The CFTR inhibitory factor (Cif) is a protein

virulence factor Virulence factors (preferably known as pathogenicity factors or effectors in botany) are cellular structures, molecules and regulatory systems that enable microbial pathogens (bacteria, viruses, fungi, and protozoa) to achieve the following: * c ...

secreted by the

Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...

Pseudomonas aeruginosa ''Pseudomonas aeruginosa'' is a common Bacterial capsule, encapsulated, Gram-negative bacteria, Gram-negative, Aerobic organism, aerobic–facultative anaerobe, facultatively anaerobic, Bacillus (shape), rod-shaped bacteria, bacterium that can c ...

'' and '' Acinetobacter nosocomialis''. Discovered at

Dartmouth Medical School The Geisel School of Medicine is the medical school of Dartmouth College located in Hanover, New Hampshire. The fourth oldest medical school in the United States, it was founded in 1797 by New England physician Nathan Smith. It is one of the sev ...

, Cif is able to alter the trafficking of select

ABC transporters The ABC transporters, ATP synthase (ATP)-binding cassette transporters are a transport system superfamily that is one of the largest and possibly one of the oldest gene families. It is represented in all extant phyla, from prokaryotes to huma ...

in eukaryotic

epithelial cell Epithelium or epithelial tissue is a thin, continuous, protective layer of Cell (biology), cells with little extracellular matrix. An example is the epidermis, the outermost layer of the skin. Epithelial (Mesothelium, mesothelial) tissues line ...

s, such as the

cystic fibrosis transmembrane conductance regulator Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane protein and anion channel in vertebrates that is encoded by the ''CFTR'' gene. Geneticist Lap-Chee Tsui and his team identified the ''CFTR'' gene in 1989 as the gene lin ...

(CFTR), and

P-glycoprotein P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp or Pgp) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) or cluster of differentiation 243 (CD243) is an important protein ...

by interfering with the host deubiquitinating machinery. By promoting the

ubiquitin Ubiquitin is a small (8.6 kDa) regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ''ubiquitously''. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 19 ...

-mediated degradation of CFTR, Cif is able to phenocopy

cystic fibrosis Cystic fibrosis (CF) is a genetic disorder inherited in an autosomal recessive manner that impairs the normal clearance of Sputum, mucus from the lungs, which facilitates the colonization and infection of the lungs by bacteria, notably ''Staphy ...

at the cellular level. The ''cif'' gene is transcribed as part of a 3 gene

operon In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter. The genes are transcribed together into an mRNA strand and either translated together in the cytoplasm, or undergo splic ...

, whose expression is negatively regulated by CifR, a

TetR Tet Repressor proteins (otherwise known as TetR) are proteins playing an important role in conferring antimicrobial resistance, antibiotic resistance to large categories of bacterial species. Tetracycline (Tc) is a broad family of antibiotics to w ...

family repressor.

Cellular mechanism of action

Cif was first discovered by co-culturing ''P. aeruginosa'' with human airway epithelial cells and monitoring the resulting effect on chloride ion efflux across a polarized monolayer. After co-culture, the CFTR specific chloride ion efflux was found to be drastically reduced. This was determined to be caused by reduced levels of CFTR at the apical surface of these cells. This effect was later found to be the result of a single secreted protein produced by ''P. aeruginosa'', which was named the CFTR inhibitory factor for this initial

phenotype In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology (physical form and structure), its developmental processes, its biochemical and physiological propert ...

. Cif is secreted by ''P. aeruginosa'' PA14 as soluble protein as well as packaged into

outer membrane vesicles Outer membrane vesicles (OMVs) are vesicles released from the outer membranes of Gram-negative bacteria. While Gram-positive bacteria release vesicles as well, those vesicles fall under the broader category of bacterial membrane vesicles (MVs ...

(OMV). Cif is far more potent when applied in OMVs, likely due to efficiency of delivery. Purified, recombinant Cif protein can be applied to polarized monolayers of mammalian cells and promote the removal of CFTR and P-glycoprotein from the

apical membrane The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extra ...

. Cif accomplishes this by interfering with the host deubiquitylation system.

Epoxide hydrolase enzyme mechanism

Cif is an

epoxide hydrolase Epoxide hydrolases (EHs), also known as epoxide hydratases, are enzymes that metabolize compounds that contain an epoxide residue; they convert this residue to two hydroxyl residues through an epoxide hydrolysis reaction to form diol products. ...

(EH) with unique substrate selectivity. Cif is the first example of an EH serving as a virulence factor. Based on structural comparison, it appears that the enzyme utilizes a catalytic triad of residues Asp129, Glu153 and His297, with accessory residues His177 and Tyr239 coordinating the epoxide oxygen during ring opening. Cif is also the first example of an EH utilizing a His-Tyr pair to coordinate an epoxide substrate, rather than the canonical Tyr-Tyr pair. In the proposed enzyme mechanism, Asp129 nucleophilically attacks a carbon of the epoxide moiety of a substrate, forming an ester linked enzyme-acyl intermediate. The preference for which carbon is attacked varies depending upon the substrate. In the second step of the reaction, a water molecule is activated by the charge-relay His297-Glu153 pair, and undergoes nucleophilic attack on the Cγ of Asp129. This hydrolyzes the ester group, liberating the hydrolysis product as a vicinal diol.

Structure

Cif belongs to the α/β hydrolase family of proteins. Its structure was determined by X-ray crystallography and consists of the canonical α/β hydrolase fold with a cap domain, which it uses to constitutively homo-dimerize in solution. The active site is buried in the interior of the protein at the interface between the α/β hydrolase core and the cap.

References

{{Reflist Virulence factors Cystic fibrosis