IsdA Protects Staphylococcus aureus against the Bactericidal Protease Activity of Apolactoferrin

An important facet of the Staphylococcus aureus host-pathogeninteraction is the ability of the invading bacterium to evadehost innate defenses, particularly the cocktail of host antimicrobialpeptides. In this work, we showed that IsdA, a surface proteinof S. aureus which is required for nasal colonization, bindsto lactoferrin, the most abundant antistaphylococcal polypeptidein human nasal secretions. The presence of IsdA on the surfaceof S. aureus confers resistance to killing by lactoferrin. Inaddition, the bactericidal activity of lactoferrin was inhibitedby addition of phenylmethylsulfonyl fluoride, implicating theserine protease activity of lactoferrin in the killing of S.aureus. Recombinant IsdA was a competitive inhibitor of lactoferrinprotease activity. Reciprocally, antibody reactive to IsdA enhancedkilling of S. aureus. Thus, IsdA can protect S. aureus againstlactoferrin and acts as a protease inhibitor.

INTRODUCTION

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INTRODUCTION

Staphylococcus aureus is an important gram-positive pathogenthat is responsible for an extensive range of pathologies inhumans (41, 65) and animals (37). Successful colonization andnasal carriage of S. aureus by patients and health care workersare linked to infection, which occurs when the organism spreadsfrom its primary ecological niche, the anterior nares, to normallysterile parts of the body (64, 70, 73). In order to establishsuch an intimate association, an invading microorganism mustbe able to resist the actions of the host's innate defenses.Human nasal secretions form the first barrier against inhaledmicroorganisms and contain a cocktail of cationic proteins whichare presumed to control bacterial growth and spread (21, 22).One such protein, lactoferrin (Lf), is the second most abundantantimicrobial polypeptide (21) (after lysozyme, to which S.aureus is insensitive [12]) in airway fluid, and significantquantities of this molecule are also found in other body secretions(49).

A globular 78-kDa glycoprotein, Lf is folded into homologousN and C lobes. Each lobe can bind one metal ion, usually Fe³⁺,to make hololactoferrin (hLf) (8). Apolactoferrin (aLf) carriesno metal ions. The bactericidal activity of aLf, including activityagainst S. aureus, is well established (4, 14), but the mechanism(s)by which it kills S. aureus has not yet been elucidated. A stablepeptide known as lactoferricin (Lfcin) results from proteolysisof Lf and is itself active against a range of bacteria, includingS. aureus (11). However, its relevance to mucosa-colonizingbacteria in the absence of an inflammatory response to provideproteolytic conditions remains in question (66).

A study of S. aureus antigens expressed during human infectionidentified IsdA, an iron-regulated, covalently attached surfaceprotein (18). Otherwise healthy individuals who were nasal carriersof S. aureus had lower serum concentrations of anti-IsdA antibodiesthan noncarriers, which led to the observation that vaccinationwith IsdA could reduce nasal carriage in the cotton rat model(18). Moreover, an isogenic isdA mutant was shown to be attenuatedfor nasal colonization in this model, and its ability to binddesquamated human nasal epithelial cells was reduced (18). IsdAis covalently bound to the cell wall peptidoglycan via the activityof sortase A (43). The mature IsdA protein has two domains withdistinct functions. The N-terminal NEAT domain of IsdA bindsa broad spectrum of human extracellular matrix and serum proteins,including transferrin (Tf), a protein with extensive homologyto Lf (17, 44, 46, 47, 60, 63). The C-terminal domain of IsdAhas recently been shown to decrease the cellular hydrophobicityof S. aureus and confer resistance to hydrophobic fatty acidsand host antimicrobial peptides and thus aid survival on livehuman skin (20). IsdA is the first protein shown to have thisfunction.

In this study, we analyzed Lf killing of S. aureus and foundthat it occurs via protease activity, which can be inhibitedby binding to IsdA, leading to resistance to this bactericidalprotein.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are listedin Table 1. Escherichia coli strains were grown on 2YT medium,using selection with the antibiotics ampicillin (100 µg/ml)and kanamycin (50 µg/ml) where appropriate. S. aureusstrains were grown either on brain heart infusion medium (Oxoid)or on chemically defined CL medium (34). When included, antibioticswere added at the following concentrations: erythromycin, 5µg/ml; lincomycin, 25 µg/ml; and chloramphenicol,10 µg/ml. All bacterial cultures were grown at 37°C.

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TABLE 1. Bacterial strains and plasmids

Overexpression of recombinant IsdA. His₆-tagged recombinant IsdA was purified using the Hi-Trapsystem (Amersham Biosciences) as described previously (17).

Ligand binding ELISAs. An enzyme-linked immunosorbent assay (ELISA) was used to analyzethe ability of recombinant IsdA to bind ligands, both immobilizedand soluble, as described previously. Briefly, 100 µlof an appropriate ligand, either aLf, hLf, apotransferrin (aTf),or holotransferrin (hTf) (all purchased from Sigma), was addedto the wells of a 96-well microtiter plate (Nunc), incubatedovernight at 4°C, and probed as described previously (16).

Inhibition ELISAs were carried out by mixing recombinant IsdA(rIsdA) with an appropriate ligand or mouse anti-IsdA antibodiesprepared as described previously (17). The reactions were carriedout as described previously (16).

Western blot ligand binding assays. Ligand binding was assayed by Western blotting, using biotinylatedhuman serum proteins as described previously (16).

Bacterial killing assays. The rate of S. aureus death was determined as described previously(20). Briefly, bacteria grown in CL broth were centrifuged andwashed in sterile distilled H₂O (dH₂O) twice. Cell suspensions(ca. 1 x 10⁸ CFU/ml in dH₂O) were incubated at 37°C withvarious amounts of Lf. Portions (10 µl) were harvestedat different time points, and serial dilutions were preparedto determine the numbers of viable bacteria present. The followingcompounds were added to the killing assay when appropriate:mouse anti-IsdA antibodies prepared as described previously(17), human serum, anti-Lf antibodies (ICN), CaCl₂, NaCl, FeSO₄,and rIsdA. Lf treated with CaCl₂ or FeSO₄ was dialyzed overnightagainst phosphate-buffered saline (PBS) to remove unbound excessligand.

When appropriate, the statistical significance of the time necessaryto obtain 50% killing was determined by using Student's t test.

Treatment of Lf with protease inhibitor. Phenylmethylsulfonyl fluoride (PMSF) (final concentration, 1mM) was added to Lf (1 mg/ml), and the preparation was incubatedat room temperature with continuous mixing for 2 h. Subsequentdialysis against PBS overnight at 4°C removed residual PMSF.

Protease activity measurement. N- ${alpha}$ -Benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC)(Sigma) was used as a substrate, as described previously (42),and Lf-catalyzed hydrolysis was determined in PBS at room temperature(21°C). Lf was used at a concentration of 10 µM, andthe substrate concentration ranged from 1 to 200 µM. Thecleavage of the substrate was followed fluorometrically at 460nm with an excitation wavelength of 355 nm by monitoring theincrease with a Victor microtiter plate reader (Wallac). Proteaseactivity inhibition studies were carried out by addition ofrIsdA to the reaction mixture.

RESULTS

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RESULTS

IsdA is an Lf binding protein. The Lf binding activity of rIsdA was tested by performing ELISAs.Binding to both aLf and hLf occurred in a dose-dependent andsaturable manner. Saturation occurred at a concentration ofca. 15 µM (Fig. 1A). An apparent K_d (dissociation constant)of ca. 1.5 µM was calculated from the concentration givinghalf-maximum binding. This was an affinity similar to the affinitythat we observed for IsdA binding to aTf (ca. 6.5 µM)(17). Addition of 1 M NaCl completely inhibited the interactionbetween IsdA and aLf or aTf, implicating ionic interactionsin ligand binding. Furthermore, it was possible to inhibit bindingof IsdA to immobilized aLf and aTf with excess quantities ofboth proteins but not with bovine serum albumin (BSA). Inhibition( $≥$ 95%) was observed for both proteins at a concentration of 2µM (Fig. 1B). Additionally, the holo forms of Lf and Tfdisplayed IsdA binding properties similar to those of the respectiveapo forms (results not shown). Taken together, these data demonstratethat IsdA binds to Lf and Tf in similar ways, regardless ofthe iron status.

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FIG. 1. (A) Binding of rIsdA to microtiter plate wells coated with aLf ( ${blacksquare}$ and ${square}$ ), aTf ( ${blacktriangleup}$ and ${triangleup}$ ), or BSA (x). In some cases 1 M NaCl was added to the reaction mixture (open symbols). Increasing concentrations of rIsdA were incubated in wells for 1 h at room temperature. Bound protein was detected with anti-IsdA antibodies and anti-mouse alkaline phosphatase-conjugated antibodies. The data for aLf and aTf without NaCl overlap extensively, as do the data for aLf and aTf with NaCl and the data for BSA. (B) Inhibition of rIsdA binding to immobilized aLf. rIsdA was preincubated for 1 h at room temperature with increasing concentrations of aLf ( ${blacksquare}$ ), aTf ( ${blacktriangleup}$ ), or BSA ( ${diamond}$ ) before incubation in aLf protein-coated wells. Bound protein was detected with anti-IsdA antibodies and anti-mouse alkaline phosphatase-conjugated antibodies. The data for aLf and aTf overlap extensively. The values in panels A and B are the means for triplicate wells from three independent experiments. (C) Screening for binding of rIsdA to lobes of human aLf using the Western affinity blotting technique. Trypsin-digested aLf (20 µg) was separated by electrophoresis on a 12.5% (wt/vol) SDS-PAGE gel. rIsdA bound to aLf was detected using anti-rIsdA antibodies. The positions of the C lobe (bearing either one [C₁]or two [C₂] N-linked glycans) and the N lobe (N) are indicated on the right. Lane 1, Coomassie blue-stained aLf; lane 2, aLf probed with rIsdA.

To identify which domain of Lf contains the binding site forIsdA, a Western blot assay was used. Lf is globular and consistsof homologous N and C lobes (3, 7, 8), which can be separatedby sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) after digestion with trypsin (62). This experiment revealedbinding of rIsdA to both lobes of Lf (Fig. 1C).

Expression of IsdA inhibits killing of S. aureus by aLf. We hypothesized that expression of IsdA on the surface of S.aureus would alter the sensitivity of the organism to Lf, whichis known to be bactericidal (1, 4, 14, 66). To test this, aLfand hLf isolated from human milk were used in killing assays.As observed in other studies, the bactericidal effect was seenonly with aLf and not with hLf (results not shown). Indeed,addition of iron to aLf abrogated its bactericidal activity(Fig. 2A). Addition of anti-Lf antibodies to the killing assaymixture significantly reduced the bactericidal effect in a dose-dependentmanner (P < 0.001 for all dilutions) (Fig. 2B). Taken together,these data confirm the findings of other workers, who showedthat Lf is bactericidal only in its iron-free state (1, 5, 14)and that the bactericidal nature of the native Lf preparationsis due to Lf rather than a contaminant.

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FIG. 2. (A) aLf is bactericidal for S. aureus Newman. FeSO₄ or CaCl₂ was added to preparations of aLf used in killing assays prior to dialysis to remove excess salts. S. aureus Newman was treated with 0.5 µM aLf ( ${blacksquare}$ ), aLf plus 10 µM FeSO₄ ( ${blacktriangleup}$ ), or aLf plus 10 µM CaCl₂ (x). (B) Addition of rabbit anti-Lf antibodies inhibits Lf bactericidal activity. S. aureus Newman was treated with aLf and different dilutions of anti-Lf antibodies, including 1:10 ( ${blacksquare}$ ), 1:100 ( ${blacktriangleup}$ ), and 1:1,000 ( ${blacklozenge}$ ), as well as no anti-Lf antibody (•). (C) IsdA protects S. aureus Newman against killing by 0.5 µM aLf. The following strains were used: ${blacksquare}$ , Newman (wild type); ${blacktriangleup}$ , SRC105 (isdA); •, SRC105(pSRC001); ${blacklozenge}$ , SRC105(pMK4); and x, SRC150 (srtA). The data for strains Newman and SRC105(pSRC001) overlap extensively, as do the data for strains SRC105, SRC105(pMK4), and SRC150. (D) Addition of a molar excess of rIsdA, but not addition of a molar excess of rIsdAC, inhibits killing of S. aureus Newman by 0.5 µM aLf. Symbols: ${blacksquare}$ , aLf; ${blacktriangleup}$ , rIsdA plus aLf (5:1 molar excess); ${triangleup}$ , rIsdA plus aLf (10:1 molar excess); •, rIsdAC plus aLf (5:1 molar excess); ${circ}$ , rIsdAC plus aLf (10:1 molar excess). In all panels, the values are the means of three independent experiments.

In killing assays, the isdA mutant was significantly more sensitivethan the parental strain to aLf (P < 0.0002), and complementationof the isdA mutation fully restored the resistance phenotype(Fig. 2C). In order to determine if any other covalently attachedsurface proteins were involved in resistance to aLf, a sortaseA mutant (S. aureus SRC150), which lacked nearly all such proteins,was used and in killing assays was found to be just as sensitiveto aLf as strain SRC105 (isdA). This indicates that under thesegrowth conditions, IsdA is the major covalently attached surfaceprotein responsible for resistance to aLf (Fig. 2C). To confirmthat binding of aLf by IsdA mediated resistance, molar excessesof rIsdA and its recombinant C domain (rIsdAC) were added tokilling assay mixtures using the isdA mutant strain, and a concomitantreduction in the lethality of aLf was observed when rIsdA wasadded (P < 5 x 10^–6) but not when rIsdAC (P > 0.5)was added (Fig. 2D).

The effect which aLf had on the growth rate of S. aureus inCL broth (a chemically defined medium in which IsdA is expressed[17, 20]) at 37°C was measured. Addition of aLf to the cultureresulted in a decrease in the growth rate (Fig. 3). IsdA isinvolved in resistance to the detrimental effects of aLf asSRC105 (isdA) grew more slowly in its presence than the parentalstrain.

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FIG. 3. IsdA protects against the growth-inhibiting effect of aLf. Symbols: ${blacksquare}$ , Newman (wild type); x, SRC105 (isdA); ${blacklozenge}$ , Newman plus 1 µM aLf; ${blacktriangleup}$ , SRC105 plus 1 µM aLf. The values are the means of three independent experiments. The data for strains Newman and SRC105 overlap extensively. O.D. 600, optical density at 600 nm.

These data make a strong case for the hypothesis that IsdA isable to reduce the bactericidal efficacy of aLf. The abilityof IsdA to readily prevent killing by aLf, whether bound tothe cell surface or in solution, suggests that IsdA may blockaLf-mediated killing by neutralizing the site(s) on Lf whichis responsible for the bactericidal activity.

Anti-IsdA antibodies inhibit Lf resistance. Given that IsdA is required for nasal colonization and thatantibodies reactive to IsdA can prevent this colonization, itwas hypothesized that anti-IsdA antibodies might prevent bindingof aLf to IsdA and thus enhance the killing activity. When strainNewman was incubated with dilutions of mouse anti-IsdA, increasedkilling was observed, but no difference was observed when SRC105(isdA) was used (Fig. 4A and B). Addition of mouse anti-IsdHto the killing assay mixtures had no effect on aLf killing efficacyfor either strain (results not shown). Taken together, thesedata demonstrate that antibodies reactive to IsdA enhance thekilling activity of aLf.

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FIG. 4. Mouse anti-IsdA serum enhances aLf killing in an IsdA-dependent manner. Different concentrations of sera were added to killing assay mixtures. (A) Symbols: •, Newman plus 0.5 µM aLf (P < 0.01); ${blacksquare}$ , Newman plus 0.5 µM aLf plus anti-IsdA (1:100 dilution of antibody; P < 4 x 10^–5); ${square}$ , Newman plus 0.5 µM aLf plus anti-IsdA (1:1,000 dilution of antibody; P < 2 x 10^–5). (B) Symbols: x, SRC105 plus 0.5 µM aLf; ${blacktriangleup}$ , SRC105 plus 0.5 µM aLf plus anti-IsdA (1:100 dilution of antibody); ${triangleup}$ , SRC105 plus 0.5 µM aLf plus anti-IsdA (1:1,000 dilution of antibody). The data for all three mixtures overlap extensively. The values are the means of three independent experiments.

Protease activity of human aLf is bactericidal. The mechanism by which aLf elicits killing of S. aureus remainsuncertain. Previous reports have shown that Lfcin, like manyother antimicrobial peptides, is inactive under saline conditions,such as those described above. Conversely, the antimicrobialactivity of whole Lf has been shown to require such conditions(5). The killing efficacies of whole Lf and Lfcin were testedto confirm the previous findings, and the results showed thatbovine Lfcin was not active against S. aureus when PBS was used;however, killing did occur when the experiment was carried outwith dH₂O (Fig. 5A). Both human aLf and bovine aLf were activein PBS, but the activity was poor in dH₂O (Fig. 5b). Thus, inthe assays conditions used in this work, the killing activityof Lf is not due to Lfcin.

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FIG. 5. Effect of assay conditions on Lfcin and aLf activity. (A) Lfcin B is active in dH₂O but not in PBS. S. aureus Newman was incubated with Lfcin B in PBS ( ${blacksquare}$ ) and dH₂O ( ${blacklozenge}$ ). (B) aLf (0.5 µM) is active in PBS but only poorly active in dH₂O. S. aureus Newman was incubated with human ( ${blacksquare}$ and ${square}$ ) or bovine ( ${blacktriangleup}$ and ${triangleup}$ ) aLf. Filled symbols, incubation in PBS; open symbols, incubation in dH₂O. The data are the means of three independent experiments. (C) Serine protease activity of aLf is bactericidal. S. aureus Newman was incubated with 0.5 µM human aLf ( ${blacksquare}$ and ${square}$ ) and 0.5 µM bovine aLf ( ${blacktriangleup}$ and ${triangleup}$ ). Some preparations were pretreated with PMSF (open symbols). The values are the means of three independent experiments.

The serine protease activity of aLf has been described extensivelypreviously (33, 42, 52, 57, 68). To test whether this activitywas responsible for S. aureus killing, aliquots of bovine aLfand human aLf were treated with PMSF, a potent inhibitor ofserine proteases, and used in killing assays. Treatment of bothhuman and bovine aLf completely inhibited killing of S. aureus(Fig. 5C). Similarly, aliquots of aLf which had been boiledfor 2 min were completely inactive (results not shown). As acontrol, aLF samples which had not been treated with PMSF weresubjected to parallel dialysis, and they maintained full proteaseand killing activity (results not shown). Thus, the anti-S.aureus activity of whole aLf is due to its serine protease activity.Experiments aimed at identifying components of S. aureus releasedby aLf proteolysis by SDS-PAGE, followed by Coomassie blue orsilver staining of killing assay supernatants, failed to yieldspecific fragments (results not shown).

NEAT domain of IsdA is an inhibitor of aLf activity. To investigate the possibility that IsdA directly inhibits theserine protease activity of aLf, proteolysis of Z-Phe-Arg-AMC,a fluorescent substrate of aLf (42), was measured. The proteolyticactivity of aLf followed simple Michaelis-Menten kinetics (k_cat= 0.8 s^–1, K_m = 34 µM) (Fig. 6A) and disappearedwhen the aLf was either denatured by heating or treated withPMSF or 10 µM FeSO₄ (see above), but not when it was treatedwith 10 µM CaCl₂, another Lf ligand (53) (Fig. 6A). Additionof rIsdA to the assay mixture decreased the serine proteaseactivity of aLf in a competitive manner (Fig. 6B). Additionof anti-IsdA antiserum at 1:1,000 and 1:100 dilutions inhibitedprotease inhibitor activity (Fig. 6C). No inhibition was observedwith rIsdAC (results not shown). Recombinant NEAT domain couldnot be used in these experiments due to its insolubility. Increasingthe concentration of substrate added to the rIsdA-aLf reactionmixture did increase the reaction rate, indicating that IsdAis a classical competitive inhibitor of aLf. No proteolyticdegradation of rIsdA was observed under any conditions used(results not shown).

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FIG. 6. Protease activity of human aLf with a fluorescent substrate. (A) Effect of substrate concentration on the rate of 0.1 µM aLf-catalyzed hydrolysis of Z-Phe-Arg-AMC. Symbols: ${square}$ , 0.1 µM aLf; ${diamond}$ , 0.1 µM aLf plus 10 µM CaCl₂; ${triangleup}$ , 0.1 µM aLf plus 10 µM FeSO₄; ${circ}$ , PMSF-treated 0.1 µM aLf; x, boiled 0.1 µM aLf. The data for PMSF-treated 0.1 µM aLf, 0.1 µM aLf plus 10 µM FeSO₄, and boiled 0.1 µM aLf overlap extensively, as do the data for 0.1 µM aLf and 0.1 µM aLf plus 10 µM CaCl₂. (B) rIsdA inhibits protease activity. Symbols: ${blacklozenge}$ , 0.1 µM aLf; ${blacktriangleup}$ , 0.1 µM aLf plus 1 µM rIsdA; ${blacksquare}$ , 0.1 µM aLf plus 0.2 µM rIsdA. (C) Anti-IsdA antibodies inhibit IsdA protease inhibitor action. Symbols: ${blacklozenge}$ , 0.1 µM aLf; ${blacktriangleup}$ , 0.1 µM aLf plus 1 µM rIsdA; ${blacksquare}$ , 0.1 µM aLf plus 0.2 µM rIsdA plus 1:1,000 dilution of anti-IsdA; x, 0.1 µM aLf plus 0.2 µM rIsdA plus 1:100 dilution of anti-IsdA. The values are the means of three independent experiments.

The ligand binding activity of IsdA has been mapped to the NEATdomain. Using S. aureus strains carrying deletions of the NEATand C domains of IsdA (Fig. 7) (20), the resistance to aLf killingwas determined. When grown under iron starvation conditions,S. aureus SRC108 (isdA ${Delta}$ NEAT) was as sensitive to the action ofaLf as strain SRC105 (isdA), exhibiting the same rate of death(P > 0.5) (Fig. 8). Conversely, S. aureus SRC109 (isdA ${Delta}$ C)was partially resistant to the action of aLf (P < 0.0004)but was significantly less resistant (P < 0.001) than thewild type (Fig. 8).

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FIG. 7. Domain structure of IsdA in S. aureus. The organization and predicted mature sizes of IsdA, IsdA ${Delta}$ C, and IsdA ${Delta}$ NEAT are shown. The numbers indicate the positions of the relevant amino acid residues.

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FIG. 8. NEAT domain of IsdA protects S. aureus against the killing activity of 0.5 µM aLf in PBS. The following strains were used: ${blacklozenge}$ , Newman (wild type); ${blacksquare}$ , SRC105 (isdA); ${blacktriangleup}$ , SRC108 (isdA ${Delta}$ NEAT); and •, SRC109 (isdA ${Delta}$ C). The data for strain SRC105 and SRC108 overlap extensively. The values are the means of three independent experiments.

C domain of IsdA confers resistance to bovine Lf. Recent work has shown that the C domain of IsdA confers resistanceto various mammalian antimicrobial peptides, probably due toinhibition of hydrophobic interactions with the cell envelope(20). We tested the ability of IsdA and specifically the C domainto resist Lf. In killing assays, S. aureus SRC108 (isdA ${Delta}$ NEAT)and its parent were similarly resistant to killing by bovineLfcin compared to strains SRC105 (isdA) and SRC109 (isdA ${Delta}$ C) (P< 5 x 10^–6), which were equally more sensitive. Complementationof SRC105 (isdA) by pSRC001 (isdA⁺) specifically restored theresistance phenotype (Fig. 9). These data show that IsdA, specificallyits C domain, is able to resist the action of bovine Lfcin,possibly by inhibiting the hydrophobic interactions which arerequired between host antimicrobial peptides and their target,the cell membrane.

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FIG. 9. C domain of IsdA protects S. aureus against 1 µM bovine Lfcin killing activity. The following strains were used: ${blacklozenge}$ , Newman (wild type); ${blacksquare}$ , SRC105 (isdA); ${blacktriangleup}$ , SRC108 (isdA ${Delta}$ NEAT); •, SRC109 (isdA ${Delta}$ C); ${square}$ , SRC105(pSRC001); and ${diamond}$ , SRC105(pMK4). The data for strains Newman, SRC108, and SRC105(pSRC001) overlap extensively, as do the data for strains SRC105, SRC109, and SRC105(pMK4). The data are the means of three independent experiments.

DISCUSSION

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DISCUSSION

There is a complex interaction between S. aureus and its humanhost as the bacterium is able to colonize several host niches,both as an opportunist pathogen that has great medical significanceand as a commensal in the nares (41, 63, 65, 70, 73). In orderto defend against colonization by microorganisms, the host producesa barrage of antimicrobial peptides. In the human nose, theprimary ecological niche of S. aureus, Lf is the predominantanti-S. aureus protein (21, 22). Indeed, transgenic mice producinghuman Lf were more able to clear S. aureus from infected mucosathan mice lacking the protein (30). Moreover, in vitro studiesusing bovine mammary tissue provided evidence that productionof Lf is up-regulated in response to infection (71, 74). Theantibiotic activity of Lf against a variety of bacteria hasbeen recognized for a long time, and the antimicrobial activityof Lf was initially attributed to iron sequestration, untilthe irreversible inhibition of Streptococcus mutans in vitrowas shown not to be attributable to iron deprivation (6). Indeed,even the role of Lf in host iron homeostasis is now being reconsidered.It is well established that the host uses Tf to sequester ironand deliver it to its cells (13, 36, 38, 61), but in Lf double-knockoutmice the iron homeostasis in adults is no different from thatin wild-type mice, and indeed there is a mild iron overloadin neonates, suggesting that Lf may reduce iron uptake (67).However, numerous further functions have been ascribed to Lf(for reviews, see references 9, 15, 25, 39, 40, 66, 68, and69), including serine protease activity, which has been shownto be responsible for the removal of colonization and virulencefactors from the surface of Haemophilus influenzae (52) andPorphyromonas gingivalis (57), respectively. Furthermore, serineprotease activity could also account for Lf inhibition of virulence,cellular invasion, type III secretion, and biofilms in a varietyof pathogenic bacteria (2, 27, 50, 58). Binding of iron hasbeen shown to significantly alter the structural conformationof Lf (29), which correlates with a loss of both protease andantibacterial activities against a number of organisms, includingS. aureus (1, 4, 14, 66). In this study, we showed that inhibitionof serine protease activity abrogates killing activity. Furthermore,expression of IsdA by S. aureus confers resistance to Lf killing,and rIsdA inhibits protease activity in vitro.

Lf is globular and consists of N and C lobes connected by athree-turn ${alpha}$ -helix (3, 7, 8). The two lobes are homologous andhave very similar tertiary structures, consistent with the hypothesisthat they arose as products of gene duplication. Each lobe isfurther subdivided into two domains with a single iron bindingsite situated between the inner faces of the interdomain cleft.IsdA binds both lobes, and it therefore seems likely that thebinding site is situated in an area of homology. The serineprotease activity resides in the N lobe, but the precise locationis a matter of contention. One study proposed that it consistsof a serine-lysine catalytic dyad located in the area adjacentto the interlobe cleft of human Lf (33), but in another study,pK_a shift calculations indicated that several serine residuesof bovine Lf display the high nucleophilicity required to potentiallycatalyze substrate cleavage (42). Definitive identificationof the active site awaits further study.

Iron deprivation is a common host environmental stimulus, andin response to this stimulus S. aureus makes a cell wall-boundprotein, IsdA (17, 18, 48, 72). Previously, IsdA has been shownto be an adhesin required for nasal colonization (17, 18). Inthis paper, we describe how IsdA binds Lf, a protein similarto Tf (46), which has also been described as a binding substratefor IsdA (17, 60). Previous studies have shown that IsdA bindsa range of iron-containing ligands, including Tf, and have proposedthat it has a role in iron uptake across the cell envelope;however, there is no phenotypic evidence for such uptake fromTf. Indeed, Park et al. (51) showed that IsdA does not havethe ability to remove iron directly from Tf and that a siderophore-mediatediron acquisition system plays a dominant and, more importantly,essential role in this process. The IsdA NEAT domain has alsobeen shown to bind heme, and it has been proposed that the bacteriumuses this activity to acquire iron (28). Several S. aureus surfaceproteins which bind hemoglobin, hemin, and haptoglobin havebeen identified, which may substitute for the loss of IsdA (23,44).

The interaction between IsdA and Lf appears to be very similarto the interaction of IsdA with Tf, which is not surprisinggiven the homologous nature of the two proteins. The ligandbinding activity of IsdA resides in its single-copy NEAT domain(17), whose structure has recently been determined and shownto consist of a beta-sheet (28). It was also speculated thatthe broad-spectrum ligand binding activity of IsdA is due tononspecific ionic interactions (28). However, although we showhere that ionic interactions are important in the binding ofLf by IsdA, these interactions are unlikely to be nonspecific,as the net charges at pH 7 of Lf and Tf are positive and negative,respectively (35, 47, 55). In this study, no inhibition of Lfactivity was observed for the C domain of IsdA; only full-lengthrIsdA inhibited protease activity. We were unable to use recombinantNEAT domain due to insolubility of the polypeptide. Instead,S. aureus strains displaying mutated forms of IsdA, lackingeither the NEAT or C domain, were used in killing assays. Noresistance to killing was observed for the isdA ${Delta}$ NEAT strain,but isdA ${Delta}$ C cells were partially resistant compared to the wildtype. It has been proposed that the C domain extends the NEATdomain into the extracellular milieu (63). Thus, in the IsdA ${Delta}$ Ctruncated version of the protein, the NEAT domain may be insufficientlyexposed or oriented for full activity.

In addition to protease activity, a small peptide, Lfcin, isreleased by proteolysis from the N terminus of Lf (10, 11, 54).This cationic antimicrobial peptide has been compared with othermolecules, such as defensins, suggesting that it has a similarmode of action. However, the significance of Lfcin to bacteriacolonizing mucosal surfaces, such as the nares, is in doubtbecause this peptide is generated by proteolysis, such as theproteolysis produced by an inflammatory response, and not undernormal conditions (66). In this study, we found that IsdA, specificallythe C domain, inhibits Lfcin activity. However, this was notresponsible for the IsdA-derived resistance to Lf. It is theLf interaction with the IsdA NEAT domain that confers resistance.

The array of surface proteins possessed by S. aureus poses interestingquestions concerning their functions. This organism inhabitsa variety of environments even within the same host, and thusthere may be a requirement for more than one function for aprotein; indeed, the spectrum of ligands bound by some surfaceproteins continues to widen (for reviews, see references 19and 26). IsdA is known to be expressed upon association withthe host, most significantly in the nares. In Streptococcuspneumoniae, another upper-airway commensal also able to causelife-threatening disease, the capacity of surface protein PspAto bind Lf has been linked to its ability to inhibit killingby this protein (31, 32, 56). Thus, binding of Lf and subsequentinhibition of its proteolytic activity may represent a commonmechanism in pathogenic bacteria. Interestingly, antibodiesto both IsdA and PspA have been shown to abrogate their inhibitionof Lf killing (56; this study). It is already known that increasedtiters of anti-IsdA and -PspA antibodies are associated withdecreased rates of carriage of both organisms (18, 45). Thus,such acquired immune responses mounted to resistance factorslike IsdA and PspA may represent a mechanism for control ofbacterial carriage.

ACKNOWLEDGMENTS

This work was funded by Biosynexus Inc. (S.R.C.).

We thank Olaf Schneewind, University of Chicago, for providingthe srtA mutant.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom. Phone: 44 114 222 4411. Fax: 44 114 222 2800. E-mail: s.foster{at}sheffield.ac.uk

Published ahead of print on 28 January 2008.

Editor: V. J. DiRita

Present address: School of Biological Sciences, University ofReading, Whiteknights, Reading RG6 6AJ, United Kingdom.

REFERENCES

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