cystic fibrosis ( cf ) is an inherited disease resulting in the formation of thick , sticky mucus in the lungs and digestive pathways . pseudomonas aeruginosa is the most common bacterial isolate that persists in cf lungs by evading host defenses and clearance mechanisms , contributing to declining lung function [ 1 , 2 ] . in order to grow and thrive , however , in the case of infection , iron is not freely available in the host and is predominantly found in iron - binding proteins such as transferrin , lactoferrin , and ferritin . thus , due to the low bioavailability of free iron , bacteria produce iron - chelating small molecules known as siderophores that are secreted in apo - form and imported as ferric complexes [ 1 , 4 ] . p. aeruginosa produces two siderophores , pyoverdine ( figures 1(a ) , and 1(b ) ) and pyochelin , that are found to be important for biofilm development and bacterial virulence . as a host defense strategy , mammals have evolved strategies to limit bacterial growth by producing antibacterial iron - depleting innate immune defense proteins , such as neutrophil - gelatinase - associated lipocalin ( ngal ) , that specifically scavenge bacterial ferric and apo - siderophores , therefore preventing bacteria from establishing infection [ 5 , 6 ] . ngal is a 25  kda protein also known as siderocalin , lipocalin 2 , uterocalin , and 24p3 , that exhibits antibacterial activity . ngal - deficient mice are more susceptible to bacterial infections   and ngal is required for pulmonary host defense against klebsiella pneumoniae . lipocalins such as ngal share a common tertiary structure , the so - called  lipocalin fold ,  characterized by an eight - strand antiparallel -barrel with a calyx that contains the ligand - binding site . ngal is expressed and secreted upon stimulation by many cells and also acts as a growth and differentiation factor in various tissues , mainly in the kidney . one study has shown that ngal levels are highly elevated in cf serum and bronchoalveolar lavage fluid ( balf ) . in spite of the abundance of ngal in serum and balf of cf patients , p. aeruginosa infections persist for many years . pathogenic bacteria evolved under the pressure of iron limitation to survive and evade host immune systems by producing structurally modified siderophores that do not bind to ngal . pathogenic gram - negative bacteria harbor the iroa gene cluster that mediates bacterial evasion of ngal [ 12 , 13 ] . for example , salmonella typhimurium , a gram - negative bacterium , produces a glycosylated enterobactin known as salmochelin [ 11 , 14 ] . no homologs of the iroa gene cluster were found in the published pseudomonas genomes , although p. aeruginosa produces three types of pyoverdines . for example , bacillus anthracis , a gram - positive bacterium , produces a siderophore known as petrobactin that does not bind to ngal ; klebsiella pneumonia , a gram - negative bacterium , produces a siderophore known as yersiniabactin   that also does not bind to ngal and thereby evades the host 's antibacterial iron - depleting defense . further , vibrio cholerae produces vibriobactin , a catecholate siderophore that evades ngal recognition by unique iron coordination in the ferric vibriobactin form . it is not known if pyoverdine , the major siderophore produced by p. aeruginosa , evades this innate defense mechanism and whether this contributes to inability of cf patients to clear this bacterium from their lungs . therefore , the aim of this work was to determine if pyoverdine binds to the antibacterial protein ngal . we hypothesized that ngal does not bind pyoverdine , allowing p. aeruginosa to establish infections and persist in cf lungs . hence , this iron - depleting innate host defense mechanism may be dysfunctional and impaired in cf . in this paper we employed a fluorescence - based assay to characterize the binding of pyoverdine to human ngal and molecular modeling of ngal pyoverdine complexes to predict possible protein - ligand interactions . here we report that neither apo- nor ferric pyoverdine bind to ngal , results that are further supported by computational predictions indicating lack of interaction of pyoverdine with ngal at its conventional ligand - binding site . we conclude that pyoverdine is a stealth siderophore that evades ngal recognition , thereby allowing p. aeruginosa to establish infections . 1757-lc ) produced in a mammalian system rather than a bacterial source was purchased from r&d systems ( minneapolis , mn , usa ) . highly purified and structurally defined pyoverdines were obtained from sigma - aldrich ( st louis , mo , usa ) in apo - form , that is , aferric pyoverdine ( catalog no . 0926  kb ) , and sterile bovine serum albumin ( bsa , endotoxin free tissue culture grade , cas 9048468 ) were purchased from sigma - aldrich . human ngal contains two tryptophan residues : w31 located at the base of the hydrophobic calyx and w79 located inside the calyx ( i.e. , the ligand - binding site ) . therefore , we employed an established tryptophan fluorescence - quenching assay [ 13 , 15 ] to determine the binding of pyoverdine to ngal . since 2,3-dhba is the core functional moiety in pyoverdine that binds iron , the fluorescence assay was first optimized by testing ferric iron and 2,3-dhba complexes diluted in 0.1  m tris , ph 8.0 , as the ligand for potentially quenching intrinsic tryptophan fluorescence in rhngal and in sterile bsa . enterobactin ( ent ) , the major siderophore from e. coli and other enteric bacteria , was used as a positive control . the enterobactin stock solution was dissolved in dmso , whereas apo - pyoverdine ( pvd ) and ferric pyoverdine ( pvd - fe ) stocks were dissolved in sterile water ; then , serial dilutions of the siderophores at 100 , 50 , 25 , and 12.5  m were made in an assay buffer ( 50  mm tris , 10  mm cacl2 , 150  mm nacl ph 7.5 ) . fifty l of each dilution was then transferred into triplicate wells ( f16 black maxisorp plates from nunc ) . fifty l of rhngal was added into each well at a final concentration of 2  m and incubated for 30  min at room temperature . recombinant hngal alone was incubated with assay buffer without any siderophore and used to measure intrinsic tryptophan fluorescence . the plate was then read at excitation and emission wavelengths of 280 and 340  nm , respectively , in the endpoint mode using a microplate reader with fluorescence detection . results are presented as the percent of tryptophan fluorescence remaining compared to rhngal alone . among 18 published crystal structures of ngal , we selected two human ngal structures solved by strong and coworkers [ 18 , 19 ] to serve as the basis for the molecular modeling study files 1l6 m and 1qqs from the protein data bank ( pdb ) . for simplicity , we refer to the 1l6 m structure as the ngal trimer  and the 1qqs structure as the ngal  monomer  in this paper . both structures were solved to 2.4   resolution , the highest resolution solved to date for an ngal structure bound to enterobactin , and in the same space group ( p41212 ) . the two structures are virtually identical in terms of conformation even though differences were observed in crystal packing in spite of matching space groups . three monomers were observed in the 1l6 m structure while the 1qqs structure was a homodimer with only one monomer . moreover , ngal has been found in a 25  kd monomeric and 45  kd dimeric biological forms , as well as a 135  kd heterodimer with mmp9 [ 2123 ] . due to the numerous structural similarities , minor distinctions in crystal packing , and the availability of different quaternary forms of ngal , the 1l6 m and 1qqs structures were selected as interesting models for predicting different types of interactions that pyoverdine might be able to make with ngal and for proposing structure - activity relationships . another important structural feature relevant to this study is the presence and location of tryptophan residues in the ngal structures . however , coordinates for the ngal monomer ( 1qqs ) contained a w79a where the side chain of w79 was disordered in the structure and modeled as an alanine . otherwise , all other tryptophans present in the crystal structures were used for molecular modeling . covalent attachment of the sugars n - acetyl - d - glucosamine and -d - mannose and the lipid decanoic acid to the monomer protein were retained . polar hydrogens were added to each protein and each set of modified pdb coordinates was converted to pdbqt format using autodock tools . pdbqt format includes information about partial atomic charges and atom types in addition to the otherwise conventional pdb format . coordinates for ferric pyoverdine ( pvd - fe ) were extracted from pdb file 2w78 , which describes a complex between iron - bound pvd - atcc13535 and its transporter protein from p. aeruginosa . the iron atom was excluded from the working ferric pyoverdine file to generate the analogous coordinates for pyoverdine without iron bound ( i.e. , apo - pyoverdine ) . other heteroatoms present in the 3cmp file including the sodium and sulfate counterions were excluded . prior to the docking experiment , a grid box was selected in autodock tools to encompass all atoms within either the modified ngal monomer or trimer structure . the search space for autodock vina was defined to be the dimensions of the 3d grid box . ngal is an antibacterial innate defense protein that limits bacterial growth by binding to and sequestering siderophores . we hypothesized that ngal does not bind pyoverdine , allowing p. aeruginosa to persist in cf lungs . to test this hypothesis , we measured the direct binding of pyoverdine ( figure 1 ) to rhngal using the tryptophan fluorescence - quenching method . human ngal contains two tryptophan residues : w31 , which is located at the base of the hydrophobic calyx , and w79 , located at the ligand - binding site . if pyoverdine binds to ngal it would be expected to cause the quenching of the intrinsic fluorescence of tryptophan . the quenching of the intrinsic tryptophan fluorescence of rhngal in a dose - dependent manner was first optimized using iron complexed to 2,3-dihydroxybenzoic acid ( fe - dhba ) ( figure 2(a ) ) . as expected , increasing concentrations of fe - dhba led to increased quenching , suggesting binding of the siderophore core iron - binding structure to ngal . as another positive control , enterobactin induced a response similar to that of fe - dhba . ferric enterobactin - ngal complexes showed a fluorescence decrease as the siderophore concentration increased , indicating fluorescence quenching and binding of the ligand to ngal ( figure 2(b ) ) . in contrast to enterobactin , neither apo - pyoverdine nor ferric pyoverdine reduced the intrinsic tryptophan fluorescence of rhngal significantly ( figure 2(b ) ) . tryptophan fluorescence was only diminished by less than 10% of the ngal baseline fluorescence even when a high dose of pyoverdine was used , suggesting that pyoverdine does not bind to ngal even when complexed with iron which is known to increase the affinity of siderophore binding to ngal . thus , ngal does not exert an antibacterial function against p. aeruginosa and , therefore , pyoverdine is a stealth siderophore . docking of apo - pyoverdine ( figure 1(a ) ) to the ngal trimer predicted nine different docking sites or modes ( figure 3(a ) ) . 1 , 2 , 4 , 5 , 7 , 8 , figure 3 ) clustered at an interface between subunits within the ngal trimer . 3 , 6 , 9 , figure 3 ) were at the periphery of a subunit near the ngal calyx without being fully buried within the hydrophobic binding pocket . when apo - pyoverdine docked at the periphery of a subunit , the ligand was also near a tryptophan residue . docking modes 6 and 9 for apo - pyoverdine were in proximity to both w31 and w79 as best seen in the 180 rotational view ( figure 3 , right ) . in docking mode 3 , all nine of the docking modes for the apo - pyoverdine ligand localized to the outer surface of the ngal monomer ( data not shown ) . in eight of the nine docking modes , apo - pyoverdine docked near the opening of the calyx but not within the ligand - binding pocket . as with the apo - pyoverdine structures , autodock vina predicted nine positions in which ferric pyoverdine could dock into the ngal trimer ; however , none of the ferric pyoverdine molecules were predicted to bind near or inside ngal 's ligand binding pocket ( figure 4(a ) ) . further , autodock vina predicted nine positions in which ferric pyoverdine could dock onto the ngal monomer structure . in each case , however , the position of the docked ligand was at the surface of the protein and not near or inside the calyx ( figure 4(b ) ) . this computational data strongly suggests that ferric pyoverdine does not bind to ngal , which is consistent with the experimental results . enterobactin , the major siderophore in e. coli and many other pathogenic enteric bacteria , binds to ngal with very high affinity [ 18 , 28 ] . the binding and molecular docking of ferric enterobactin to ngal is well established and extensively investigated . in order to validate the docking results obtained for pyoverdine , ferric enterobactin ( figure 1(d ) ) was used as a positive control for computational docking to the ngal trimer ( figure 5 ) and monomer ( data not shown ) . autodock vina predicted 9 positions in which ferric enterobactin could dock into the trimeric ngal protein crystal structure pdb 1l6 m . seven different docking modes ( nos . 1 , 2 , 3 , 4 , 5 , 6 , and 9 ) indicate that ferric enterobactin could dock inside the ngal calyx as been reported in the crystal structure of ferric enterobactin - bound ngal . the remaining two docking modes ( nos . 7 and 8) predicted ferric enterobactin docking away from the calyx and between the ngal subunits ( figure 5 ) . overall , autodock vina accurately simulated the binding of ferric enterobactin to the ngal trimer at the ligand binding site ( i.e. , inside the calyx of ngal ) . further , ferric pyoverdine was superimposed on ferric enterobactin docked into ngal calyx with a transparent surface presentation . the results reflect again the inability of ferric pyoverdine to dock into the calyx ( figure 6 ) . the more negative the value , the tighter the predicted binding . for the apo - pyoverdine interaction with the ngal trimer , predicted binding affinities were very similar for the nine modes , ranging from 6.4 to 6.9  kcal / mol . predicted binding affinities for apo - pyoverdine with the ngal monomer ( 5.8 to 6.5  kcal / mol ) were slightly lower in range than those for the analogous trimer complex ( table 1 ) . data for ferric pyoverdine interactions gave slightly tighter predicted binding than that for apo - pyoverdine interactions : predicted binding energies of 8.2 to 9.2  kcal / mol for the trimer and 7.6 to 8.9  kcal / mol for the monomer . consistent with biological trends , iron - bound siderophores were predicted to have stronger binding interactions with ngal monomer or trimer than their corresponding apo - forms . in all permutations , docking mode 1 represented the highest affinity conformation of the ligand interacting with the ngal . in every case , docking mode 1 was computed to have a higher binding energy in interaction with the trimer over the monomer , although even this mode for pyoverdine gave low predicted affinity . in contrast , the tightest predicted binding affinities were computed for the ferric enterobactin interactions with the ngal trimer ( 9.7 to 11.8  kcal / mol ) . while all of the predicted binding affinities are moderate in scale , none of the pyoverdine values are comparable to the range predicted for ferric enterobactin binding with ngal . p. aeruginosa is an opportunistic pathogen that causes severe infection not only in cf patients but also in critically ill patients in intensive care units where it can cause ventilator - associated infections . it is a major nosocomial pathogen associated with significant morbidity and mortality ( 4060% ) in immunocompromised patients and in certain hospital units like burn and palliative care . p. aeruginosa possesses several virulence factors , one of which is pyoverdine , the major siderophore that sequesters iron from the host to facilitate bacterial growth [ 30 , 31 ] . pyoverdine is a virulence factor required for establishing infection and biofilm formation . in response to infection , the host secretes ngal that sequesters bacterial siderophores thereby preventing bacteria from establishing infection . the role of ngal in innate immunity is well established and although ngal - deficient mice develop normally , they are more susceptible to bacterial infections . ngal is required for pulmonary host defense against klebsiella pneumonaie , indicating that ngal is an important mediator in innate host defense . however , not all bacterial siderophores are recognized by ngal and some pathogenic bacteria modify their siderophore structure to evade ngal recognition [ 1113 , 16 , 17 ] . it has been shown that salmochelin , a major siderophore in pathogenic salmonella , is a glucosylated form of enterobactin [ 11 , 12 , 33 ] . the glycosylation renders the molecule bulky such that it would not fit within the ngal calyx , thus evading innate immune defense . p. aeruginosa does not contain the iroa gene cluster responsible for the glycosylation of catecholate - based siderophores that do not bind to ngal [ 1 , 3 ] . compared to other dihydroxybenzoic - acid-(dhba- ) based catecholates that bind to ngal such as enterobactin , pyoverdine is a structurally bulky molecule due to its dihydroxyquinoline chromophore . we postulated that pyoverdine is a stealth siderophore that does not bind to ngal . here we show that pyoverdine evades ngal recognition and therefore likely facilitates iron acquisition by p. aeruginosa which supports establishing infection . , we found that pyoverdine did not bind to ngal , neither in the apo - form nor when complexed with iron . in contrast , enterobactin bound to ngal causing strong tryptophan quenching . using molecular docking we showed that pyoverdine did not dock into the calyx of ngal while identical approaches indicate that enterobactin docks to ngal with high affinity . we conclude that pyoverdine is a virulence factor and a stealth siderophore which allows p. aeruginosa to establish infection . lipocalins act as carrier proteins for a variety of ligands including small - molecule drugs [ 34 , 35 ] . other members of the lipocalin family have also been shown to act as siderocalins , that is , binding and sequestering bacterial siderophores [ 36 , 37 ] . tear lipocalin , also called lcn1 , has been shown to sequester enterobactin but not pyoverdine which lends strong support to our results [ 38 , 39 ] . unlike ngal that preferably binds catecholate - based siderophores , tear lipocalin can bind wide range of bacterial and fungal siderophores including catecholate - based and hydroxamate - based siderophores . in contrast to ngal and tear lipocalin , galline ex - fabp , a lipocalin expressed in chicken serum and egg white , has been reported to sequester both glucosylated enterobactin and pyoverdine . it is also a sensor for lysophosphatidic acid and is reported to have dual ligand specificities . the recently published crystal structure of galline ex - fabp explained the dual ligand specificity due its expanded multichambered cavity . ngal is reported to be a rigid and quite stable lipocalin with a smaller calyx that does not undergo conformational changes upon ligand binding [ 18 , 19 ] which explains why pyoverdine binds to ex - fabp but not to ngal . similar to ngal , galline ex - fabp was shown to exert strong bacteriostatic activity against e. coli and b. subtilis but not against pseudomonas aeruginosa due to low affinity of interaction between ex - fabp and ferric pyoverdine . we also performed similar bacterial growth inhibition assay with ngal and p. aeruginosa and found only 10% reduction in bacterial growth ( data not shown ) ; therefore we concluded that ngal is not bacteriostatic for p. aeruginosa . p. aeruginosa produces another siderophore called pyochelin which is a small chatecholate - based molecule with iron - binding affinity . recently , strong and coworkers crystallized ngal bound to pyochelin ( pdb 3u03 , deposited at the rcsb protein data bank ) and the crystal structure clearly shows that pyochelin docks inside the calyx of ngal but without binding avidly . however , pyochelin is not essential for establishing infection since a p. aeruginosa mutant that lacks pyochelin successfully established infection and formed a biofilm . chronic infections caused by p. aeruginosa as seen in cf are associated with biofilms that protect the bacteria against the host killing mechanisms as well as increase resistance to antibiotics [ 4 , 30 ] . in established biofilms of cf airways , p. aeruginosa thrives and downregulates pyoverdine since iron is abundant in the airway surface liquid due to chronic inflammation [ 41 , 42 ] . therefore , pyoverdine is important for establishing infections but not for well - established infections like that in biofilms .   in summary , we show that pyoverdine , the major siderophore in p. aeruginosa , is a stealth siderophore that evades ngal recognition .