Evolution of the ΔF508 CFTR mutation: Response

Guggino makes several cogent points about the impact of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) and evolutionary selective pressures. However, the questions raised by Guggino about our hypothesis may not have considered important aspects of epithelial cell responses to bacteria, heterozygote selection and evolutionary theory. There is nothing in our hypothesis or data about protection against Salmonella typhi infection that excludes protection against diarrheal disease as an additional aspect of the heterozygote selective advantage in cystic fibrosis (CF). CFTR is clearly a multifunctional molecule, and the ability to dissociate chloride ion conductance from bacterial binding suggests that heterozygotes with one mutant CFTR gene might have advantages against multiple undesirable pathological consequences.Two concerns about the diarrheal disease hypothesis are that it was experimentally tested with cholera toxin1xGabriel, S.E. et al. Science. 1994; 266: 107–109Crossref | PubMedSee all References1 and was not confirmed in a subsequent study2xCuthbert, A.W. et al. J. Physiol. Lond. 1995; 482: 449–454See all References2. Vibrio cholerae was not present in Europe until 1832 and could not have been the selective force accounting for the current level of the ΔF508 allele in European populations, unless two-thirds of Europeans of reproductive age died from cholera in the 19th century3xSee all References3. Certainly, the related labile toxin (LT) synthesized by other enteric pathogens could behave similarly, but no reports of the specific effect of LT on heterozygote CF tissues have been published.Our hypothesis about increased resistance to S. typhi infection is not restricted to the ΔF508 allele; all mutant CFTR alleles resulting in reduced CFTR expression, including nonsense mutations, frameshifts and splicing variants, could provide a selective advantage against S. typhi. Even the G551D allele, which results in a dysfunctional surface CFTR protein, could fit into our hypothesis, as it is not merely the expression of the Salmonella entry domain on the epithelial surface but the ability of the cell to internalize the pathogen and orchestrate a response that determines host susceptibility and resistance to S. typhi (and to Pseudomonas aeruginosa). Salmonella internalization into gastrointestinal (GI) epithelial cells results in either bacterial passage through the cell and entry into the lamina propria from the basolateral side, or cellular desquamation, notably of the M cells, creating a hole in the epithelium through which non-ingested bacteria gain access to the submucosa4xJones, B.D., Ghori, N., and Falkow, S. J. Exp. Med. 1994; 180: 15–23Crossref | PubMed | Scopus (572)See all References4. There is no reason to assume that binding of S. typhi to the G551D or similar mutant CFTR proteins initiates this series of cellular responses permitting S. typhi access to the submucosa. Indeed, one might assume from the usually severe course of P. aeruginosa lung infection in G551D homozygote CF patients that this molecule is defective in its ability to orchestrate cellular responses to bacteria, even if microorganisms can initially bind to this CFTR variant. Moreover, there is no reason to assume that the G551D CFTR protein expresses amino acids 103–117 on the extracellular side of a cell, as wild-type CFTR appears to do. Even if G551D CFTR appropriately expresses this Salmonella entry domain, it would only be necessary, but not sufficient, for bacterial uptake. Finally, our preliminary results from G551D mutant mice derived by Delaney et al.5xDelaney, S.J. et al. EMBO J. 1996; 15: 955–963PubMedSee all References5 indicate that GI uptake of S. typhi by these mice is compromised in a manner identical to that of ΔF508 CFTR mice.Guggino's contention that one might expect more mutations in the first extracellular loop of CFTR if this provided protection against S. typhi entry represents the untenable Lamarckian view of evolution that natural selective forces can influence genetic mutations. In the Darwinian model of evolution, randomly occurring genetic variation is acted upon by natural selective forces providing for reproductive advantages in affected individuals and their offspring that inherit the variant allele. Thus, the random appearance of the ΔF508 CFTR allele in an individual living in an environment where the selective force was operative would allow a potential heterozygote advantage to develop. This allele arose over 50 000 years ago, probably in a single individual6xMorral, N. et al. Nat. Genet. 1994; 7: 169–175Crossref | PubMed | Scopus (240)See all References6. The increased resistance of the heterozygote offspring of this individual (which might include 20 000 000 to >50 000 000 individuals alive today with at least one ΔF508 CFTR allele) to a selective force such as S. typhi infection could account for essentially all of the selective advantage, resulting in the level of CF that occurs today.Finally, it should be noted that the series of events involved in P. aeruginosa lung infection in CF described by Guggino are not all well established by experimental testing. Adherence of P. aeruginosa to asialoGM1 moieties, which is reportedly increased on CF cells7xSaiman, L. and Prince, A. J. Clin. Invest. 1993; 92: 1875–1880Crossref | PubMedSee all References7, has only been documented in vitro using cell cultures; a careful examination of the differences in P. aeruginosa binding to CF and non-CF cells reveals the differences to be modest7xSaiman, L. and Prince, A. J. Clin. Invest. 1993; 92: 1875–1880Crossref | PubMedSee all References7. Moreover, in studies reporting that P. aeruginosa strains deficient in pili and flagella (the reported bacterial ligands for asialoGM1) have decreased epithelial cell adhesion, no compensation for the loss of twitching or swimming motility was made and no isogenic strains complemented for the genetic defect were evaluated. Thus, decreased epithelial cell binding of these mutants cannot be attributed to the adherence properties of pili or flagella. No confirmation of this aspect of P. aeruginosa infection in vivo has been published, and several investigators have failed to confirm these in vitro findings8xde Bentzmann, S. et al. Infect. Immun. 1996; 64: 1582–1588PubMedSee all References, 9xPlotkowski, M.C. et al. J. Med. Microbiol. 1992; 36: 104–111Crossref | PubMedSee all References.Similarly, data supporting a role for antimicrobial peptides, particularly salt-sensitive defensins, in hypersusceptibility of CF patients to P. aeruginosa infection are limited to in vitro findings. In addition, recent studies on the salt composition of airway surface liquid indicate that no compositional differences exist between CF and non-CF individuals10xKnowles, M.R. et al. J. Clin. Invest. 1997; 100: 2588–2595Crossref | PubMedSee all References10; in both situations, the salt content is inhibitory to the function of salt-sensitive defensins. The antimicrobial peptide hypothesis further suffers from the lack of specificity of this defect for P. aeruginosa and therefore does not explain the overwhelming predilection of CF patients for mostly chronic P. aeruginosa lung infections. In spite of the isolation of Staphylococcus aureus, Haemophilus influenzae and other pathogens from CF patients, studies have clearly shown it is essentially only mucoid P. aeruginosa lung infection that gives rise to long-term morbidity and most of the ultimate mortality in CF patients11xDemko, C.A., Byard, P.J., and Davis, P.B. J. Clin. Epidemiol. 1995; 48: 1041–1049Abstract | Full Text PDF | PubMed | Scopus (179)See all References11.While our hypothesis about the binding and entry of P. aeruginosa into epithelial cells via CFTR as a critical host response for clearance of this organism from the lung needs further study and confirmation, it is the only hypothesis put forward to date that accounts for the finding that 70–90% of CF patients are infected with, and die from, a single pathogen, P. aeruginosa, and only rarely from infection with other pathogens. P. aeruginosa is the only major lung pathogen we have found that binds to CFTR, and lack of this critical host defense for P. aeruginosa might be a key component of the pathogenesis of CF in the 20th century.