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Cystic fibrosis transmembrane conductance regulator and lung clearance of Pseudomonas aeruginosa

Submitter: Giulio Cabrini | cbg@unife.it

Laboratory of Molecular Pathology, Cystic Fibrosis Center, Verona, Italy

Published October 24, 2002

Giulio Cabrini
Laboratory of Molecular Pathology, Cystic Fibrosis Center, Verona, Italy
Department of Experimental Medicine and Diagnostics, Section of General Pathology, University of Ferrara, Ferrara, Italy
and Luisa Zanolla
Department of Cardiology, University of Verona,Verona, Italy
Address correspondence to: Giulio Cabrini, Department of Experimental Medicine and Diagnostics, Section of General Pathology, Via Borsari 46, 44100 Ferrara, Italy. Phone: +39-0532-291780; Fax: +39-0532-247278; E-mail: cbg@unife.it
The airways of patients with cystic fibrosis (CF) become precociously colonized with a variety of bacteria, mainly Staphylococcus aureus or Haemophilus influenzae. In the course of their lifetime, over 80% of CF patients are infected with Pseudomonas aeruginosa resulting in progressive loss of lung function and early death (Cystic Fibrosis Foundation Patient Registry, Annual Data Report 1999). As P. aeruginosa infection is rarely associated with lung diseases other than CF, the reason that patients with CF initially acquire and fail to eliminate environmental strains of P. aeruginosa from the lungs is enigmatic.
Mutations in the CF transmembrane conductance regulator (CFTR) gene are responsible for CF. The gene codes for a multifunctional chloride channel and conductance regulator that is localized to the apical plasma membrane of different epithelia, including the airway surface cells (1-3). More than 900 different CF gene mutations are known, although the most frequent is the DF508 alteration, which accounts for 50-80% of the diseased alleles. A direct connection has been proposed between mutated CFTR in lung epithelial cells and the initiation of P. aeruginosa infection. Pier and colleagues have provided evidence that epithelial cells use CFTR as a receptor for binding, internalization, and subsequent clearance of P. aeruginosa (4-7). Binding takes place between the lipopolysaccharide of P. aeruginosa and the first extracellular domain of CFTR (amino acids 103-117). In experimentally infected mice, competitive inhibition of CFTR-mediated binding of P. aeruginosa with free bacterial lipopolysaccharide or CFTR peptide 103-117 resulted in increased bacterial loads in the lung. Moreover, in recombinant cell lines in which a mutant CFTR is present that is not localized to the plasma membrane, as in the case of the most frequent mutation DF508, internalization does not take place. Additional experiments performed on both in vitro cell models and transgenic mice suggested that CFTR acts as a clearing receptor for P. aeruginosa. CFTR has been reported to play the same role for Salmonella typhi, but not for Staphylococcus aureus (4,8). On the contrary, in a recent issue of the JCI, Worlitzsch and colleagues propose that P. aeruginosa infection in CF patients occurs in the airway mucus, therefore in the luminal rather than the epithelial cell surface compartment (9). This study was conducted in CF patients in vivo and thus seems to exclude a direct role of CFTR as a clearing receptor for P. aeruginosa.
In an attempt to distinguish between these two conflicting hypotheses, we investigated the effect of the molecular defects at the basis of mutated CFTR on colonization of P. aeruginosa in the lungs of CF patients. There are at least five mechanisms by which alterations in CFTR may affect the chloride conductance of the CFTR protein (10,11). Class I mutations lead to absence of protein synthesis, while class II mutations (e.g. DF508) give rise to improper protein processing. Both class I and II mutations result in incorrect subcellular localization of CFTR. Class III-IV-V mutations are rare and lead to defective chloride transport, although the protein is localized in the plasma membrane. In particular, class III mutations result in defects in regulation, class IV in defective chloride conductance, and class V mutations reduce protein synthesis. Therefore, if the hypothesis of Pier and colleagues is correct, it would be expected that individuals in which the CFTR protein is completely absent from plasma membrane would be colonized by P. aeruginosa. Such individuals include homozygotes for class I-II mutations. On the other hand, CF patients carrying class III-IV-V mutations should be protected by infection from this bacterium. To test this hypothesis, all patients with CF followed at the Center of Verona since 1974 were grouped together according to specific CF genotypes. The first group included 202 homozygotes for class I-II mutations (all DF508 homozygotes), while the second group was composed of 37 patients carrying class III-IV-V mutations (heterozygous carriers of G551D, G1244E, R117H, R334W, R347P, 3849+10Kb C>T). Sputum samples were taken during routine follow-up of these patients and were subjected to microbiological analysis. The fraction of infected patients was plotted as a function of age, according to a Kaplan-Meier survival estimate model and statistical significance was analyzed by log-rank test. As expected, patients homozygous for the DF508 mutation are infected by P. aeruginosa and the fraction of positive subjects increases progressively up to 75% (Fig. 1 ). CF patients carrying class III-IV-V mutations were free of P. aeruginosa infection for significantly longer times with respect to the first group of patients (p = 0.0005), although they eventually succumb to infection (Fig. 1 ). The trend of bacterial infection was also examined for the other two most common pathogens of CF lung, namely S. aureus and H. influenzae, which are precociously found in a large fraction of DF508 homozygous patients (Fig. 2 ). Similarly to P. aeruginosa infection, the presence of class III-IV-V CFTR mutations is associated with a statistically significant delay in bacterial colonization (p<0.0001) (Fig. 2 ).
Thus, CF patients carrying class III-IV-V mutation are neither specifically nor completely protected from P. aeruginosa colonization, as would be expected if the CFTR protein acts as a receptor that binds, internalizes, and removes this microorganism from the airway surface. The most striking difference between the two groups of CF patients compared in this study is a significant delay in the age of onset of pulmonary bacterial colonization. This can be explained by considering that cells expressing CFTR with class III-IV-V molecular defects maintain a residual ion conductance that is completely missing when the protein is localized to the wrong cellular compartment. A minimal conductance activity could postpone both CFTR-related impairment of mucociliary clearance and eventual changes in the airway surface microenvironment that favor bacterial infection (3). In conclusion, our observations contradict thesupposition that CFTR is an epithelial cell receptor for internalization of P. aeruginosa and provide indirect support for the most recent alternative hypothesis (9) that CFTR plays a different role in the predisposition of local conditions leading to pulmonary colonization of P. aeruginosa in patients with CF.
Acknowledgments
We wish to thank the personnel of the Laboratory of Molecular Pathology for mutation analysis and the Laboratory of Microbiology for sputum culture, with special mention to Valentino Stanzial for help in database analysis. This work is dedicated to the memory of Rossella Rolfini and to her commitment to CF research.
1. Sheppard, D.N. and M.J. Welsh. 1999. Structure and function of the CFTR chloride channel. Physiol.Rev. 79:S23-S45
2. Schwiebert, E.M., D.J. Benos, M.E. Egan, M.J. Stutts, and W.B. Guggino. 1999. CFTR is a conductance regulator as well as a chloride channel. Physiol.Rev. 79:S145-S166
3. Pilewski, J.M. and R.A. Frizzell. 1999. Role of CFTR in airway disease. Physiol.Rev. 74:S215-S255
4. Pier, G.B., M. Grout, T.S. Zaidi, J.C. Olsen, L.G. Johnson, J.R. Yankaskas, and J.B. Goldberg. 1996. Role of mutant CFTR in hypersusceptibility of cystic fibrosis patients to lung infections. Science 271:64-67.
5. Pier, G.B., M. Grout, and T.S. Zaidi. 1997. Cystic fibrosis transmembrane conductance regulator is an epithelial cell receptor for clearance of Pseudomonas aeruginosa from the lung. Proc.Natl.Acad.Sci.USA 94:12088-12093.
6. Pier, G.B. 2000. Role of cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections. Proc.Natl.Acad.Sci.USA 97:8822-8828.
7. Schroeder, T.H., N. Reiniger, G. Meluleni, M. Grout, F.T. Coleman, and G.B. Pier. 2001. Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract. J.Immunol. 166:7410-7418.
8. Pier, G.B., M. Grout, T. Zaidi, G. Meluleni, S.S. Mueschenborn, G. Banting, R. Ratcliff, M.J. Evans, and W.H. Colledge. 1998. Salmonella typhi uses CFTR to enter intestinal epithelial cells. Nature 393:79-82.
9. Worlitzsch, D., R. Tarran, M. Ulrich, U. Schwab, A. Cekici, K.C. Meyer, P. Birrer, G. Bellon, J. Berger, T. Weiss, K. Botzenhart, J.R. Yankaskas, S.H. Randell, R.C. Boucher, and G. Doring. 2002. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients. J.Clin.Invest. 109:317-325.
10. Welsh, M.J. and A.E. Smith. 1993. Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73:1251-1254.
11. Zielenski, J. and L.-C. Tsui. 1995. Cystic fibrosis: genotypic and phenotypic variations. Annu.Rev.Genetics 29:777-807.

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Reduced oxygen in cystic fibrosis airways and interactions of P. aeruginosa with epithelial cells.

Submitter: Gerald B. Pier | gpier@channing.harvard.edu

Harvard Medical School

Published February 19, 2002

In their report on the effects of reduced oxygen concentration in airway Pseudomonas aeruginosa infections in cystic fibrosis (J. Clin. Invest. 2002;109:317-325), Worlitzsch et al. write in the discussion that their findings "?contradict recent hypotheses emanating from in vitro model systems that focus on high-salt/defensin inactivation, or luminal epithelial cell binding, which predict bacterial infection of CF airway epithelial cells themselves. They cite a paper from myself and colleagues (Pier GB et al. 1996. Science, 71:64-67) relevant to the last part of this statement regarding predictions of CF airway epithelial cells. However, our work, published not only in the cited reference, but in numerous references since then, clearly predict that CF airway epithelial cells would not show evidence of bacterial infection, a finding consistent with that of Worlitzsch et al. We have proposed that the cystic fibrosis transmembrane conductance regulator (CFTR) serves as an epithelial cell receptor mediating internalization of P. aeruginosa, a process that promotes bacterial clearance. The lack of CFTR expression in CF patient's airway epithelial cells obviously would preclude bacterial interaction with these cells through CFTR. Our confidence in this hypothesis stems from earlier observations, confirmed by Worlitzsch et al., that P. aeruginosa is not observed on or in CF epithelial cells as viewed in lung sections taken at autopsy or from lung transplants. Also, in the cited reference from 1996 we showed that clinical isolates of P. aeruginosa lose the bacterial receptor for CFTR, further diminishing the interaction with CF epithelial cells. Finally, our work has not been merely in vitro; we have demonstrated in infections of live mice and monkey trachea that P. aeruginosa invades epithelial cells on an intact epithelium expressing CFTR, but not in transgenic CF mice lacking this ion channel. Our work on P. aeruginosa-CFTR provides a molecular explanation for the extraordinarily high rate of infection with this one pathogen in CF. Worlitzsch et al. findings, while clearly important in elucidating a critical component of pathogenesis, fail to provide any explanation for the specificity of P. aeruginosa and CF. Thus, while the CF airway surface liquid factors promoting infection are undoubtedly important components of the disease process, the findings here do not contradict, but rather support, our hypothesis that the failure of epithelial cells in CF patients to interact with P. aeruginosa via CFTR also represent an important component of the pathologic process.