ThePseudomonas aeruginosaSecreted Protein PA2934 Decreases Apical Membrane Expression of the Cystic Fibrosis Transmembrane Conductance Regulator

Abstract

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl ؊ ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in ␣/␤ hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0–1

“…2A) (15). Little to no c-di-GMP was bound by heat-denatured protein, resin alone, or an unrelated, histidinetagged protein (PA2934his) (21). The addition of unlabeled c-di-GMP blocked [ 32 P]-c-di-GMP binding, whereas 1000-fold excess of GTP had no effect, demonstrating a specific interaction between LapD and c-di-GMP (Fig.…”

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

“…The effects of Cif virulence factors on CFTR levels were monitored after applying purified Cif or aCif protein to the apical surface of polarized cells, followed by domain-selective cell-surface biotinylation using EZ-Link TM Sulfo-NHS-LC-Biotin (Pierce), pull-down using streptavidin-conjugated agarose, and quantitative Western blot for CFTR, as described previously (20,43,44).…”

“…Sequence and structural homologies indicate that Cif adopts an ␣/␤ hydrolase-fold, a scaffold commonly associated with hydrolytic enzymes (20,27). Based on alignments to other ␣/␤ hydrolase family members, it was originally proposed to act as an epoxide hydrolase (EH).…”

“…They also deploy a variety of highly sophisticated virulence factors with more specialized targets. Among these is the CFTR inhibitory factor (Cif), a recently characterized P. aeruginosa protein expressed both in the PA14 strain and in clinical isolates (20,21). Originally characterized as an extracellular factor that inhibited CFTR-mediated chloride currents across polarized epithelial monolayers (22), Cif is both directly secreted and is also packaged into outer membrane vesicles that dramatically enhance delivery to airway epithelial cells (23).…”

Pseudomonas aeruginosa – Pathogenesis and Pathogenic Mechanisms

“…It is dominant pathogen in chronic lung infection in CF. In the majority of cases, colonization of the CF airway by P. aeruginosa leads to a chronic infection that is resistant to antimicrobial therapy (MacEachran et al, 2007;Döring et al, 2014). Chronic colonization and infection with P. aeruginosa is an inevitable reality for the majority of adults with CF, as over 80% of adults over the age of 18 years return positive cultures for P. aeruginosa (Hodson, 2000).…”