ABCMdb

PMID: 19386787

Bajmoczi M, Gadjeva M, Alper SL, Pier GB, Golan DE
Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa.
Am J Physiol Cell Physiol. 2009 Aug;297(2):C263-77. Epub 2009 Apr 22., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Trp1282* To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (⌬F508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)- CFTR with an NH2-terminal green fluorescent protein (GFP) tag. Login to comment 10 ABCC7 p.Trp1282* ABCC7 p.Trp1282* To investigate the molecular bases for the hypersusceptibility of CF patients to P. aeruginosa, we used the IB3-1 cell line with two defective CF transmembrane conductance regulator (CFTR) genes (⌬F508/W1282X) to generate isogenic stable, clonal lung epithelial cells expressing wild-type (WT)- CFTR with an NH2-terminal green fluorescent protein (GFP) tag. Login to comment 12 ABCC7 p.Trp1282* P. aeruginosa internalization, a component of effective innate immunity, required functional CFTR and caveolin-1, as shown by: 1) direct correlation between GFP-CFTR expression levels and P. aeruginosa internalization; 2) enhanced P. aeruginosa internalization by aminoglycoside-induced read through of the CFTR W1282X allele in IB3-1 cells; 3) decreased P. aeruginosa internalization following siRNA knockdown of GFP-CFTR or caveolin-1; and 4) spatial association of P. aeruginosa with GFP-CFTR and caveolin-1 at the cell surface. Login to comment 32 ABCC7 p.Trp1282* To explore in detail the properties of CFTR that are essential to mount a rapid innate immune response to P. aeruginosa and to identify the spatial interactions of CFTR, caveolin-1, and P. aeruginosa throughout the course of bacterial attachment and internalization, we have developed a series of nominally isogenic, clonal bronchial epithelial cell lines stably expressing a green fluorescent protein (GFP)-NH2-terminal- labeled WT-CFTR, using as a parental cell line the IB3-1 cells initially obtained from a CF patient with ⌬F508/W1282X cftr alleles. Login to comment 34 ABCC7 p.Trp1282* To explore in detail the properties of CFTR that are essential to mount a rapid innate immune response to P. aeruginosa and to identify the spatial interactions of CFTR, caveolin-1, and P. aeruginosa throughout the course of bacterial attachment and internalization, we have developed a series of nominally isogenic, clonal bronchial epithelial cell lines stably expressing a green fluorescent protein (GFP)-NH2-terminal- labeled WT-CFTR, using as a parental cell line the IB3-1 cells initially obtained from a CF patient with ⌬F508/W1282X cftr alleles. Login to comment 49 ABCC7 p.Trp1282* IB3-1 cells (⌬F508/W1282X), derived from the bronchial epithelium of a CF patient, have been immortalized by stable infection with adeno-12-SV-40 (78). Login to comment 51 ABCC7 p.Trp1282* IB3-1 cells (⌬F508/W1282X), derived from the bronchial epithelium of a CF patient, have been immortalized by stable infection with adeno-12-SV-40 (78). Login to comment 149 ABCC7 p.Trp1282* A series of stable transfectants (C clones), each originating from a single transfected cell, was derived by introducing a GFP-CFTR expression vector into the human CF bronchial epithelial cell line IB3-1 (⌬F508/W1282X) and selecting for resistance to the G418 aminoglycoside antibiotic. Login to comment 151 ABCC7 p.Trp1282* A series of stable transfectants (C clones), each originating from a single transfected cell, was derived by introducing a GFP-CFTR expression vector into the human CF bronchial epithelial cell line IB3-1 (⌬F508/W1282X) and selecting for resistance to the G418 aminoglycoside antibiotic. Login to comment 246 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Treatment of IB3-1 cells with an aminoglycoside (G-418 or gentamicin) increases levels of W1282X-CFTR mRNA, with increased production of CFTR due to read through of the W1282X stop mutation and parallel induction of cAMP-dependent Cl- efflux in treated cells (6, 40). Login to comment 248 ABCC7 p.Trp1282* ABCC7 p.Trp1282* Treatment of IB3-1 cells with an aminoglycoside (G-418 or gentamicin) increases levels of W1282X-CFTR mRNA, with increased production of CFTR due to read through of the W1282X stop mutation and parallel induction of cAMP-dependent Cl-efflux in treated cells (6, 40). Login to comment 252 ABCC7 p.Trp1282* In IB3-1 cells, the G-418-induced increase in functional CFTR correlated with a statistically significant increase in P. aeruginosa invasion levels (Supplemental Fig. 5B), providing further evidence for the importance of functional CFTR in mediating the efficient uptake of P. aeruginosa in IB3-1 cells. Although G-418 was included in the medium for the routine growth of C clones, the antibiotic was removed 3-4 days before experimentation to minimize unintended expression of endogenous CFTR protein from the native W1282X allele at the time of the experiment. Login to comment 254 ABCC7 p.Trp1282* In IB3-1 cells, the G-418-induced increase in functional CFTR correlated with a statistically significant increase in P. aeruginosa invasion levels (Supplemental Fig. 5B), providing further evidence for the importance of functional CFTR in mediating the efficient uptake of P. aeruginosa in IB3-1 cells. Although G-418 was included in the medium for the routine growth of C clones, the antibiotic was removed 3-4 days before experimentation to minimize unintended expression of endogenous CFTR protein from the native W1282X allele at the time of the experiment. Login to comment 275 ABCC7 p.Trp1282* DISCUSSION We have examined the mechanism of P. aeruginosa entry into an isogenic set of clonal cell lines derived from the human CF bronchial epithelial cell line IB3-1 (⌬F508/W1282X). Login to comment 277 ABCC7 p.Trp1282* DISCUSSION We have examined the mechanism of P. aeruginosa entry into an isogenic set of clonal cell lines derived from the human CF bronchial epithelial cell line IB3-1 (⌬F508/W1282X). Login to comment