, X. Q. Pham, A. L. Erwin, S. D. Mizoguchi, P. Warrener, M. J. Hickey, F. S. L. Brinkman, W. O. Hufnagle, D. J. Kowalik, et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PAO1, and opportunistic pathogen. Nature. 406, 959-964. Encyclopedia Britannica Online. Point mutation. Accessed 17 Oct. 2012 http://www. britannica. om/EBchecked/topic/54744/point-mutation Manfield, I. (2009)
6. Biacore surface plasmon resonance. Univ. of Leeds, Astbury Centre for Structural Molecular Biology. Accessed 18 Oct. 2012 http://www. astbury. leeds. ac. uk/facil/SPR/spr_intro2004. htm
7. Jackson, J. H. (1999) Terminologies for gene and protein similarity. Michigan State Univ. , Dept. of Microbiology. Accessed 19 Oct. 2012 https://www. msu. edu/~jhjacksn/Reports/similarity. htm Bevan, D. R. (2003)
8. Homology modeling. Virginia Tech, Dept. of Biochemistry. Accessed on 19 Oct. 012 http://www. biochem. vt. edu/modeling/homology. html Sasse, F. , Steinmetz, H. , Schupp, T. , Petersen, F. , Memmert, K. , Hofmann, H. , Heusser, C. , Brinkmann, V. , von Matt, P. , Hofle, G. , and Reichenbach, H. (2002)
9. Argyrins, immunosuppressive cyclic peptides from myxobacteria. I. Production, isolation, physico-chemical and biological properties. J. Antiobiot. 55, 543-551. Rape, M. , and Jentsch, S. (2002)
10. Taking a bite: proteasomal protein processing. Nat. Cell Biol. 4, 113-116. Bielecki, P. , Lukat, P. , Husecken, K. , Dotsch, A. Steinmetz, H. , Hartmann, R. W. , Muller, R. , and Houssler, S. (2012)
11. Mutation in elongation factor G confers resistance to the antibiotic Argyrin in the opportunistic pathogen Pseudomonas aeruginosa. Chembiochem. 13, 2340. Simonovic, M. and Steitz, T. A. (2009)
12. A structural view on the mechanism of the ribosome-catalyzed peptide bond formation. BBA Gene Reg. Mech. 1789, 612-623. Abedon, S. T. (2009)
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Introduction

Cystic fibrosis is a genetic disorder currently affecting over 9000 people living in the United Kingdom alone, with millions of people carrying the faulty recessive gene responsible for the disease. This essay is split into 4 distinct sections, firstly looking at the faulty gene and its effects on the organs of the body, followed by an in-depth look at the symptoms of patients suffering with cystic fibrosis, whereas the third section will look at the treatments available to sufferers. The forth section will contain potential future cures and treatments for the cystic fibrosis.

Molecular Mechanisms

The faulty gene that codes for cystic fibrosis affects organs such as the lungs and pancreas. This fault causes high mucosal build up in these organs. Noticeably with regards to the lungs as the high volume of mucous can cause severe breathing difficulties.

The cystic fibrosis transmembrane regulator (CFTR) protein is coded for by the CFTR gene, in chromosome 7 of the human genome. CFTR is a glycoprotein made up of 1480 amino acids consisting of 5 domains. The CFTR protein is responsible for a variety of functions in the apical membranes of cells including the transport of chloride ions, regulation of the sodium ion channels and the regulation of hydrogen carbonate ion transport across the apical membrane[3]. However, it seems that the main contributing factor to cystic fibrosis is the transportation of chloride ions across the apical membrane and sodium ion regulation.

Mutations in the CFTR gene can be categorized into 6 classes; depending on the effect they have on the production of the CFTR protein. The mutations are listed below in Figure 1, with the consequence of the mutation on the CFTR protein.

Mutation NumberConsequence of Mutation
INot synthesised
IIInadequately processed
IIINot regulated
IVShows abnormal conductance
VPartially defective production
VIAccelerated degradation

Figure 1 – Table showing the Mutation Number and the Consequence of the mutation on the CFTR protein

The mutations stated in figure 1, either cause the CFTR protein to become ineffective or prevents synthesis altogether. Classes I and III prevent synthesis of the CFTR protein, whereas other mutations cause problems in the production of the protein. Class II mutations effects can vary from the CFTR protein being completely dysfunctional to significantly reduced function depending on the patient. Class IV and Class V mutations do not cause the CFTR protein to cease working, but do have a derogatory effect on their function. Class VI mutations cause before-time degradation of the protein meaning reduced function.

One of the consequences of the CFTR protein not functioning in cystic fibrosis patients is a high concentration of chloride ions developing in the intracellular space, as well as little regulation of sodium ions entering the cell. Under normal circumstances water would diffuse out the cell and contribute to the airway surface liquid as the concentrations of chloride and sodium ions would be higher in the extracellular space. However with a defective CFTR gene the osmotic gradient is reversed. Thus leading to a high ion concentration within the cell and depletion in the airway surface liquid.

Cilia are small hair like projections in the respiratory tract which are responsible for wafting the mucous up the respiratory tract so it can be swallowed and infection averted. Mucous is one of the body’s primary physical defences against bacterial infection. Pathogens capable of causing respiratory disease are caught on the mucous in the respiratory tract and eventually swallowed with the aid of cilia thus avoiding the entry of pathogens into the lungs and causing bacterial infection. Airway surface liquid also prevents infection by facilitating the movement of mucous up the respiratory tract. However when there is depletion in airway surface liquid, the cilia are also affected. The mucous therefore needs to be of low viscosity so it can be easily moved up the respiratory tract. However, due to the lack of airway surface liquid in a patient with cystic fibrosis the mucous becomes static and more viscous leading to bacterial infections in the lungs.

Cystic Fibrosis does not just affect the lungs in some cases. Occasionally, the pancreas and in the case of men, reproductive organs can be affected. This all depends on which class of mutation of the CFTR gene the patient has as patients with Class I, II and III are prone to pancreatic insufficiency3. In normal pancreatic exocrine secretion, the digestive enzymes secreted from pancreatic gland cells, are mixed with a bicarbonate-rich fluid, secreted from duct cells and released into the small intestine to aid in the digestion of food. The function of the pancreatic gland cells remains fairly constant, but there is a noticeable difference in secretion of the bicarbonate-rich fluid from the duct cells. Thickening secretions causes the duct releasing fluids into the small intestine becomes blocked by enzymes precipitating as well as mucosal build up. With the ducts becoming more blocked, the pressure inside the pancreas increases and as the pancreas lacks structures aiding in support is therefore very prone to damage.

Symptoms

Cystic fibrosis effects different organs around the body and therefore gives rise to a vast range of symptoms. The organs most heavily affected by cystic fibrosis are the lungs and the pancreas, and in males, the reproductive organs are affected.

The most obvious indication that a patient may have cystic fibrosis would be a family history of the disease. As cystic fibrosis is a genetic disorder, the faulty recessive gene can be passed to children. Due to the gene being recessive, it is possible for parents to be a carrier for cystic fibrosis but not express any symptoms themselves. Therefore if both parents are carriers of the gene, then there is a 25% chance of the offspring having cystic fibrosis. From 2009 onwards it is required for new-born babies in the USA to be screened for genetic disorders like cystic fibrosis. If positive, it gives doctors an opportunity to act quickly and maybe prevent other more serious problems, related to cystic fibrosis developing later in life[7]. Other common symptoms that are shown by most ages are salty tasty skin, clubbing of the fingers and toes, coughing with sputum production, mucoid Pseudomonas aeruginosa isolated from airway secretions and hypochloraemic metabolic alkalosis[8].

The symptoms shown by the patient are also different depending on their age. Figure 2 shows a table listing the symptoms by age they become prevalent.

NeonatalInfancyChildhoodAdolescence and Adulthood

Meconium ileusPersistent infiltrates on chest radiographs Chronic pansinusitis or nasal polyposisAllergic bronchopulmonary aspergillosis
Protracted jaundiceFailure to thriveSteatorrhoea Chronic pansinusitis or nasal polyposis
Abdominal or scrotal calcificationsAnasarca or hypoproteinaemiaRectal prolapseBronchiectasis
Intestinal atresiaChronic diarrhoeaDistal intestinal obstruction syndrome or intussusceptionHaemoptysis
Abdominal distentionIdiopathic recurrent or chronic pancreatitisIdiopathic recurrent pancreatitis
CholestasisLiver diseasePortal hypertension
Staphylococcus aureus pneumoniaDelayed puberty
Idiopathic intracranial hypertension (vitamin A deficiency)Azoospermia secondary to congenital bilateral absence of the vas deferens
Haemolytic anaemia

Figure 2 – Depending on the age of the patient, different symptoms for Cystic Fibrosis will be apparent shown in the table above8

As shown in Figure 2, cystic fibrosis has a big effect on many parts of the body. However, the main problem for a patient with cystic fibrosis remains pulmonary disease and the effect of the gastrointestinal problems which arise.

Innate defence mechanisms like the physical barrier provided by the mucous, lining the respiratory tract, is inefficient at its function in a patient with cystic fibrosis. This therefore leads high levels of bacterial infection and inflammation.

The bacterial infections begin soon after birth with Staphylococcus aureus and Haemophilus in?uenzae usually being the pioneer bacteria causing primary infection in the lungs of a patient. It has been suggested that these bacteria are responsible for damaging the epithelial surface cells and therefore aiding other bacteria bind to the surface, however this is still under debate by scientists. However, Pseudomonas aeruginosa is the organism responsible for the later, fatal infections that cause the highest mortality rate in patients with cystic fibrosis. The CFTR protein not only has functions transporting ions, but it is also thought to have a role in binding molecules of Pseudomonas aeruginosa. In a normal individual, Pseudomonas aeruginosa binds to the CFTR protein, and a rapid and self-limiting in?ammatory response9 occurs removing the infection from the respiratory tract. This explains why Pseudomonas aeruginosa is the main causative agent of pulmonary disease in cystic fibrosis sufferers.

Symptoms of cystic fibrosis caused by gastrointestinal problems are mainly caused by the inability to digest food. As mentioned above the ducts leading to the small intestine, which would carry a liquid, containing digestive enzymes is blocked. This causes the pancreas to come pressure and gets damaged. The symptoms caused by this inability to digest food are greasy stools, flatulence, abdominal bloating, and poor weight gain8. At the time of its discovery, malnutrition was the main cause of death due to the inability to produce the enzymes in the pancreas to digest food. Malnutrition can now be treated using pancreatic enzyme replacement therapy8, however other factors like the poor adsorption of fat soluble vitamins can lead to acrodermatitis, anaemia, night blindess, neuropathy, osteoporosis and bleeding disorders8.

A high percentage of Cystic fibrosis patients can develop Cystic Fibrosis related Diabetes Mellitus (CFRD) due to the pancreatic damage that is done by the blocking of the ducts within in the pancreas. The Islet of Langerhans produces insulin and glucagon to regulate blood glucose concentrations. Insulin stimulates the formation of glycogen, removing glucose from the blood stream whereas glucagon stimulates the breakdown of glycogen. With the pancreas undergoing autolysis, it is inevitable that these cells will become damaged and unable to produce a sufficient amount of insulin. However, CFRD is different to diabetes mellitus I and II. The specific symptoms affected by cystic fibrosis are glucose metabolism, acute and chronic infection, glucagon deficiency, liver dysfunction, decreased intestinal transit time, and increased work of breathing8.

Cystic fibrosis also affects male reproduction. In the male reproduction organs, the vas deferens is responsible for the transfer of sperm from the epididymis in anticipation of ejaculation8. Male patients with cystic fibrosis lack this muscular tube and therefore there is no sperm in their ejaculate. Women however are fertile, but careful control of nutritional intake must be taken to ensure the full term of pregnancy and subsequent birth can be achieved. I can be possible for parents to pass the gene for cystic fibrosis onto their children. As a man expressing the disease being infertile the recessive gene must come from a carrier of cystic fibrosis but not expressing any symptoms. If the female sufferers from cystic fibrosis then there is a 50% chance that the child will also have the disease. However if the female is also a carrier of the recessive gene then there is a 25% chance that the child will have cystic fibrosis.

Current Treatments

As it stands at the moment, cystic fibrosis cannot be cured. Cystic fibrosis is a genetic disease, and therefore there is an error in the DNA of cells of an individual apart from their gametes. This means that the only available option to sufferers is to find drugs to treat the various symptoms. However in recent years there have been successful attempts to find drugs to resolve the original defects.

Patients with cystic fibrosis often suffer from severe pulmonary infections, as they are less efficient at swallowing mucous containing pathogens. The airway surface liquid and cilia, as discussed above are responsible for the movement of mucous up the respiratory tract in a normal individual. Cystic fibrosis sufferers lack the required volume of airway surface liquid. One such treatment, looking to solve the problem caused by the faulty gene is hypertonic saline. Hypertonic saline is the current drug used to bring about an increase in the volume of airway surface liquid in the lungs of the patients. It has been shown by researchers that in vitro, hypertonic saline is effective with rehydrating and providing more airway surface liquid8. If from an early age there is aid with mucosal clearance from the respiratory tract, it would reduce the chances of the patient developing severe bacterial infections from pathogens such as pseudomonas aeruginosa.

Antibiotics are also important to control pulmonary bacterial infections that occur from poor mucosal clearance. Macrolide antibiotics are cheap antibiotics used to treat cystic fibrosis sufferers. They work by inhibiting the bacteria protein biosynthesis; however the precise mechanism they use is currently not fully understood. Macrolides have a dual function in the cystic fibrosis treatment. They affect cytokine production of many cell types and are therefore effective as anti-inflammatory agents8. One study showed that taking azithromycin three times a week reduced the virulence factor production, decreased biofilm production, bactericidal effects on pseudomonas aeruginosa growing in stationary phase8.

Patients with gastrointestinal symptoms brought about by cystic fibrosis require other forms of treatment, as they unable to produce enough enzymes to digest their food. Enzymes are usually secreted from the pancreas into the small intestine, allowing the individual to digest their food. However a patient with pancreatic insufficiency is unable to secrete the enzymes into the small intestine due to the intrapancreatic ducts being blocked. This needs to be treated as patients are at risk of severe malnutrition if food cannot be fully digested. Pancreatic Enzyme Replacement Theory (PERT) is used to treat this problem. The enzymes used in PERT are taken with every meal to aid in the absorption of food in the small intestine. This does not solve the underlying problem caused by cystic fibrosis, and can only treat the symptoms. This is because the enzymes do not last for a great deal of time in the body; the reason why enzymes must be taken with every meal.

Chest physiotherapy can aid in the treatment of a sufferer as it can help in mucosal clearance from the pulmonary tract[11]. Some methods focus on the way the patient breathes which include active cycle of breathing techniques, and autogenic drainage11. However some techniques focus on actual mucosal clearance like positive expiratory pressure (PEP) oscillating positive expiratory pressure11.

The final and most severe treatment used to use the chronic pulmonary disease is lung transplantation. This procedure comes with high risk with only 50% children surviving 5 years after the transplant and 50% of adults surviving 6 years after the transplant8.

Future Treatments

As of yet there is no cure for cystic fibrosis, however there are promising new drugs on the horizon which could one day all but cure the genetic disease.

A drug that is combatting the “basic defect” of cystic fibrosis is VX-770 made by Vertex pharmaceuticals. This drug is currently undergoing trials in the United States of America and is showing promise for improving lung function in those affected by cystic fibrosis. The drug itself targets the chlorine channels in the apical membrane, opening them to allow chlorine to leave the epithelial cells. Results from the recent Phase 3 clinical trials showed that there was a 10% increase in lung function in people aged 12 and above, and a 12.5% increase in lung function for those from ages 6 to 11. The drug also decreased the concentration of chlorine in sweat and allowed the patients taking the drug to gain on average 7 pounds12. Pancreatic insufficiency usually makes it hard for the cystic fibrosis patient to gain weight, meaning the drug has an effect on gastrointestinal symptoms as well as respiratory symptoms.

The idea of gene therapy is a concept that has been around for a while. The Cystic fibrosis gene therapy consortium was set up in 2001 to focus on developing new ways to combat cystic fibrosis and the symptoms. In February 2009, the consortium made a step closer to find a “gene therapy cure” for cystic fibrosis. They managed to insert successfully, a working CFTR gene into a viral vector, which was then transported into the lungs of a cystic fibrosis patient. Whereas the idea itself is good, there are some drawbacks. The viral vector itself comes under attack from the immune system as well as viral vectors being poor at inserting DNA into epithelial cells8. Therefore the The Cystic fibrosis gene therapy consortium has been looking at using lipid vectors currently with little success. They have reported that currently gene expression in the cells that do take up the gene is currently temporary and they are looking into methods to provide sustained gene expression in the epithelial cells.

Not all mutations cause the CFTR protein not to be synthesised. Some mutations cause the CFTR protein to be marked for degradation as the chaperones, aiding with folding the protein, do not dissociate from the protein8. There have been tests in labs using chemicals such as phenylbutyrate8, however any further developments in this field of research.

Conclusion

It is remarkable how such a small difference in the DNA of an individual can have so much effect on the patients’ health. However the future looks bright for those patients suffering with cystic fibrosis. There is a huge amount of research taking place in order to find new treatments and potential cures for the disease. Recently, research has shifted from looking for ways to treat the symptoms, to methods of treating the underlying problems behind the disease. For example a drug called VX-770 made by Vertex Pharmaceuticals, focuses on the inefficient chlorine channels in the epithelial cells. If cystic fibrosis remains a continually financially backed area of research, there is no reason why significant progress cannot be made sooner rather than later.

Abstract

Cystic fibrosis (CF) is a genetic disease that causes a dysfunctional cystic fibrosis transmembrane conductance regulator protein (CFTR) to be produced. This essay firstly will focus on the mutations of this defective protein and the intracellular effects. It will then consider the symptoms of the disease that can be observed including pulmonary, gastrointestinal, endocrine and reproductive problems. Then focus will be on the current treatment methods which target the consequences of the CFTR dysfunction such as phlegm retention and infection and the new treatment methods which treat the underlying CFTR defect such as targeting the trafficking of the protein.

Introduction

Cystic fibrosis is an autosomal recessive inherited disease caused by a gene defect on chromosome seven that is responsible for the cystic fibrosis transmembrane conductance regulator protein (CFTR). This is found in the apical plasma membrane of epithelial cells in the lungs, sweat glands, pancreas amongst other tissues. This causes dysfunctional CFTR to be produced leading to a thick sticky mucus causing a recurrent cough, frequent lung infections by bacteria such as Psuedomonas aeruginosa and digestive problems. More than 1,500 mutations have been found including DF508 which is the most common, caused by a deletion of phenylalanine. The mutant allele was first isolated in 1989 and since then life expectancy has improved greatly to between 31 and 37 years old and is still increasing today. Numerous mutations have been identified which are classed differently (class I – VI) depending on how the dysfunctional protein is handled within the cell.

Molecular mechanisms

Cystic fibrosis is an autosomal recessive disease which means both parents must be heterozygous carriers of the CF allele in order for the offspring to have a 25% chance of inheriting the disease, or 50% of being a carrier (figure 1). There are over 1,500 observed mutations of the CFTR protein but the majority of these are rare. The most common mutation is caused by a deletion of phenyl-alanine in position 508 (DF508) which accounts for 66% of CF cases.

The DF508 for example produces a misfolded CFTR and is recognised within the cell endoplasmic reticulum as an abnormal protein, leading to it being proteolytically degraded in the proteasome. This results in only small amounts of CFTR reaching the plasma membrane but this has a short half life leading to an insufficiency of chloride transport. The misfolded CFTR leads to a protein trafficking problem, hence new drugs that aim to rescue the protein from ER degradation could be therapeutic strategies to re-develop intracellular protein movement. Since different mutations lead to different problems with the CTFR protein, certain treatment strategies may only work on a small proportion of patients.

There are several hypotheses as to how this CFTR mutation causes the disease known as cystic fibrosis. The first is the low-volume hypothesis. The loss of inhibition of sodium channels causes excess sodium and water reabsorption causing dehydration of airway surface materials and lack of a compensatory mechanism. This lower water volume causes inhibition of normal ciliary and cough clearance of the mucus and plaques form that harbour bacteria. Secondly, the salt hypothesis believes excess sodium and chloride are retained in airway surface liquid and the increased concentration of chloride disrupts the innate antibiotic molecules so bacteria persist. Thirdly, it is hypothesised disease is due to the dysregulation of host inflammatory response which is backed up by the abnormally high concentration of inflammatory mediators found in children as young as 4 weeks who appear disease free. Finally, the increased presence of asialo-GM1 receptors in apical membranes allow increased binding of P. aeruginosa and S. aureus without the rapid self-limiting innate immune response since in normal patients it is believed the binding of bacteria to functioning CFTR generates an innate immune response which would not function in CF patients. This is made worse by the combination of increased bacterial binding. The CFTR gene defect causes absent or malfunctioning CTFR protein causing abnormal chloride conductance on apical membrane of epithelial cells in the lungs.

CFTR belongs to a family of transmembrane proteins called adenosine triphosphate binding cassette transporters and is a chloride channel. It also has several other functions such as inhibition of sodium transport through sodium channels in the epithelium, regulation of ATP channels, regulation of intracellular vesicle transport, acidification of intracellular organelles and inhibition of endogenous calcium activated chloride channels. In the lungs, this dysfunctional CFTR causes airway surface liquid depletion leading to decreased ciliary stability and ciliary collapse with decreased mucociliary transport causing phlegm retention, infection and inflammation of the airways.

Increased cAMP levels leads to phosphorylation of CFTR causing chloride transport but since this is not functioning in CF patients the chloride channel fails to open and respond to cAMP (a second messenger). This causes a decreased secretion of Cl? into the lumen airway so excessive water and sodium is absorbed. This cannot cross the epithelial membrane due to the osmotic gradient created leading to increased viscosity of mucus. Local mediators that are secreted onto airway surface liquid help regulate the surface liquid volume as they induce CFTR dependent and independent chloride secretion.

The alternative chloride channel mediates chloride secretion since the P2Y receptor is activated by ATP in both CF and non CF epithelium which is triggered by movement. Respiratory syncytical viruses that may infect the airways have increased ATPase activity so more ATP is broken down; the loss of this compensatory mechanism that would activate the alternative chloride channel has a negative effect on airway clearance becoming a problem in CF patients.

Symptoms

Cystic fibrosis can be diagnosed at different stages of a child’s life; newborn testing occurs as standard since all babies are tested by a heel-prick blood sample as part of the Guthrie test and antenatal testing is carried out on women considered to be high risk of having a child with CF. Carrier testing is a mouthwash test to establish if each parent is a carrier and a genetic test via a swab on the inside of the cheek probes for 40 of the most common CF mutations which correctly diagnoses 90% of cases. One further test is to test the sweat on the skin of babies or children since patients with CF have a high salt concentration in the sweat and CF can be diagnosed if the salt concentration is above 60 mmol/L – this is because CFTR resorbes chlorine into cells of sweat glands and if this is dysfunctional this cannot occur.

General symptoms that lead to a diagnosis include a family history, salty-skin, clubbing of the toes and fingers, a cough with sputum production, mucoid Pseudomonas aeruginosa isolated – repeated chest infections, diarrhoea and poor weight gain. The further symptoms can be grouped into the organ they affect from pulmonary to gastrointestinal, digestive system, endocrine and reproductive symptoms.

Pulmonary symptoms are perhaps the most obvious and commonly associated with the disease. A thick secretion of high levels of mucus into the lungs occurs which leads to frequent bronchial infections and a recurrent cough. Pseudomonas aeruginosa and Staphylococcus aureus are the most commonly isolated bacteria and can be found at high affinities in CF lungs. It is the failure of the mucosal defence system to clear these organisms that is the issue. Early studies suggested P. aeruginosa binds to CF epithelial cells at higher density than normal individuals due to more asialo-GM1 receptors in CF patients, however other theories hypothesised CFTR itself is a receptor for the bacteria that mediates intracellular uptake of the bacteria and killing of it that would be absent in patients with defective CFTR protein. Current studies however suggest the bacteria are present on the mucus layer on respiratory epithelial cells rather than the cell membranes making it unlikely this is the case. It was hypothesised salt-sensitive cationic antimicrobial peptides called defensins could not function in CF patients if the luminal side of the epithelium has an increased sodium chloride concentration. This seems unlikely though as not all defensins are salt sensitive. It is now thought dehydration of the airway surface liquid impairs cilia functioning and mucociliary clearance so inhaled bacteria colonise. Furthermore CF sputum has below normal oxygen levels that switch P. aeruginosa from non-mucoid to mucoid form that is resistant to host defences. “The persistence of chronic P. aeruginosa infections in cystic fibrosis patients is due to biofilm growing mucoid strains.”  These biofilms exhibit increased tolerance to antibiotics and resist phagocytosis as well as parts of the innate and adaptive immune system. This leads to complex-mediated chronic inflammation which can cause lung damage. The bacteria are also so persistent as the mutate and have low metabolic rates and increased doubling times.

In the gastrointestinal tract, several problems occur throughout life. At the newborn stage, some babies may need an operation to remove mucus that is obstructing the bowel – a condition known as meconium ileus. Pancreatic insufficiency is also seen causing symptoms such as greasy stools, flatulence, abdominal bloating, poor weight gain and fat soluble vitamin deficiency with malnutrition. Since it is hard to digest food, malnutrition can occur which causes poor growth, physical weakness and delayed puberty. This requires a pancreatic enzyme therapy with high calorie intake to manage. Older patients’ may develop an intestinal obstruction and the lack of absorption of vitamins A, D, E and K can lead to conditions such as anaemia, neuropathy and osteoporosis.

The endocrine system can sometimes be affected in later life due to obstruction of the pancreatic ducts due to thickened secretions. As pancreatic disease develops the proportion of islet cells declines leading to a lack of insulin production where the blood sugar cannot be controlled which is then diagnosed as CF related diabetes mellitus, with symptoms such as constant thirst, hunger, weight loss and urination, however CF diabetes is not the same as type 1 and 2 diabetes. The reproductive system in women patients does not seem to be affected and they still produce healthy eggs, in men however the sperm ducts are blocked leading to male infertility.

Some other symptoms include frequent sinusitis and hay fever that requires nasal spray or antibiotics and adults may develop nasal polyps. Incontinence can sometimes develop and in some patients bile ducts in the liver become blocked by mucus and the patient may require a liver transplant.

Treatment

Current

Treatment of cystic fibrosis currently focuses on the consequences of the CFTR dysfunction such as phlegm retention, infection and inflammation though new strategies target the underlying gene defect. Currently, physiotherapy is one main treatment strategy used in combination with other management techniques. The thick sticky mucus secretions that block the airway in CF patients causing infections and coughing can be dislodged either by mechanical chest thumps or autogenic drainage and positive expiratory pressure. Physiotherapy is needed every day from between 15 and 50 minutes depending on the level of mucus present. Physical activity is also important as it prevents deterioration of the lungs and increases bulk and strength.

Medication is used to treat cystic fibrosis such as lung medication including bronchiodilator drugs to open airways by relaxing the surrounding muscles, relieving tightness and shortening of breath and can be taken by being inhaled in nebulisers, taken orally or intravenously. Other medication includes antibiotics to treat persistent pulmonary infections, steroids to reduce inflammation of the airways and DNase to break down the mucus making it easier for the body to digest. Repeated pulmonary infections and thick mucus secretions can become so severe that the patient may need a lung transplant and possibly a heart or liver transplant also.

Due to the nutritional problems associated with the disease, enzyme pills are taken with every meal and snack to replace pancreatic enzymes so more energy is gained from the food since there is a lack of digestive enzymes hence less nutrients can be absorbed from the food. These problems occur due to blocking of the small channels carrying digestive juices by mucus causing enzymes to build up in the pancreas that damages it over time. Nutritional supplements may also be given such as high energy drinks, and insulin may be necessary if the patient develops CF related diabetes mellitus. A suitable diet that is high in calories is also required to ensure adequate energy is gained. The lack of mineral absorption can lead to osteoporosis – weakening of the bones – which can be treated with bisphosphonates.

Future

There are a variety of new treatment possibilities targeting the underlying gene defect in the transmembrane receptor rather than downstream effects. Anti inflammatory drugs are one option due to persistent endobronchial inflammation in patients. The first main possibility is CF transmembrane regulator replacement therapy. This has already been tested using a variety of vectors such as adneoviruses, adeno-associated viruses and cationic lipids to transfect the functioning gene into epithelial cells. Some successful gene transfer has been seen into airway epithelial cells however it was short-lived CFTR expression and was hard to prove the link between improvement in CFTR function and clinical manifestations. The issue is it is yet unknown how much improvement in CTFR function is needed in order to make a big difference. The current research now focuses on the correct vector to use to minimise adverse effects and increase expression time – this is difficult as viral vectors have good transfection rates but more adverse effects and as multi dose therapy would be needed, virus-specific immune responses would devleop whereas liposomal vectors have less negative effects but worse transfection rates.

A second option being researched currently is CFTR pharmacotherapy involving drugs with affect intracellular trafficking of CFTR. This would not work for all patients due to the specific classes of mutations so it is of limited benefit. Class I mutations are stop mutations that decrease or eliminate production of CFTR. Aminoglycosides induce read through of premature stop codons so would produce a full length functioning CFTR protein, these can be topically applied and an improvement in CFTR functioning has been seen however the concentration needed is high and adverse effects mean they are not clinically suitable. An alternative to this includes PTC 124 – premature termination codon – which acts in a similar way but lacks toxicity. Class II mutations have misfolded CFTR and the trafficking of these is impaired due to proteosomic degradation; this CFTR does have chloride transport function however it is prematurely degraded and most does not reach the membrane. This gives a new target – drugs which reduce degradation of the misfolded protein and increase trafficking to the membrane – and libraries of chemical agents are being screened for applicants. Class III mutations have a reduced probability of opening the CFTR channel but these are rarer. Compounds which activate CFTR would aid class III mutations such as VX770 (a potentiator) that is being used in trials for patients with the G551D mutation that could show improvements in function of the CFTR as well as reduced sweat chloride concentration. However effects in class II may also be seen if used in combination with a corrector compound that brings CFTR to the surface and then the potentiator can activate it.

Option three involves opening alternative chloride channels to compensate for the lack of function of the CFTR channel. CFTR is not the only chloride transport channel in a membrane, a calcium-dependent chloride channel also secretes chloride in epithelial cells and increasing the activity of this may be an option so enough chloride transport occurs in the cell. Two drugs have shown to have an ability to do this via the P2Y receptor. First of these is denufusol, which bypasses the defective channel and activates the alternative chloride transporter – “This activation results in an increase in airway surface epithelial hydration, and through these actions and effects on cilia beat frequency, increases mucociliary clearance” and has been shown to be an early intervention strategy when inhaled. The second of these drugs is lacovutide (Moli990) increases intracellular calcium level and activates alternative chloride channels, it does not bind with receptors but instead interacts with phospholipids on the plasma membrane.

The CFTR protein has several functions – chloride transport, inhibiting sodium transport as well as regulation of ATP channels. Inhibition of sodium absorption was hypothesised as a treatment option however amiloride (an epithelial sodium channel blocker with a short half life) was shown to have no clinical benefit and a tendency to decreased lung function. Studies on mice have shown when given as an early intervention the disease progression was prevented, however there is little evidence to show this in humans. An improvement may be seen in a blocker with a longer half life.

Finally, airway surface liquid rehydration could improve the inadequate water content of the surface liquid by increasing the airway fluid layer with an inhaled osmotic agent. Hypertonic saline was found to have a positive effect on mucociliary transport and lung function due to induction of coughing and hydrating the mucus and new evidence has shown it also increased depth of the airway surface liquid. Inhaled powdered mannitol is an alternative. Effectiveness is limited to those with established lung disease but again, early intervention may prove more effective.

Conclusion

Cystic fibrosis is a lifelong eventually fatal disease caused by a genetic defect in the CFTR protein. How this protein functions and which factor is responsible for all the symptoms seen in CF patients is not yet confirmed though it is clear the dehydration if airway surface liquid causing the thick mucus that is hard to dislodge and harbours biofilms of bacteria leading to frequent infection is a major factor. Current treatment strategies target the downstream effects of CF such as the phlegm retention and make the disease manageable. The new development of drugs targeting the underlying defect is occurring with some in clinical trials though the benefit to each patient is unknown. This is because of the diversity of mutations and varying symptoms within each patient making this a difficult disease to treat.

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