The Capsule Sensitizes Streptococcus pneumoniae to {alpha}-Defensins Human Neutrophil Proteins 1 to 3

Streptococcus pneumoniae is a major cause of morbidity and mortalityworldwide. Its polysaccharide capsule causes resistance to phagocytosisand interferes with the innate immune system's ability to clearinfections at an early stage. Nevertheless, we found that encapsulatedpneumococci are sensitive to killing by a human neutrophil granuleextract. We fractionated the extract by high-performance liquidchromatography and identified ${alpha}$ -defensins by mass spectrometryas the proteins responsible for killing pneumococci. Analysisof sensitivity to the commercial ${alpha}$ -defensins human neutrophilproteins 1 to 3 (HNP1-3) confirmed these findings. We analyzedthe sensitivities of different pneumococcal strains to HNP1-3and found that encapsulated strains are efficiently killed atphysiological concentrations (7.5 µg/ml). Surprisingly,nonencapsulated, nonvirulent pneumococci were significantlyless sensitive to ${alpha}$ -defensins. The proposed mechanisms of ${alpha}$ -defensinresistance in nonencapsulated pneumococci is surface chargemodification, e.g., by introduction of positive charge by D-alanylationof surface-exposed lipoteichoic acids. These mechanisms aresurmounted by the presence of the capsule, which we hypothesizeis masking these charge modifications. Hence, ${alpha}$ -defensins inthe phagolysosome of neutrophils possibly contribute to intracellularkilling after antibody-mediated opsonophagocytosis of encapsulatedpneumococci.

INTRODUCTION

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INTRODUCTION

Streptococcus pneumoniae (the pneumococcus) is a major humanpathogen and the cause of community-acquired pneumonia, sinusitis,otitis media, and meningitis. Despite access to antibiotic treatment,pneumococci remain a major cause of morbidity and mortalityworldwide. Pneumococci colonize the nasopharynx of up to 60%of healthy children (21) and the microbial and host factorsthat determine if carriage leads to disease or not need to bebetter characterized. The role of the effectors of the innateimmune system, such as neutrophils and antimicrobial peptides(AMPs), which could affect early clearance of pneumococci, isnot well understood.

Neutrophils are professional phagocytes that have cytoplasmicgranules (azurophilic, specific, and small storage) that containAMPs (see below) and enzymes (e.g., neutrophil elastase andcathepsin G). Upon encountering microbes, neutrophils are activatedand kill bacteria by phagocytosis (22, 24). In this process,microbes are engulfed into a phagosome that fuses with granuleswhere reactive oxygen species are produced and AMPs and enzymesare present. Neutrophils also degranulate, killing microbesextracellularly through the release of AMPs from granules thatfuse with the cytoplasmic membrane. Finally, neutrophils canform neutrophil extracellular traps (NETs), extracellular structuresconsisting of DNA, histones, and granule proteins that captureand kill microbes by means of a high local concentration ofAMPs and histones (3, 35).

Typically, AMPs are small (12- to 100-amino-acid), amphiphilic,and cationic proteins. Two major groups of mammalian AMPs arethe cathelicidins and the defensins. Defensins are small (15-to 20-residue), cysteine-rich cationic peptides with a characteristicβ-sheet-rich fold. They constitute 30 to 50% of the contentof azurophilic granules and are active against a wide rangeof bacteria, fungi, and enveloped viruses. Due to their netpositive charge and hydrophobicity, defensins as well as otherAMPs are thought to exert their antimicrobial effects by permeabilizingthe bacterial cytoplasmic membrane (15). There are three subfamiliesof defensins described for mammals, namely, ${alpha}$ , β, and ${theta}$ ,which were classified based on their cysteine pairing and structure(9, 15). Human neutrophil proteins 1 to 4 (HNP1-4) are ${alpha}$ -defensins,expressed exclusively by neutrophils.

Gram-positive pathogens can be resistant to phagocytosis, AMPs,and NETs, preventing efficient clearance by innate immune mechanisms.The pneumococcal polysaccharide capsule, of which there aremore than 90 different types, makes pneumococci resistant tocomplement-mediated opsonophagocytosis (5). The capsule alsohampers trapping by NETs (34). Since most AMPs are cationic,the introduction of positive charge via D-alanylation of surface-exposedlipoteichoic acids (LTAs) (13, 17, 18, 25) makes microbes resistantto AMPs. Indeed, we recently showed that D-alanylation in nonencapsulatedpneumococci rendered them resistant to killing by antimicrobialcomponents present in NETs (34). The combination of capsule,DNase expression, and D-alanylation of LTA makes pneumococciresistant to NETs (1, 34, 35).

Other surface proteins modifying pneumococcal surface chargeand thereby potentially the susceptibility to positively chargedAMPs include LytA and PgdA. The murein hydrolase LytA, a choline-bindingprotein bound to phosphocholine residues on LTA, is involvedin autolysis and drug-induced lysis (32). Pneumococcal strainslacking choline-binding proteins, such as LytA, are more net-negativelycharged than wild-type strains (30). PgdA removes negativelycharged acetyl groups from the GlcNAc sugar moieties on cellwall peptidoglycan (33). The inactivation of pgdA is thereforeexpected to increase the net negative surface charge.

Since many pneumococcal infections are controlled by the innateimmune system, we set out to identify individual AMPs from neutrophilsthat kill pneumococci. We fractionated human neutrophil granuleextracts (hNGE) by high-performance liquid chromatography (HPLC)and tested the effect on different pneumococcal serotypes andmutants.

MATERIALS AND METHODS

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MATERIALS AND METHODS

hNGE isolation. Neutrophils were purified from human blood from healthy donorsas approved by the local ethics committee. A protocol using3% dextran (MP Biomedicals, Illkirch, France) and Ficoll-Hypaque(GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was used asdescribed in reference 1. The isolation of hNGE was performedas previously described (6, 23).

Chromatography. hNGE was fractionated with a C₄ reverse-phase HPLC column (Vydacprotein C₄ column; column length of 250 mm, with particle sizeof 5 µm). Proteins were eluted with a gradient of increasingconcentrations of acetonitrile containing 0.1% (vol/vol) trifluoroaceticacid (TFA) at a flow rate of 1 ml/min. Fractions were lyophilized,dissolved in 20 mM sodium acetate buffer (pH 4.0), and testedfor antimicrobial activity (see below). Separation was performedwith a Waters 626 LC system connected to a photodiode arraydetector (Waters, Milford, MA).

Mass spectrometry. The identity and purity of the antimicrobial component wereanalyzed by matrix-assisted laser desorption ionization massspectrometry (Proteomics 4700 workstation; Applied Biosystems,Foster City, CA). The lyophilized sample was digested with 50mM NH₄HCO₃, 5% acetonitrile, 2% (wt/vol) trypsin (sequencing-grademodified trypsin [Promega, Madison, WI]), and 0.15 M dithiothreitolfor 4 h at 37°C. The reaction was stopped with 0.2% TFAand the reaction mixture was further mixed with matrix alpha-cyano-4-hydroxycinnamicacid (CHCA) solubilized in 50% acetonitrile-0.3% TFA at a concentrationof 5 mg/ml.

Analysis of peptide mass fingerprints was obtained with thefollowing parameters: reflectron mode, 20-kV accelerating voltage,and a low mass gate of 800 Da. Tandem mass spectrometry spectrawere obtained without collision gas. The parameters for databasesearches (MASCOT; Matrix Science) were as follows: 30-ppm peptidemass tolerance for peptide mass fingerprint and 0.3 Da for tandemmass spectrometry spectra. The uncleaved protein was analyzedin linear mode with CHCA as the matrix with an internal marker(M_r, 2465.21).

Bacterial strains used. Pneumococci were grown on blood agar plates overnight at 37°Cin 5% CO₂. The bacteria were subcultured in semisynthetic mediumc+y (19) to an optical density at 620 nm of 0.5 and dilutedin Dulbecco's phosphate-buffered saline (PBS) without Ca²⁺ andMg²⁺ (Invitrogen, Lidingö, Sweden). The following strainswere used: the serotype 1 strain BHN32, the serotype 2 strainD39, the serotype 4 strain TIGR4 (31), and the serotype 9V strainI95 (8), as well as their nonencapsulated derivatives, type1R (34), type 2R (R6) (14), type 4R (TIGR4R) (8), and type 9VR(8), respectively. The TIGR4 ${Delta}$ lytA mutant (with an erythromycincassette replacing lytA) was obtained from E. Tuomanen (12).The TIGR4R ${Delta}$ lytA mutant was created by transforming TIGR4R withgenomic DNA from the TIGR4 ${Delta}$ lytA mutant and selecting for nonencapsulatedmutants on erythromycin plates. We used PCR ligation mutagenesis(20, 29) to inactivate dltA (34) or pgdA in TIGR4 and TIGR4R.For pgdA, primers used for construction and screening of deletionalleles were 5'-taagactttctttcctgctg-3' and 5'-TTGGGCCCggtctgttagatatttgacag-3'flanked with ApaI for the upstream fragment and 5'-gactatccaacagagaggag-3'and 5'-TTGGATCCgcaatccacaattcctctag-3' flanked with BamHI forthe downstream fragment (lowercase letters indicate the genesequence, and underlining indicates the restriction enzyme site).The PCR products were ligated to an erythromycin cassette (GenBank;AB057644) containing ApaI and BamHI sites and transformed intothe recipient pneumococcal strain as previously described (2).The resulting transformants were selected on blood agar platescontaining erythromycin (1 µg/ml) and confirmed by PCRand sequencing of the insertion area.

Shigella flexneri (strain M90T) and Escherichia coli (Top10;Invitrogen, Lidingö, Sweden) were grown in tryptic soybroth and LB broth, respectively, overnight at 37°C andsubcultured at a 1/100 dilution on the day of the experiment.

Killing assays. The bactericidal activities of hNGE, HPLC fractions, and HNP1-3(Hycult Biotechnology b.v., Uden, The Netherlands), all of themin sodium acetate buffer (pH 4.0), were determined by incubatingbacteria (1.0 x 10⁶/ml) in Dulbecco's PBS (yielding a pH of7.0) at 37°C with the indicated doses for 1 h. Survivorswere counted by plating serial dilutions on blood agar plates.Bacterial killing was measured as percentages of control values(obtained using bacteria incubated with sodium acetate bufferand PBS alone). Low-binding, siliconized tubes and pipette tips(Biozym, Hessich Oldendorf, Germany) were used for handlingHNP1-3, hNGE, and the individual HPLC fractions. Experimentswere performed at least three independent times.

Defensin binding assay. Pneumococci were grown to mid-logarithmic phase and dilutedto gain a 1.7 x 10⁶/ml solution and incubated together withHNP1-3 (HyCult Biotechnology) at concentrations of 0.15, 1.5,and 15 µg/ml for 1 to 2 min. After centrifugation, thepellet was lysed using lysozyme (10 mg/ml) for 30 min at 37°C.An HNP1-3 enzyme-linked immunosorbent assay (ELISA) (HyCultBiotechnology) was performed with dilution series of the lysedbacteria as outlined in the manufacturer's protocol. Bindingof defensins (femtograms per single bacterium) was calculated.Bacteria incubated with sodium acetate buffer were used as thecontrol.

Statistical analyses. The nonparametric Mann-Whitney U test was used for analysis.A P value of $≤$ 0.05 was considered significant.

RESULTS

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RESULTS

Killing of pneumococci by hNGE. To determine the effect of neutrophil granule components onpneumococci, we prepared a crude extract from human neutrophils(hNGE) and tested its activity on the encapsulated S. pneumoniaeTIGR4 strain (Fig. 1). After 1 h, a 1/10 dilution of the 6.4-mg/mlprotein-containing hNGE fraction killed 27% of the pneumococcalculture. These data demonstrate that encapsulated pneumococciare sensitive to components present in human neutrophil granules.

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FIG. 1. Killing of encapsulated pneumococci by a hNGE and its individual HPLC fractions. The antimicrobial activity in hNGE (white bar) was determined by incubating encapsulated TIGR4 pneumococci with a 1/10 dilution of hNGE in sodium acetate buffer and determining survivors by plating serial dilutions. The extract was fractionated on a C₄ reverse-phase HPLC column into 40 fractions and incubated with pneumococci to determine killing of individual fractions (black bars). The mean percentage of killing plus standard error of the mean (SEM) for each fraction is shown. The elution profile at A₂₈₀ from the HPLC is shown (black curves). Encapsulated TIGR4 pneumococci are killed by the hNGE and efficiently by fraction 20.

Identification of active substances in hNGE. To identify the components in hNGE active against pneumococci,we fractionated this extract with C₄ HPLC columns into 40 fractions.The killing activity of each fraction was tested (Fig. 1) byincubation with TIGR4 pneumococci as described in Materialsand Methods. Sodium acetate buffer, the vehicle in the fractions,was used as a control. Killing was calculated as the percentCFU recovered after incubation with individual fractions comparedto that obtained after incubation with buffer alone. The elutionprofile (Fig. 1) at A₂₈₀ shows the elution of major proteinpeaks in fractions 20 to 30. Fraction 20, with a protein concentrationof 1.6 mg/ml, showed the most pronounced antimicrobial effect(Fig. 1). Mass spectrometry analysis showed that this fractioncontained ${alpha}$ -defensins HNP1 (gi:228797) and HNP3 (gi:229858).We also observed antimicrobial activity in fractions 10 to 16,but this was not further investigated.

The killing activity of fraction 20 on pneumococci was dosedependent (Fig. 2A). A 1/10 dilution of the 1.6-mg/ml protein-containingfraction killed 100% of the inoculum, while a 1/100 dilutionresulted in more than 20% killing. We used commercial HNP1-3to confirm that ${alpha}$ -defensins kill encapsulated pneumococci (Fig.2B). A concentration as low as 3.75 µg/ml killed 90% ofthe TIGR4 inoculum, and this activity was also dose dependent.The gram-negative bacteria Shigella flexneri and Escherichiacoli were significantly less susceptible to HNP1-3 than wereencapsulated pneumococci. Fifteen micrograms of HNP1-3 per milliliterkilled only 25% of either of these two gram-negative organisms,a sensitivity similar to that seen for the nonencapsulated derivativeTIGR4R strain (Fig. 2B). The nonencapsulated TIGR4R strain wasless sensitive not only to HNP1-3 but also to hNGE (data notshown). We conclude that human HNP1-3 is the most effectivecomponent of hNGE against TIGR4 pneumococci, and the presenceof the type 4 capsule confers this high sensitivity.

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FIG. 2. Killing of pneumococci and gram-negative bacteria by defensins. Bacterial killing was determined by incubating bacteria with increasing concentrations of fraction 20, which contains defensins (A), or with commercial HNP1-3 (B) and enumerating survivors. The mean values plus SEM are shown. (A) Killing of encapsulated TIGR4 by fraction 20 diluted 1/10 to 1/100 in sodium acetate buffer. Sodium acetate buffer only was used as a control. (B) Killing of encapsulated TIGR4 and nonencapsulated TIGR4R pneumococci, Shigella flexneri, and Escherichia coli by HNP1-3. Effective concentrations ranged from 0 to 15.0 µg/ml of HNP1-3. Encapsulated pneumococci are more sensitive to HNP1-3 than are nonencapsulated pneumococci or gram-negative bacteria.

Differences in pneumococcal sensitivity to HNP1-3 depend on capsule and charge. To test if capsular types other than type 4 sensitize pneumococcito HNP1-3, we incubated sets of encapsulated (serotypes 1, 2,4, and 9V) and the corresponding nonencapsulated ("rough"-morphologyserotypes 1R, 2R, R6, 4R, and 9VR) strains with HNP1-3 at 3.75(Fig. 3, left) and 7.5 (Fig. 3, right) µg/ml. Killingwas determined for either HNP1-3 concentration after a 1-hourincubation period at 37°C. Controls were incubated withsodium acetate buffer alone and were used as a reference todetermine the percentage of killing. All strains were killedin a dose-dependent manner, and encapsulated strains were (withthe exception of serotype 2 [see below]) killed more efficiently(Fig. 3A). We conclude that pneumococcal strains expressingcapsule polysaccharide are more efficiently killed by HNP1-3than are their nonencapsulated derivatives. These results contradictthe common view that the capsule is a mediator of AMP resistance.

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FIG. 3. Dose-dependent killing of different pneumococci by HNP1-3. Bacterial killing was determined by incubating bacteria with 3.75 µg/ml (left) and 7.5 µg/ml (right) of HNP1-3. The mean values plus SEM are shown. (A) Encapsulated strains (black bars) are more sensitive than the corresponding nonencapsulated strains (white bars) to HNP1-3. (B) The absence of LTA D-alanylation ( ${Delta}$ dltA) or of the LytA amidase ( ${Delta}$ lytA) sensitizes nonencapsulated pneumococci (white bars) but not encapsulated pneumococci (black bars) to HNP1-3. The absence of peptidoglycan deacetylation ( ${Delta}$ pgdA) has a less pronounced effect on determining ${alpha}$ -defensin sensitivity in nonencapsulated pneumococci. **, P $≤$ 0.01; ***, P $≤$ 0.001.

Strain R6 is the exception to the rule outlined above (Fig.3A). However, R6 is not an isogenic capsule mutant of parentalstrain D39; rather, it also harbors a spontaneous dltA mutation(17). To further investigate this relation, we analyzed surfacecharge-affected dltA (D-alanylation) mutants in the TIGR4 background(Fig. 3B). Deletion of dltA from the encapsulated TIGR4 ${Delta}$ dltAmutant did not increase sensitivity to HNP1-3. However, uponinactivating dltA in the nonencapsulated TIGR4R background (theTIGR4R ${Delta}$ dltA mutant), we observed a marked increase in sensitivityto HNP1-3, yielding a result similar to that for the R6 strain.Hence, modification in surface charge by abolishing the introductionof positively charged D-alanine to LTAs sensitizes nonencapsulatedstrains (R6, the TIGR4R ${Delta}$ dltA mutant) to the positively chargedHNP1-3.

We also used lytA and pgdA mutants to determine their effecton defensin-mediated killing (Fig. 3B). The presence or absenceof LytA and PgdA had no effect on the killing of encapsulatedTIGR4. However, the nonencapsulated TIGR4R ${Delta}$ lytA mutant was moresensitive than the parental strain, TIGR4R. Also, the TIGR4R ${Delta}$ pgdA mutant was killed more efficiently than the wild-type strainbut not in a fashion as pronounced as that for the TIGR4R ${Delta}$ dltAand TIGR4R ${Delta}$ lytA mutants.

Taken together, these data suggest that the introduction ofpositive surface charge (via D-alanylation) or the reductionof negative surface charge (proteins decreasing exposure ofcholine residues or removal of acetyl groups) leads to resistanceto HNP1-3. However, this mechanism is inactivated in the presenceof the capsule.

We did not find a correlation between negatively charged capsules(types 1, 2, 4, and 9V) and noncharged capsules (types 7F and14) and susceptibility to HNP1-3 (Fig. 4). Hence, the exactmechanism by which the capsule interferes with protective surfacecharge modifications remains to be elucidated.

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FIG. 4. Dose-dependent HNP1-3-mediated killing of pneumococci with different capsular charges. Bacterial killing was determined by incubating bacteria with 3.75 µg/ml (A) and 7.5 µg/ml (B) of HNP1-3. The mean values plus SEM are shown. There was no correlation between negatively charged capsules (types 1, 2, 4, and 9V; black) or noncharged capsules (types 7F and 14; white) and susceptibility to HNP1-3.

In order to determine whether differences in defensin killingare dependent on differences in binding, we analyzed the bindingof defensins to different pneumococcal strains (Fig. 5A). Forthis, the TIGR4 strain, its isogenic capsular mutant, and thetwo dltA mutants were used and incubated shortly together withHNP1-3. After lysis, binding was determined by using a commerciallyavailable ELISA kit (see Materials and Methods). We did notobserve any significant difference in HNP1-3 binding betweenthe individual strains. However, incubating the TIGR4 strainwith different concentrations of HNP1-3 clearly showed a dose-dependentbinding (Fig. 5B).

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FIG. 5. Binding of HNP1-3 to different pneumococcal strains. Binding was determined using an ELISA, as outlined in Materials and Methods. Binding was calculated as femtograms of HNP1-3 per single bacterium. One representative experiment is shown. (A) Binding of HNP1-3 (1.5 µg/ml) to TIGR4 pneumococci and isogenic capsular and dltA mutants. No significant difference between the strains in the binding of defensins was detected. (B) Dose-dependent binding of HNP1-3 (0.15, 1.5, and 15 µg/ml) to TIGR4 pneumococci.

DISCUSSION

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DISCUSSION

Pneumococci are major human pathogens that produce an antiphagocyticpolysaccharide capsule. Nonencapsulated pneumococci are virtuallyavirulent in murine models. Interestingly, we found here thatencapsulated strains are killed more efficiently by ${alpha}$ -defensinsthan are nonencapsulated strains in vitro. This difference inkilling is not due to differences in binding of defensins tothe bacteria. According to the widely accepted model, positivelycharged host AMPs bind to and insert into the microbial cellmembrane, thereby killing the microbe (27). An increased netpositive surface charge (mediated, for example, by D-alanylationof LTAs) repels cationic peptides. This model is supported byour data showing that nonencapsulated strains become sensitiveto cationic ${alpha}$ -defensins when net negative surface charge is increasedby genetic inactivation of dltA. The sensitizing effect of thecapsule brings in a novel aspect to this well-accepted modelby putative mechanisms discussed below.

Apart from the removal of D-alanylation, the removal of theamidase LytA, a choline-binding protein, also sensitized nonencapsulatedpneumococci against ${alpha}$ -defensins. A less pronounced effect ofprotection against HNP1-3 could be observed for PgdA, a peptidoglycandeacetylase that confers protection from lysozyme by removingacetyl groups from N-acetylglucosamine (33). The resistancemechanism toward lysozyme was described to be due to alteredsubstrate specificity. Here we found that this deacetylation,potentially via charge modification, also affects sensitivitytoward charged AMPs but not to the same extent as D-alanylation.

The question remains as to why the above-mentioned mechanismsmanifest only in the absence of the polysaccharide capsule.We found no reports addressing potential differences in D-alanylation,peptidoglycan deacetylation, or levels of choline binding proteinsin encapsulated versus nonencapsulated strains that might explainwhy nonencapsulated strains are less sensitive to human defensins.Nevertheless, Swiatlo et al. showed that the removal of choline-bindingproteins in a nonencapsulated background results in a significantalteration in the hydrophobic character of the pneumococcalcell surface (30). These findings do not exclude the possibilitythat the overall level of choline-binding proteins or othersurface molecules is changed upon the inactivation of capsularexpression.

Another explanation for the observed capsule-mediated effectmight be a direct binding of AMPs to polysaccharides. Camposand coworkers described a mechanism of resistance to AMPs dependenton the Klebsiella pneumoniae capsule polysaccharide (4). Thus,a capsule polysaccharide mutant was shown to be more sensitivethan the wild-type strain to human neutrophil defensin 1, β-defensin1, lactoferrin, protamine sulfate, and polymyxin B. A highersusceptibility of encapsulated strains to defensins was alsoobserved for Neisseria meningitidis (28). Possibly, the directbinding of AMPs to the polysaccharide moiety might lead to ahigher local concentration, effectively killing encapsulatedbacteria.

A third possibility is that the capsule masks the charge effectsof D-alanylation, peptidoglycan deacetylation, and choline bindingand thereby abrogates the protective effect of increased positiveor decreased negative surface charge.

Thus, paradoxically, the pneumococcal capsule provides resistanceagainst neutrophil phagocytosis and prevents capture in NETs(1, 34) but increases susceptibility to killing by ${alpha}$ -defensins,which are abundant in neutrophil granules and the phagolysosome(16). We demonstrated previously that neither encapsulated nornonencapsulated pneumococci are killed in AMP-rich NETs uponcapture (1, 34) despite the presence of ${alpha}$ -defensins (C. Urbanand A. Zychlinsky, unpublished observations). It has previouslybeen demonstrated, however, that ${alpha}$ -defensins released from neutrophilsare rapidly inactivated by serum proteins and by the ion concentrationin the extracellular environment (9). The physiological concentrationof ${alpha}$ -defensins HNP1-3 within the neutrophil phagolysosome canbe in the mg/ml range (7, 11), but extracellular concentrationsdue to degranulation are significantly lower ( $~$ 6 µg/ml)(10). The inactivating effect of salts and serum in vivo clearlyreduces activity; however, the extent to which this occurs cannotbe determined, and hence the in vitro concentrations (up to15 µg/ml) used here need to be treated with caution whenextrapolating into the in vivo setting. Our data, however, areconsistent with the literature: in vitro individual defensinsfrom humans kill bacteria, fungi, and enveloped viruses at peptideconcentrations in the 1- to 50-µg/ml range and gram-positivebacteria at concentrations as low as 1 µg/ml (for a review,see reference 26).

We conclude that ${alpha}$ -defensins are active against pneumococci ingeneral; however, the capsule increases susceptibility toward ${alpha}$ -defensins and interferes with the protective effect accomplishedby D-alanylation. How this increased killing of encapsulatedpneumococci translates into human infections remains to be elucidated.

ACKNOWLEDGMENTS

We thank Björn Eilers for help with the hNGE preparation.

This research project was supported by two Marie Curie EarlyStage Research Training Fellowships of the European Union'sSixth Framework Programme (called IMO-train and EIMID-EST),the Torsten and Ragnar Söderberg Foundation, the SwedishRoyal Academy of Sciences, and the Swedish Research Council.

FOOTNOTES

* Corresponding author. Mailing address: Swedish Institute for Infectious Disease Control, Nobels Väg 18, SE-171 82 Solna, Sweden. Phone: 4684572413. Fax: 468302566. E-mail: Birgitta.Henriques{at}smi.ki.se

Published ahead of print on 12 May 2008.

Editor: J. N. Weiser

These authors contributed equally to this work.

REFERENCES

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