Human Tear Lipocalin Exhibits Antimicrobial Activity by Scavenging Microbial Siderophores

Human tear lipocalin (TL; also known as Lcn1) is a secretoryprotein present in large amounts in fluids that cover epithelialsurfaces such as tears and respiratory secretions. It is supposedto act as a physiological scavenger of hydrophobic, potentiallyharmful molecules, but there is evidence that it also inhibitsbacterial growth. In the present study, we reconsidered thepossibility that TL might interfere with microbial growth byscavenging of siderophores, as described for human neutrophilgelatinase-associated lipocalin (NGAL). Indeed, our experimentsrevealed that TL binds to microbial siderophores with high affinities.In contrast to NGAL, which was shown to have some specificityfor bacterial catecholate-type siderophores, TL binds to a broadarray of siderophores, including bacterial catecholate-typeenterobactin and hydroxamate-type desferrioxamine B, and allmajor classes of fungal siderophores. By adding exogenous TL,bacterial and fungal growth could be inhibited under iron-limitingconditions. Thus, TL might be a novel member of the innate immunesystem especially involved in mucosal defense against fungalinfections.

INTRODUCTION

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Introduction

Several epithelial surfaces are continuously exposed to potentialpathogens but rarely become infected because they are protectedby specific secretions. These secretions contain a number ofantimicrobial components and are constantly cleared by mechanicalaction. For instance, the human cornea has an elaborate systemof defense provided by the tear film, which contains an assortmentof proteins that derive antimicrobial activity by a varietyof mechanisms. Human tear lysozyme hydrolyzes the glycosidiclinkages in the polysaccharide components of the cell wall andkills Micrococcus leisodeikticus (6) and Bacillus subtilis (36).Secretory phospholipase A₂ hydrolyzes phospholipids found mainlyin microbial membranes and is the principal bactericide in tearsfor staphylococci and other gram-positive bacteria (32). Lactoferrininduces bacteriostasis by competing for free iron, but it alsohas bactericidal activity against several bacteria (2). Secretoryimmunoglobulin A inhibits the adhesion of specific bacteriaand protozoa to the corneal epithelium (1). Defensin peptides,which have a wide range of potential antimicrobial activitiesas a result of their formation of pores in the microbial cellmembrane (16), have also been found in human tear fluid (14).

There is circumstantial and somewhat controversial evidencefor the antibacterial activity of another protein found in highconcentrations in tear fluid and other secretions, namely, tearlipocalin (TL), also called von Ebner's gland protein or Lcn1(33, 35). TL is a member of the protein superfamily of lipocalinsand is typically characterized by its ability to bind to a largevariety of hydrophobic compounds, including fatty acids, phospholipids,glycolipids, cholesterol, retinol, arachidonic acid, and otherlipid peroxidation products (10, 20, 33). By scavenging thesemolecules, TL may act as a general protection factor for epithelialsurfaces (35).

Several facts prompted us to reconsider the antimicrobial activityof TL. First, there were some indications that its antibacterialactivity depends on the ligands bound (10). TL purified by gelfiltration showed antibacterial activity, which was not presentafter purification by reversed-phase high-pressure liquid chromatography(7, 36). Second, two other properties of TL might be relevantfor antibacterial activity: cysteine proteinase inhibition andnonspecific magnesium-dependent endonuclease activity (38, 42).Third, very recently it was found that human neutrophil gelatinase-associatedlipocalin (NGAL), another member of the lipocalin superfamily,avidly binds to the bacterial siderophore enterobactin and inhibitsbacterial growth by interference with siderophore-mediated ironacquisition by members of the family Enterobacteriaceae (12).

Here we show that TL exhibits its antimicrobial activity bybinding to microbial siderophores similarly to NGAL. However,in contrast to NGAL, TL was found to bind to a broad spectrumof siderophores, including all major classes of fungal siderophores.Growth inhibition tests showed that this siderophore bindingresults in a strong reduction of bacterial and fungal growthunder iron-limiting conditions.

MATERIALS AND METHODS

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Materials and Methods

Chemicals, siderophores, strains, and media. The fluorescent fatty acid derivative 11-({[5-(dimethylamino)-1-naphthalenyl]sulfonyl}amino)undecanoic acid (DAUDA) was purchased from Molecular Probes(Leiden, The Netherlands). The recombinant TL used for the bindingstudies was produced in Escherichia coli as described previously(15) and was delipidated by chromatography on Lipidex 1000 (Sigma,Vienna, Austria) (11). The siderophores enterobactin, pyoverdine,aerobactin, and coprogen were purchased from Biophore ResearchProducts (Tübingen, Germany). Ferricrocin and triacetylfusarinineC (TAFC) were purified from Aspergillus nidulans by high-pressureliquid chromatography by standard methods (18). DesferrioxamineB, rhodotorulic acid, and ferrichrome were obtained from Sigma.

The following organisms were used for microbial growth inhibitiontests: E. coli (XL1-Blue), Pseudomonas aeruginosa (ATCC 9027),and A. nidulans ${Delta}$ sidA (29). All bacteria were cultivated in M9minimal medium (which contained the following per liter: 200ml of 5x M9 minimal medium [64 g of Na₂HPO₄ · 7H₂O, 15g of KH₂PO₄, 2.5 g of NaCl, 5 g of NH₄Cl], 2 ml of 1 M MgSO₄,20 ml of 20% glucose, and 0.1 ml of 1 M CaCl₂) at 37°C.The fungi were cultivated as described by Pontecorvo et al.(30) at 30°C in minimal medium (MM) containing 1% glucoseas the carbon source and 20 mM glutamine as the nitrogen source.

Microbial growth inhibition tests. To test the bacteriostatic activity of TL, 500 µl of M9minimal medium in 48-well dishes (Nunc, Wiesbaden, Germany)was inoculated with a 1% overnight culture of E. coli or P.aeruginosa. TL was added at 5 µM, and the optical densityat 600 nm (OD₆₀₀) was measured after 10, 25, and 36 h. Assaysfor antifungal activity were performed on MM agar in six-wellplates (Techno Plastic Products, Trasadingen, Switzerland) containing50 nM TAFC or 50 nM TAFC that had been preincubated with 500nM TL in potassium phosphate (pH 7.2) for 15 min and then addedto the agar. Afterwards, 5 µl of a spore suspension (10⁵conidia/ml) was point inoculated. The plates were incubatedat 30°C.

Ligand binding assays. Binding of siderophores to TL was analyzed by displacement ofthe fluorescent fatty acid DAUDA as described previously (8).Experiments were carried out in a Hitachi F4500 fluorescencespectrophotometer with slits set at bandwidths of 1 and 5 nmfor emission and excitation, respectively, and at a constanttemperature (22°C). The excitation wavelength was 345 nm,and fluorescence was measured at 490 nm. The assay was carriedout in a final volume of 250 µl (20 mM potassium phosphate[pH 7.2]) containing 5 µM TL and 4 µM DAUDA. Exactly10 µl of an appropriately diluted competitor (from a 0.5mM stock solution of siderophores dissolved in potassium phosphate)was added. Determination of the kinetics of binding of TAFCto TL was performed by internal tryptophan fluorescence quenchinganalysis essentially as described previously for other lipocalins(25). Experiments were carried out in the same fluorescencespectrophotometer described above. Tryptophan fluorescence ofTL was excited at 295 nm, and emission was recorded at between330 and 350 nm. For each measurement 250 µl of proteinsolution (5 µM) in 20 mM potassium phosphate (pH 7.2)was used, and the solution was mixed with 1 µl of a dilutedligand. The set of datum points was analyzed by standard mathematicalmethods, and a curve was fitted by nonlinear hyperbolic regression.

Endpoint saturation. M9 minimal medium (500 µl) was inoculated with a 1% overnightE. coli culture containing 5 µM TL. Iron (FeCl₃) was addedin increasing concentrations: 0.1, 1, 5, and 50 µM. TheOD₆₀₀ was measured after incubation for 16 h at 37°C.

Atomic absorption measurements. All atomic absorption measurements were performed with a Unicam939 QZ atomic absorption spectrophotometer. Before the ironcontent of the probe was measured, a solution containing 2.4nM TAFC, 0.8 nM Fe (FeCl₃), and 2.4 nM TL was dialyzed in aSlide-A-Lyzer dialysis cassette (molecular weight cutoff, 3,500)obtained from Pierce (Rockford, Ill.) to remove the free ironin the buffer. Probes containing 2.4 nM TL in buffer and 2.4nM TAFC in buffer (20 mM potassium phosphate buffer [pH 7.2])were used as a control.

RESULTS

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Results

Binding of bacterial siderophores to TL. In general, the binding of siderophores was measured by competitiveDAUDA displacement assays. DAUDA, a fluorescent fatty acid analogue,exhibits little fluorescence in buffer but shows enhanced fluorescenceand a blue shift when it is bound to TL. It is known that DAUDAbinds to TL entirely within the hydrophobic pocket, at a singlebinding site, with a calculated dissociation constant (K_d) of2.8 µM (10). When DAUDA is displaced from TL, there isa corresponding decrease in intensity and a redshift of themaximum peak.

Most bacterial siderophores are divided into two groups on thebasis of their chemical compositions: hydroxamates and catecholates(27). Therefore, we tested enterobactin (26), the major catecholate-typesiderophore of E. coli, and desferrioxamine B (13), a hydroxamate-typesiderophore of Streptomyces (Fig. 1). In addition, aerobactin(9), another hydroxamate-type siderophore of enterobacteria,and pyoverdine (31), the hydroxamate-type siderophore of P.aeruginosa, were included in this study. Pyoverdins differ fromother hydroxamates by the additional attachment of a conserveddihydroxyquinoline chromophore (31).

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FIG. 1. Schematic structures of bacterial and fungal siderophores. Italicized siderophores are the desferri forms.

The displacement curves for various concentrations of ferricenterobactin, ferric aerobactin, and ferrioxamine B (iron-saturateddesferrioxamine B) are shown in Fig. 2, top panel. All threesiderophores were found to bind to TL. Ferric enterobactin anddesferrioxamine B showed similar affinities, which were higherthan that of ferric aerobactin. Pyoverdine could not be investigatedby this assay due to the intrinsic fluorescence of the attachedchromophore. Therefore, we investigated the binding of pyoverdineby obtaining atomic absorption measurements with Fe-saturatedpyoverdine. Whereas the binding of Fe-saturated enterobactincould be easily detected by this method, no binding of pyoverdinewas found (data not shown). Pyoverdine does not bind to TL dueto the additional bulky chromophore. In addition, no significantbinding of free iron to TL could be detected by atomic absorption.

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FIG. 2. (Top panel) Competitive binding assays of bacterial siderophores determined by measurement of displacement of DAUDA (4 µM) from TL (5 µM) by increasing concentrations of aerobactin ( ${diamondsuit}$ ), enterobactin ( ${blacksquare}$ ), and desferrioxamine B ( ${blacktriangleup}$ ). (Bottom panel) Competitive binding assays of fungal siderophores determined by measurement of displacement of DAUDA (4 µM) from TL (5 µM) by increasing concentrations of ferricrocin ( ${diamondsuit}$ ), ferrichrome ( ${circ}$ ), rhodotorulic acid (*), coprogen ( ${blacktriangleup}$ ), and TAFC ( ${blacksquare}$ ).

Binding of fungal siderophores to TL. All of the most relevant fungal siderophores belong to the groupof hydroxamates (Fig. 1). Therefore, rhodotorulic acid fromRhodotorula pilimanae (24), TAFC from A. nidulans (4, 29), coprogenfrom Neurospora crassa (37), and two ferrichromes, namely, ferrichromefrom Ustilago maydis and ferricrocin from A. nidulans (4, 21,29), were used in our binding studies. From the DAUDA displacementcurves in Fig. 2 (bottom panel), it is evident that all fungalsiderophores tested bind to TL. Rhodotorulic acid showed thehighest affinity to TL; TAFC, coprogen, and ferrichrome showedslightly lower affinities; and ferricrocin showed the lowestaffinity.

Since it is known that fatty acids are physiological ligandsof TL, we compared the K_d of TAFC, which shows an affinity toTL in the medium range, to the K_ds of fatty acids. By analysiswith internal tryptophan fluorescence quenching, a K_d of 0.5± 0.1 µM was obtained for TAFC (Fig. 3). One ofthe most strongly TL-binding fatty acids is stearic acid; however,its affinity to TL was not measured directly but was calculatedfrom relative displacement assays (10). Therefore, we performeda direct comparison of TAFC and stearic acid by displacementanalysis in the same series of experiments. The results of ourexperiments demonstrate that TAFC has only a slightly loweraffinity than the strongly binding ligand stearic acid (Fig.4).

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FIG. 3. Determination of the binding affinity of TL for TAFC by internal tryptophan fluorescence quenching experiments. The ligand concentration was plotted against the relative decrease in fluorescence.

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FIG. 4. Comparison of DAUDA displacement from TL by increasing concentrations of TAFC ( ${blacksquare}$ ) and stearic acid ( ${blacktriangleup}$ ).

We found no significant difference in the binding of TL to iron-depletedor iron-saturated siderophores either for desferrioxamine Bor for TAFC. Together with the results from atomic absorptionanalysis that no free iron is bound by TL, it is evident thatiron is bound to TL only in a complex with a siderophore andthat siderophore binding to TL is independent of iron.

Inhibition of bacterial growth by TL. In order to test the interference of TL with siderophore-mediatediron uptake by microorganisms, we first explored its effecton the growth of E. coli in M9 minimal medium under iron-limitingconditions. The M9 minimal medium was not supplemented withiron, but trace amounts present in glassware and medium weresufficient for the bacteria to grow to saturation. However,after the addition of exogenous TL at a concentration of 5 µM,drastic inhibition of growth was observed (Fig. 5). This growthinhibition could be reversed by adding free iron, as indicatedfrom endpoint saturation experiments (Fig. 5). In addition,no growth inhibition could be detected in non-iron-limited medium(data not shown). Therefore, our experiments clearly suggestthat the bacteriostatic effect of TL is directly related tothe inhibition of siderophore-mediated iron sequestration.

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FIG. 5. Results of endpoint saturation experiments of bacterial growth. Hatched columns, growth (OD₆₀₀) of E. coli XL-1 Blue cultures grown in M9 minimal medium at 37°C after 36 h, without and with 5 µM TL; black columns, growth in M9 minimal medium containing 5 µM TL supplemented with increasing concentrations of iron.

The same experiments were performed with P. aeruginosa, butno inhibition of growth could be detected (data not shown),thus supporting the results of our binding studies.

Inhibition of fungal growth by TL. Investigations concerning the inhibitory effects of TL on fungalgrowth were performed with a mutant strain of A. nidulans thatcontains a deletion of the L-ornithine N⁵-monooxygenase-encodinggene sidA ( ${Delta}$ sidA), which completely blocks siderophore biosynthesis.Thus, this mutant strain is unable to grow on minimal mediumwithout supplementation with siderophores (5). A. nidulans ${Delta}$ sidAwas grown on a medium supplemented with TAFC or a medium containingTAFC preincubated with TL. As shown in Fig. 6, significant inhibitionof growth was apparent after 48 and 72 h of growth on the mediumcontaining the TL-preincubated TAFC compared to the growth onthe TAFC-supplemented medium. TL does not have any antifungalactivity beyond siderophore sequestration, since no growth inhibitionwas observed when a surplus of TAFC was used (data not shown).It is evident from the data for the 72- and 96-h time pointsthat growth is not completely blocked but is retarded. The growthafter prolonged incubation on the medium containing TL-preincubatedTAFC was probably due to protease activity, since increaseddegradation of TL could be detected between 72 and 96 h by Westernblot analysis (data not shown).

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FIG. 6. Effect of TL addition on growth of siderophore-deficient strain A. nidulans ${Delta}$ sidA. Conidia (5 x 10²) were point inoculated on MM supplemented with 50 nM TAFC (left column) or TL preincubated with TAFC (right column) and incubated for the indicated times.

DISCUSSION

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Discussion

In the present study we demonstrated that the antimicrobialactivity of TL depends on its ability to bind to microbial siderophores,thereby interfering with the iron acquisition systems of microorganisms.Since iron is an essential nutrient for microorganisms but isstrongly bound to host proteins in mammals, microorganisms mustcompete with these proteins for this essential ion. Microbeshave evolved powerful acquisition systems by producing highlyspecific iron chelators (siderophores) in response to iron deficiency.Thus, inhibition of this iron acquisition process by scavengingmicrobial siderophores is a powerful mechanism in the defenseagainst microbial infections. Indeed, two human proteins havebeen found to interfere with microbial siderophores so far.The first one is serum albumin, which was described to inhibitthe transfer of iron from ferric enterobactin to E. coli (19).The other one is NGAL, a member of the lipocalins produced byneutrophiles and epithelial cells during inflammation and enhancedcell proliferation (17). NGAL could be isolated and crystallizedwith bound ferric enterobactin (12). Our results show that TLis an additional member of this group.

Similar to NGAL, siderophores seem to be bound within the hydrophobiccalyx of TL, since they are able to displace DAUDA, which isknown to have a specific binding site within this pocket (10).Nevertheless, TL shows some significant differences from NGALin siderophore binding. In contrast to NGAL, which binds tobacterial catecholate type siderophores, TL also binds to bacterialand fungal hydroxamate-type siderophores. NGAL has an extremelyhigh affinity for ferric enterobactin, with a K_d of about 0.4nM (12). TL has a much lower affinity (K_d, about 0.5 µMfor TAFC) but binds to a broad spectrum of siderophores, consistentwith a general scavenger function (20). The action of TL againstfungal organisms would complement the numerous bacterial defensesalready described for the eye and would be valuable in the mouth,trachea, and other locations where TL is expressed (3, 34).

Growth inhibition tests showed that TL has strong bacteriostaticand fungistatic effects in vitro. However, since TL is not asiderophore-specific binding protein but, rather, a scavengerwith a broad ligand specificity, several aspects concerningits antimicrobial action in vivo should be considered. Mostof the TL-containing secretions also contain other potentialligands of TL, e.g., fatty acids. Therefore, the affinity ofTL for siderophores must be at least as high as the affinityfor the other ligands. 16-(9-Anthroyloxy)palmitic acid, a derivativeof palmitic acid, has been shown to bind to TL, with a K_d of0.8 µM (11). The K_d of stearic acid, which was describedto be one of the ligands that binds to TL most strongly in vivo,was not measured directly but was calculated from relative displacementassays, which suggested a K_d of about 0.3 µM (10). Ourresults demonstrated that TL indeed has an affinity for siderophorescomparable to that of strongly binding fatty acids. Next, theconcentration and affinity of TL must be high enough to competewith microbial siderophore uptake systems. TL is constantlyproduced by several glands and is present in large amounts intear fluid, nasal mucus, and tracheal secretions, e.g., tearfluid contains up to 3 mg of TL per ml (7, 22). In addition,its concentration increases during inflammation and/or infection(22, 34). The K_d values of fungal siderophore transporters werefound to range from 0.9 to 2.3 µM, which were for ferrichromesand TAFC, respectively (23, 41). Since we found comparable affinitiesof TL for these fungal siderophores, TL should be specific enoughto efficiently outcompete all of the fungal siderophore receptors.Bacterial siderophore transporters are known to have higheraffinities; e.g., enterobactin binds to its receptor with aK_d in the range of 0.4 to 100 nM (28). Therefore, TL might bea more efficient inhibitor of fungal growth than of bacterialgrowth in vivo.

Recent investigations demonstrated that TL binds to LIMR, anovel endocytic receptor (39) which targets the TL-ligand complexand places them into specific intracellular vesicles, probablylate endosomes or lysosomes (40). Targeting of the TL-siderophore-ironcomplex to these cellular compartments will be an importantmechanism in the clearance process; furthermore, the acidicconditions of these vesicles might lead to ligand removal anddegradation of the siderophores.

ACKNOWLEDGMENTS

We thank E. Artner-Dworzak for providing access to atomic absorptionanalysis, G. Gstraunthaler for advice on fluorescence measurements,and F. Marx for helpful discussions.

M.F. and B.R. were supported by Austrian Science Foundation(FWF) grant P14850, and B.J.G. was supported by NIH grant EY-11224.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology, Innsbruck Medical University, Peter-Mayr Strasse 4b, A-6020 Innsbruck, Austria. Phone: (43) 512-507-3603. Fax: (43) 512-507-2866. E-mail: bernhard.redl{at}uibk.ac.at.

REFERENCES

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