Specific Resistance to Pseudomonas aeruginosa Infection in Zebrafish Is Mediated by the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

Cystic fibrosis (CF) is a genetic disease caused by recessive mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is associated with prevalent and chronic Pseudomonas aeruginosa lung infections. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease, particularly infection by P. aeruginosa. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, but this phenomenon was not observed with the other bacterial species. Bacterial burden in Cftr morphants infected with a P. aeruginosa ⌬LasR mutant, a quorum sensing-deficient strain, was comparable to that in control fish, indicating that the regulation of virulence factors through LasR is required for enhancement of infection in the absence of Cftr. The zebrafish system provides a multitude of advantages for studying the pathogenesis of P. aeruginosa and for understanding the role that innate immune cells, such as neutrophils, play in the host response to acute bacterial infections commonly associated with cystic fibrosis.Cystic fibrosis (CF) is a multiorgan genetic disorder characterized by chronic pulmonary infections as well as gastrointestinal and reproductive abnormalities. In 1989, it was discovered that CF results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (25), which encodes a chloride ion channel present in most epithelial cells. Today, 80 to 95% of CF patients succumb to respiratory failure resulting from chronic lung infections that begin in early childhood and that are caused primarily by Pseudomonas aeruginosa (7, 32). Chronic infection with the bacterium leads to hyperinflammation characterized by the excessive recruitment of neutrophils to the airways and the subsequent release of oxidants and enzymes from activated neutrophils, which damages the lung tissue over time (32).Pseudomonas aeruginosa is a flagellated Gram-negative opportunistic pathogen of plants and animals and is found naturally in soil and water. As a nosocomial pathogen, it is the third most commonly isolated organism, leading primarily to urinary tract or wound infections in immunocompromised patients (17). Due to its production of alginate, the low permeability of its outer membrane and the presence of multidrug efflux pumps, P. aeruginosa exhibits a high degree of ...

Does the ΔF508-CFTR mutation induce a proinflammatory response in human airway epithelial cells?

“…An indolent response to P. aeruginosa associated with the absence of CFTR has also been observed in mice and zebrafish. Murine models of CF reveal reduced bacterial clearance of P. aeruginosa in CFTR (Ϫ/Ϫ) mice (34), whereas, in zebrafish morpholino, knockdown of CFTR suppressed the innate immune response to P. aeruginosa, including decreasing the production of reactive oxygen species and reducing neutrophil migration, resulting in an increase in the load of P. aeruginosa (29). Thus both in vivo models recapitulate the hyperinflammatory response to P. aeruginosa observed in the human CF lung compared with the non-CF lung.…”

“…Approximately 90% of patients with CF have at least one ⌬F508 CFTR allele, which codes for a protein that misfolds in the endoplasmic reticulum and is subsequently degraded by the proteasome, preventing it from trafficking to the plasma membrane, where it functions as a secretory chloride ion channel in a variety of tissues including the lung, pancreas, and intestine (32). CFTR is also expressed in immune cells including neutrophils and macrophages, and reduction in CFTR expression in zebrafish reduces the ability of neutrophils to phagocytose and kill Pseudomonas aeruginosa (P. aeruginosa) (29). Individuals with the ⌬F508 CFTR mutation exhibit defects in pulmonary host defense mechanisms that lead to chronic bacterial infection, primarily P. aeruginosa, and inflammation, resulting in a progressive decline in lung function and death before an average age of 38 (27).…”

CFTR: cystic fibrosis and beyond

“…neutrophils and macrophages, that accumulate in CF airways and are thought to drive chronic inflammation have been shown to express CFTR mRNA and protein and may utilise CFTR for proper phagolysosomal function and intracellular bacterial killing [117][118][119]. While our current understanding predicts that CFTR malfunction in airway epithelia is critical for triggering the disease, emerging studies with conditional deletion of CFTR in myeloid cells in mice [120] and studies with morpholino knock-down technology in zebrafish models [121] support the notion that CFTR dysfunction in myeloid cells may contribute to the pathogenesis and outcome of P. aeruginosa infection in CF lung disease. Beyond phagocytes, a potential role of CFTR in innate immunity includes other immune cell types, such as dendritic cells and natural killer T-cells, a topic discussed in more depth previously [115].…”

mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish

“…Detailed examination of inflammation and host-pathogen interactions at the cellular level has been enabled by several transgenic strains marking embryonic and adult neutrophils, such as Tg(mpx:EGFP) 3,4 and lyz-driven transgenes. 5,6 Such lines have been used to study neutrophil responses and fluxes during infection 7,8 and during acute and chronic inflammation. 3,4,9 Combinations of these approaches have provided some important new insights (eg, regarding the role of leukocytes in mycobacterial infection).…”