of March 26, 2018.This information is current as

pyogenesStreptococcus and the Pathogen magna

FinegoldiaInactivated by the Commensal Epithelium Are Differentially Processed andAntimicrobial Peptides Produced by Constitutive and Inflammation-Dependent

Arne EgestenMatthias Mörgelin, Ole E. Sørensen, Anders I. Olin and Inga-Maria Frick, Sara L. Nordin, Maria Baumgarten,

http://www.jimmunol.org/content/187/8/4300doi: 10.4049/jimmunol.1004179September 2011;

2011; 187:4300-4309; Prepublished online 14J Immunol 

Referenceshttp://www.jimmunol.org/content/187/8/4300.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2011 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Constitutive and Inflammation-Dependent AntimicrobialPeptides Produced by Epithelium Are DifferentiallyProcessed and Inactivated by the Commensal Finegoldiamagna and the Pathogen Streptococcus pyogenes

Inga-Maria Frick,* Sara L. Nordin,† Maria Baumgarten,* Matthias Morgelin,*

Ole E. Sørensen,* Anders I. Olin,*,† and Arne Egesten†

Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the

bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the

anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both

MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is

upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibac-

terial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial b-defensins human b-defensin (hBD)-2

and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar

concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage

was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and

BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In

contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released

by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being

the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions

of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and

a virulent pathogen, the latter operating during the limited time course of invasive disease. The Journal of Immunology, 2011,

187: 4300–4309.

Epithelial linings constitute a physical barrier against en-vironmental microbes that present potential threats to thehost (for instance, pathogenic bacteria) but are also col-

onized by bacteria that are part of the normal microbiota. In ad-dition to being a physical barrier, epithelial cells have a constitutiveproduction of antimicrobial agents; for example, antimicrobialpeptides (AMPs) such as the antibacterial chemokine BRAK/CXCL14 (1–3). Another example is midkine (MK), an antibac-terial growth factor that is constitutively expressed in the basal

parts of the epidermal layer (4, 5). It is likely that there is a deli-cate balance among constitutively produced AMPs, the ecologicalniche inhabited by bacteria of the normal microbiota, and theprevention of infection caused by pathogenic bacteria. In the caseof injury and invasion of pathogenic bacteria, the expression ofseveral AMPs of epithelial cells is induced, resulting in productionof, for example, the b-defensins human b-defensin (hBD)-2 andhBD-3 (6, 7).The normal human microbiota includes several bacterial species

that inhabit the skin without causing any inflammation. An exam-ple of a skin commensal is the anaerobic Gram-positive coccusFinegoldia magna. This bacterium is also an opportunistic path-ogen causing several clinical conditions, such as soft tissue in-fections, wound infections, and bone/joint infections in immuno-compromised hosts (8). In addition, a number of recent studiesreported the presence of F. magna in chronic wounds, includingdiabetic ulcers (9–11). F. magna resides in the lower parts of theepidermal layer, where it binds to BM-40, which is part of thebasal membrane (BM), via the surface-associated protein FAF(12). It is therefore likely that F. magna encounter the constitu-tively expressed AMPs MK and BRAK/CXCL14 during normal,noninflamed conditions. Most strains of F. magna express a sub-tilisin-like enzyme, subtilase of F. magna (SufA), which is asso-ciated with the bacterial surface (13). Both FAF and SufA areproduced at substantial amounts during early logarithmic phase,and, in addition to being surface associated, they are released tothe environment (12, 13). Thus, it is likely that high local con-centrations of these proteins can be reached in vivo. Studies on theproteolytic activity of SufA demonstrated that the enzyme cleaves

*Section for Infection Medicine, Department of Clinical Sciences, Lund University,Skane University Hospital, SE-221 85 Lund, Sweden; and †Section for RespiratoryMedicine and Allergology, Department of Clinical Sciences, Lund University, SkaneUniversity Hospital, SE-221 85 Lund, Sweden

Received for publication December 23, 2010. Accepted for publication August 16,2011.

This work was supported by grants from the Swedish Research Council (Projects20674 and 7480), the Swedish Heart and Lung Foundation (Project 20080246), theMedical Faculty at Lund University, the Swedish Government Funds for ClinicalResearch, and the foundations of Bergh, Crafoord, Greta, and Johan Kock, Marianneand Marcus Wallenberg, and Alfred Osterlund.

The microarray data presented in this article have been submitted to the MinimumInformation About a Microarray Experiment database (http://www.ebi.ac.uk/arrayex-press) under accession number E-MEXP-3305.

Address correspondence and reprint requests to Dr. Arne Egesten, Section for RespiratoryMedicine and Allergology, Department of Clinical Sciences, Lund University, BMC B14,Tornavagen 10, SE-221 84 Lund, Sweden. E-mail address: Arne.Egesten@med.lu.se

Abbreviations used in this article: AMP, antimicrobial peptide; BM, basal membrane;Ct, threshold cycles; hBD, human b-defensin; MK, midkine; MS/MS, tandem massspectrometry; SufA, subtilase of Finegoldia magna; TH, Todd–Hewitt broth.

Copyright� 2011 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/11/$16.00

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and inactivates antibacterial molecules such as LL-37 and MIG/CXCL9 (13). In addition, F. magna can neutralize the activity ofAMPs by release of protein FAF from its surface (12, 14).Streptococcus pyogenes is a highly virulent pathogen causing

both superficial and deep severe infections, such as pharyngitis,erysipelas, necrotizing fasciitis, and septic shock (15). Duringinfection and dissemination in tissues, S. pyogenes releases thecysteine-protease SpeB that can degrade several molecules of thehost response (16). In addition, S. pyogenes releases protein SICthat inhibits complement activation and neutralizes AMPs (17–21). Both SpeB and SIC are produced in high amounts duringearly growth phase (20, 22), and both proteins are important forthe virulence of S. pyogenes (22–27).In this study, we show that the commensal F. magna can cir-

cumvent the bactericidal activity of MK, BRAK/CXCL14, andhBD-3 by releasing SufA and protein FAF. In contrast, the virulentpathogen S. pyogenes inactivates MK, BRAK/CXCL14, hBD-2,and hBD-3 by release of SpeB and protein SIC. The findingsexplain how F. magna can create a protected ecological nicheduring noninflamed conditions, whereas S. pyogenes possessespotent means to avoid a broad range of host defense moleculesproduced in response to inflammation during invasive infection.These properties are likely to reflect different strategies used bya permanently colonizing commensal and a highly virulent path-ogen, the latter operating during the limited time course of in-vasive disease.

Materials and MethodsProteins and peptides

Native SufA and SpeB were purified as previously described (13, 28).Recombinantly expressed protein FAF (aa 28–616) was obtained as a fu-sion protein with GST as described (12). Human recombinant MK andBRAK/CXCL14 were from PeproTech (Rocky Hill, NJ). hBD-2 and hBD-3 were from Creative Peptides (Shirley, NY).

Human skin samples

Samples from human skin wounds were obtained under protocols approvedby the Ethics Committee at Lund University (LU 509-01 and LU 708-01).Nonwounded human skin was obtained by taking punch biopsies fromhealthy donors, and skin wound samples were obtained by making newpunch biopsies from the edges of the initial biopsies. Parts of the sampleswere fixed in formalin (10%) for immunohistochemistry. For cDNAmicroarray analysis, as much dermal tissue as possible was removed bydissection from the biopsies. The remaining tissue was washed thoroughlyin sterile sodium chloride (0.9%) to remove infiltrating inflammatory cells.This procedure ensured that the samples mainly consisted of epidermis.RNA was isolated from these samples and used for cDNA microarrayanalysis.

To obtain ex vivo infestation, skin biopsies from healthy skin wereincubated with F. magna bacteria (strain ALB8) as described (12). In brief,punch biopsies were incubated with F. magna (2 3 108 bacteria) in PBSfor 1 h, washed, and incubated under anaerobic conditions at 37˚C for anadditional 48 h. Following a washing step with PBS plus 0.05% Tween 20,samples were fixed in 2.5% glutaraldehyde in 0.15 M sodium cacodylate(pH 7.4), postfixed in 1% osmium tetroxide, and prepared for transmissionelectron microscopy (see below).

A skin biopsy from a patient presenting with erysipelas (S. pyogenesinfected), a kind gift from Dr. Adam Linder, Lund University, SkaneUniversity Hospital, was processed as described (29).

Immunohistochemistry for light microscopy

After fixation in formalin (10%), the specimens were dehydrated, embed-ded in paraffin, placed on Superfrost Plus glass slides (Menzel-Glazer,Braunschweig, Germany), incubated for 2 h at 60˚C, deparaffinized inTissue Clear (Sakura Finetek, Zoeterwoude, The Netherlands), and rehy-drated in graded alcohol and water. The slides were then treated with DakoAg Retrieval Solution (DakoCytomation, Glostrup, Denmark) for 40 minat 97˚C, followed by blocking in TBS supplemented with 0.05% Tween 20,1% BSA, for 45 min at room temperature. After three 15-min washings,the specimens were incubated at room temperature for 1 h with polyclonal

Abs diluted (5 mg/ml) in TBS supplemented with 0.05% Tween 20, 1%BSA. After three 20-min washings in TBS with 0.05% Tween 20, theslides were incubated with a MACH 3 Probe alkaline phosphatase Polymerkit (Biocare Medical, Concord, CA) for 10 plus 10 min, followed by three15-min washes. Color was developed using Vulcan Fast Red chromogen(Biocare Medical, Concord, CA), and the slides were counterstained withHarris Hematoxylin (Histolab, Goteborg, Sweden).

Cell culture and stimulation of cells

Primary human keratinocytes were obtained from Cascade Biologics(Portland, OR) and cultured in serum-free medium (KGM2 Bullet kit) fromCambrex (Karlskoga, Sweden). Cells were stimulated, beginning 24 h aftercomplete confluence was reached with conditioned medium from PBMCsthat had been stimulated with LPS (100 ng/ml, Escherichia coli O55:B5;Sigma-Aldrich, St. Louis, MO) for 4 d as described (27). After stimulationof the keratinocytes for 48 h, the cells were solubilized in TRIzol (Invi-trogen, Carlsbad, CA) and RNA purified according to the instructions fromthe manufacturer.

RNA isolation and microarray analysis

Total RNAwas isolated with TRIzol (Invitrogen, Carlsbad, CA) accordingto the recommendations of the supplier and resuspended in 0.1 mM EDTA.The concentration was determined by spectrophotometric measurement.

For gene expression analysis, total RNAwas biotinylated and hybridizedto HumanGenomeU133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA)according to the instructions by the manufacturer. The microarray fluo-rescence signals were normalized using the GeneChip Operation Software(GCOS ver. 1.4; Affymetrix). The analysis was performed essentially aspreviously described (30). To calculate fold change in mRNA expression,the cDNA hybridization signals for MK and BRAK/CXCL14 obtainedfrom wounds on day 4 were compared with the signals obtained fromnonwounded skin. Microarray data are available through the MinimumInformation About a Microarray Experiment database (http://www.ebi.ac.uk/arrayexpress; accession number E-MEXP-3305; username: Reviewer_E-MEXP-3305; password: 18dxiehx).

Quantitative real-time RT-PCR

cDNA was synthesized from 400 ng purified RNA from the keratinocyteculture using iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) accordingto the instructions given by the manufacturer. The expression of MK andBRAK/CXCL14 together with G3PDHwas analyzed using iQ SYBRGreenSupermix (Bio-Rad). Primers for real-time PCRwere:MK forward, 59-GCTTCC TCC TCC TCA CCC TC-39 and reverse, 59-TTA TCT TTC TTTTTG GCG ACC G-39; and BRAK/CXCL14 forward, 59-CTC AAG TTTGGT TGC CAG AA-39 and reverse, 59-GAA ATC CTC AGG TTG GGAAA-39. Amplification was performed at 55˚C for 40 cycles in the iCyclerThermal Cycler (Bio-Rad) and data analyzed using iCycler iQ OpticalSystem Software (Bio-Rad). To calculate fold change in mRNA levels, thethreshold cycles (Ct) of MK and BRAK/CXCL14, respectively, weresubtracted from the Ct of the G3PDH housekeeping gene, thus obtainingnormalized Ct. To calculate the difference in mRNA levels for MK andBRAK/CXCL14, values of resting keratinocytes were subtracted fromthose of stimulated keratinocytes and expressed as ddCT. Negative value ofddCT denotes downregulation, and positive ddCT denotes upregulation.

Bacterial strains and growth conditions

F. magna strains 505 and ALB8 were isolated at Lund University Hospital(Lund, Sweden). S. pyogenes strain AP1 (40/58) of serotype M1 was fromthe World Health Organization Collaborating Centre for Reference andResearch on Streptococci, Prague, Czech Republic. AP1 was grown inTodd–Hewitt broth (TH; BD/Difco, Franklin Lakes, NJ) at 37˚C with 5%CO2. F. magna bacteria were grown in TH supplemented with 0.5%Tween-80 at 37˚C under strict anaerobic conditions (Anaerobic Worksta-tion; Elektrotek, Shipley, U.K.).

Bactericidal assay

F. magna bacteria (505 strain) were cultivated for 24 h (midexponentialgrowth phase, OD620 � 0.4), washed in incubation buffer (10 mM Tris/HCl, 5 mM glucose [pH 7.5]), and adjusted to a concentration of 2 3 106

CFU/ml. Fifty microliters bacterial solution (∼105 CFU) was incubated inbuffer or with various concentrations of MK, BRAK/CXCL14, hBD-2, orhBD-3 for 1 h under strict anaerobic conditions. To quantify the bacteri-cidal activity, serial dilutions of the mixtures were plated on TH-agar(supplemented with Tween-80) in duplicates and incubated at 37˚C for3 d under anaerobic conditions. The number of CFUs was then determined.

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S. pyogenes (AP1 strain) was cultivated to midexponential growth phase(OD620 � 0.4). The bacteria were washed, diluted in incubation buffer, and∼105 CFU were incubated in buffer or with various concentrations of MK,BRAK/CXCL14, hBD-2, or hBD-3 for 1 h at 37˚C. The mixtures werediluted, plated on TH-agar, incubated at 37˚C overnight, and the number ofCFUs were counted. All dilutions were performed in incubation buffer.Bacteria were also incubated with SufA- and SpeB-degraded peptides for 1h at 37˚C, and the bactericidal activity was quantified as described above.

Cleavage of AMPs with SufA and SpeB

Digestion of MK, BRAK/CXCL14, hBD-2, or hBD-3 with SufA and SpeBwas performed at 37˚C for 1 and 18 h, respectively. For cleavage withSufA, 5 mg peptides were incubated with 0.5 mg enzyme in PBS. Thereactions were terminated by the addition of PMSF (Sigma-Aldrich) toa final concentration of 5 mM. In the case of SpeB, 5 mg peptides wereincubated with 0.5 mg enzyme in PBS supplemented with DTT (finalconcentration 10 mM). The reactions were terminated by the addition ofthe cysteine-protease inhibitor E64 (final concentration 10 mM). Thecleaved peptides were separated and analyzed by Tricine SDS-PAGE.

SDS-PAGE

SDS-PAGE was performed as described by Laemmli using a total poly-acrylamide concentration of 10% and 3.3% cross-linking. Analysis ofpeptide degradation was performed with Tricine SDS-PAGE (31) usinga polyacrylamide concentration of 16.5% and 3.3% cross-linking. Gelswere stained with Coomassie R-250.

Mass spectrometry

Obtained Coomassie-stained gel protein bands after SufA digestion wereselected, cut, washed with 40% (v/v) acetonitrile, and evaporated to dryness.Reduction was performed with 10 mM DTT at 56˚C for 30 min, alkylationwith 30 mM iodoacetamide, and digestion with Asp-N endopeptidase(Roche Diagnostics, Mannheim, Germany; 25 mg/ml) in 25 mM ammo-nium bicarbonate buffer (pH 7.8) for 16 h at 37˚C. After extraction of gelpieces with 75% acetonitrile in 5% trifluoroacetic acid, peptides weresubjected to reversed-phase HPLC separation (CapLC; Waters, Man-chester, U.K.) and subsequent sequencing on a Q-TOF Ultima API MS/MS(Waters, Manchester, U.K.). MK and BRAK/CXCL14 incubated withSufA were also taken directly to liquid chromatography tandem massspectrometry (MS/MS) for analysis of obtained shorter peptide products.The MS/MS results were interpreted using the Peptide Mass Fingerprinttool of the Mascot search engine (http://www.matrixscience.com). Settingsof fixed and variable modifications were used. Experimental data werecompared with Asp-N cleavage of the MK and BRAK/CXCL14 sequencesusing the PeptideCutter tool (http://www.expasy.org).

Transmission electron microscopy

For negative staining, bacteria were incubated with intact AMPs or pro-teolytic fragments as described above, followed by processing for trans-mission electron microscopy as previously described (32). In short, samples(5 ml) were absorbed for 2 min onto the grids followed by two washes withdistilled water. The samples were stained for 3 s with 0.75% uranyl for-mate droplets, followed by staining for an additional 30 s with 0.75%uranyl formate.

For immunolabeling, fixed and washed samples were subsequentlydehydrated in ethanol and further processed for epon embedding. Sectionswere cut with an LKB ultratome (LKB, Bromma, Sweden) and mounted ongold grids. For immunostaining, the grids were floated on top of drops ofimmune reagents displayed on a sheet of parafilm. For inactivation ofglutaraldehyde and osmium tetroxide used during fixation, saturated so-dium metaperiodate was applied, and for elimination of unspecific binding,the grids were then incubated with 50 mM glycine, and 5% (v/v) goatserum in incubation buffer (0.2% Aurion-BSA [Aurion] in PBS [pH 7.6]).This blocking procedure was followed by overnight incubation withpolyclonal rabbit Abs: anti-SufA, anti-FAF, anti-SpeB, anti-SIC, and

FIGURE 1. MK and BRAK/CXCL14 expression in skin. A, Bound

specific Abs against MK and BRAK/CXCL14, respectively, were detected

by secondary alkaline phosphatase-conjugated Abs, reacting with a sub-

strate that is deposited as a red stain. MK and BRAK/CXCL14 are detected

in epidermis of normal skin. MK shows a stronger staining in the basal

parts, whereas BRAK/CXCL14 is more evenly distributed (top panel). In

skin from an acute wound (day 4; bottom panel), dispersed expression of

MK is seen in the epidermal layer, whereas BRAK/CXCL14 expression

is decreased. Replacement of the specific Abs with rabbit control IgG

resulted in loss of staining. B, Electron micrograph showing overview with

basal cell at the epidermal/dermal junction (stratum germinativum, aster-

isk; top left panel) and prickle cell in stratum spinosum in the epidermis of

healthy skin. Scale bar, 5 mm. Subcellular localization of MK and BRAK/

CXCL14 in keratinocytes was determined using immunogold where bound

specific Abs were detected using secondary Abs conjugated with colloidal

gold (10 nm). Both MK and BRAK/CXCL14 are seen in association with

the BM (lamina densa; asterisk), at the nuclear membrane (NM), and in

association with the plasma membrane (PM) in keratinocytes of healthy

skin, whereas only weak labeling for BRAK/CXCL14 was seen in kera-

tinocytes of skin wounds after 4 d. Scale bar, 1 mm. C, To compare MK

and BRAK/CXCL14 gene expression, mRNAwas isolated from epidermis

of healthy skin and of epidermis obtained by biopsy at the wound edges

4 d postinjury. cDNA microarray revealed an ∼3-fold upregulation of MK

gene expression, whereas BRAK/CXCL14 was downregulated (left panel).

Primary keratinocytes were stimulated with LPS-conditioned medium

from PBMC for 48 h. MK and BRAK/CXCL14 expression were de-

termined by quantitative RT-PCR. The chart depicts the difference in

mRNA levels (ddCt) in normalized Ct (dCt) (calculated by subtracting the

MK and BRAK/CXCL14 Ct from the Ct of the G3PDH housekeeping gene

that was used as reference) between stimulated wells compared with

nonstimulated controls. A negative ddCT value depicts downregulation and

a positive value upregulation. Aweak upregulation of MK gene expression

was found, whereas BRAK/CXCL14 was downregulated by ∼12 threshold

cycles (right panel).

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polyclonal goat anti-MK, and anti-BRAK/CXCL14 at 4˚C. Signal forbinding was detected with EM goat anti-rabbit IgG 10 nm Gold (BBIn-ternational, Cardiff, U.K.) and EM rabbit anti-goat IgG 20 nm Gold(BBInternational), respectively. Samples were finally stained with uranylacetate and lead citrate before being observed and evaluated as above.They were examined with a Jeol JEM 1230 EX transmission electronmicroscope (Jeol) operated at 60 kV accelerating voltage. Images wererecorded with a Gatan Multiscan 791 charge-coupled device camera(Gatan, Pleasanton, CA).

To evaluate the concentrations of the AMPs and the bacterial proteins inskin, the gold probe density on specific primary Abs and on normal rabbitIg controls was quantified by taking 30 random electron micrographs onindividual skin samples. Micrographs were masked with overlays repre-senting 1 mm2. The number of gold particles within different 1-mm2 win-dows was determined manually by point counting in each micrograph.Molar Ag concentrations were then calculated on characteristics of Agretrieval procedures (33–35) and predictions on immunolabeling effi-ciency, surface relief of thin sections, and surface accessibility of randomlydispersed molecules as described (36).

Surface plasmon resonance spectrometry

Protein FAF, protein SIC, or hBD-3 was immobilized via amine couplingin flow cells of a CM5 sensorchip (BIAcore, Uppsala, Sweden). The im-mobilization levels were ∼1000 resonance units. A flow cell subjected tothe coupling reaction without added protein was used as a control for bulkrefraction index changes. For affinity measurements, binding and disso-ciation were monitored in a BIAcore 2000 instrument (BIAcore). Differentconcentrations of proteins were injected over the coated surface at 10 ml/min and 25˚C (in running buffer: 10 mM HEPES [pH 7.5], 150 mM NaCl,0.005% surfactant P20, and 3.4 mM EDTA). The FAF, SIC, or hBD-3surfaces were regenerated by injection of a 200-ml 2-min pulse of run-ning buffer containing 2 M NaCl followed by an extensive wash procedure.After X and Y normalization of data, the blank curves from the controlflow cell of each injected concentration were subtracted. The association(ka) and dissociation (kd) rate constants were determined simultaneouslyusing the equation for 1:1 Langmuir binding in the BIA Evaluation 3.1software (BIAcore). The binding curves were fitted locally, and the equi-librium dissociation constants (KD) were calculated from mean values ofthe obtained rate constants.

ResultsExpression of MK and BRAK/CXCL14 in the epidermis duringhealthy and inflamed conditions

Using immunohistochemistry, both MK and BRAK/CXCL14could be detected in the epidermis of skin obtained fromhealthy donors (Fig. 1A). Labeling for MK was most intense inthe basal parts of epidermis. However, in biopsies from woundedges 4 d postinjury, a more dispersed staining for MK was ob-served, whereas the staining for BRAK/CXCL14 was less intense,suggesting a reciprocal gene regulation for these genes duringinflammation.Subcellular localization of MK and BRAK/CXCL14 in kerati-

nocytes was determined using immunoelectron microscopy. BothMK and BRAK/CXCL14 are seen in association with the BM(lamina densa; asterisks, Fig. 1B), at the nuclear membrane, and inassociation with the plasma membrane in keratinocytes of healthyskin, whereas only weak labeling for BRAK/CXCL14 was seen inkeratinocytes of skin wounds after 4 d (Fig. 1B).Using cDNA microarray, an ∼3-fold increase in MK expression

was seen 4 d after injury, whereas BRAK/CXCL14 was down-regulated (Fig. 1C). To investigate the influence of inflammatorymediators on MK and BRAK/CXCL14 expression in vitro, PBMCwere stimulated with LPS (100 ng/ml) for 4 d. The conditionedmedium was then used to stimulate primary human keratinocytesfor 48 h. The conditioned medium doubled the MK expression,whereas BRAK/CXCL14 expression was downregulated by 12threshold cycles (Fig. 1C). Downregulation of BRAK/CXCL14expression in inflammatory skin disorders has been describedpreviously (3).

Bactericidal activity of MK, BRAK/CXCL14, hBD-2, andhBD-3 against F. magna and S. pyogenes

Previous studies have demonstrated that MK and BRAK/CXCL14both possess antibacterial activity (3, 5). Having seen the strongupregulation of MK and the downregulation of BRAK/CXCL14during inflammation, we therefore investigated whether the ac-tivity of the proteins differ depending on the bacterial speciesbeing a commensal or a pathogen. In these experiments, wecompared the sensitivity of the commensal F. magna (strain 505)and the pathogenic S. pyogenes (strain AP1) in a viable countassay. Interestingly, MK was more potent in killing S. pyogenesbacteria, whereas BRAK/CXCL14 killed F. magna bacteria moreefficiently (Fig. 2A, 2B). Thus, at a MK concentration of 0.1 mM,∼100% of S. pyogenes bacteria were killed, whereas 1 mM of thepeptide was required to achieve 100% killing of F. magna bac-teria. In contrast, 0.1 mM BRAK/CXCL14 killed ∼80% of F.magna bacteria, but only 20% of the S. pyogenes bacteria. Duringinfection and inflammation, the b-defensins hBD-2 and hBD-3 areupregulated and secreted by keratinocytes, and the bactericidalactivity of these peptides was also compared against F. magna(strain 505) and S. pyogenes (strain AP1). Of these peptides, hBD-3 was the most potent and efficiently killed both strains at 1 mM(Fig. 2D). At this concentration, hBD-2 only slightly affected F.magna bacteria (Fig. 2C) and, to achieve 100% killing of both F.magna and S. pyogenes bacteria with this peptide, concentrationsof 10 mM were required.

Proteolytic activity from SufA of F. magna and SpeB of S.pyogenes against MK, BRAK/CXCL14, hBD-2, and hBD-3 andcharacterization of residual bactericidal activity

SufA of F. magna has proteolytic activity against LL-37 andthe chemokine MIG/CXCL9 (13). Also, SpeB of S. pyogenes isknown to cleave LL-37 (37) and several chemokines (38). Thus,we incubated MK, BRAK/CXCL14, hBD-2, and hBD-3 withSufA and SpeB, respectively, for 1 and 18 h, and the resultingfragments were analyzed by Tricine SDS-PAGE (Fig. 3). SpeB

FIGURE 2. Bactericidal activity of MK (A), BRAK/CXCL14 (B), hBD-

2 (C), and hBD-3 (D) against F. magna and S. pyogenes. Bacteria were

grown to midlogarithmic phase and incubated with peptides at the in-

dicated concentrations for 1 h. To determine bactericidal activity, the

number of CFUs was determined and compared with that obtained after

incubation in buffer alone. The data shown represent mean6 SEM of three

to five separate experiments.

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rapidly cleaved all polypeptides, and after 1 h of incubation, theywere almost completely degraded (Fig. 3A–D). In contrast, SufAcleavage of MK generated several smaller fragments, and twostable fragments were observed over time (Fig. 3A), whereasBRAK/CXCL14 was completely degraded after 18 h of in-cubation (Fig. 3B). To identify the cleavage sites in MK andBRAK/CXCL14, the protein bands, indicated in Fig. 3A and 3B,were excised and subjected to MS/MS analysis. In addition,cleaved peptides in solution were also analyzed by MS/MS. Thepredicted SufA cleavage sites are indicated in the amino acidsequences of MK and BRAK/CXCL14 (Fig. 3E, 3F). Further-more, in the models of these molecules, the amino acid residuesprone to cleavage are indicated (Fig. 3E, 3F). The bactericidalactivity of MK has been located to a region in the N-terminaldomain and to the C-terminal tail (5). As shown in Fig. 3E, sev-eral cleavage sites for SufA are identified in these regions. Cat-ionic amino acids (i.e., arginine and lysine) are characteristicfeatures of AMPs, and the majority of cleavage sites included

either of these amino acids. SufA did not degrade the defensinshBD-2 and hBD-3, neither as seen after SDS-PAGE (Fig. 3C, 3D)nor as determined by MS/MS.Next, the bactericidal activity of the SufA-generated fragments

of MK and BRAK/CXCL14 was tested to compare the activityagainst F. magna (505 strain) and S. pyogenes (AP1 strain). Thefragments generated after 1 h of digestion with SufA were stillable to efficiently kill S. pyogenes, but killed F. magna to a sig-nificantly lesser degree. The bactericidal activity was lost fol-lowing further digestion (Fig. 4A, 4B). Electron microscopyrevealed rupture of bacterial membranes and leakage of in-tracellular contents in the presence of the intact polypeptides, butto a lesser extent in the case of F. magna, when incubated withSufA-cleaved MK and BRAK/CXCL14, respectively (Fig. 4C).This suggests that the activity of SufA against these constitutivelyexpressed AMPs will provide survival advantages for F. magna,at least during healthy conditions and during early stages of in-fection.

FIGURE 3. Proteolytic activity of SufA and SpeB, respectively, against MK (A), BRAK/CXCL14 (B), hBD-2 (C), and hBD-3 (D). The peptides (5 mg)

were incubated with the respective enzymes (0.5 mg) for 1 and 18 h at 37˚C, and cleavage products were analyzed by Tricine SDS-PAGE. The fragments of

MK and BRAK/CXCL14 generated by SufA after 1 h were characterized by MS/MS. The primary amino acid sequences and models of MK and BRAK/

CXCL14 with predicited SufA cleavage sites indicated in red are shown in E and F, respectively. No cleavage of hBD-2 or hBD-3 with SufA occurred as

determined by MS/MS.

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Neutralizing capacity from protein FAF of F. magna andprotein SIC of S. pyogenes against MK and BRAK/CXCL14

Most strains of F. magna express the surface protein FAF, whichalso is released into the surrounding environment, partly throughaction of SufA (12, 14). Thus, we asked whether FAF could bindand inhibit the activity of the AMPs described above. First, pos-sible interference of FAF with the killing of F. magna bacteria byMK and BRAK/CXCL14 was investigated. The F. magna 505strain was incubated with bactericidal concentrations of MK (1mM), BRAK/CXCL14 (1 mM), and hBD-3 (1 mM), respectively,in presence of various ratios of FAF. As shown in Fig. 5A, FAFdose dependently blocked the activity of all three peptides. No-tably, the defensin hBD-3, which was resistant to cleavage bySufA, was the peptide most efficiently blocked by FAF. To in-vestigate binding characteristics between FAF and the variouspeptides, surface plasmon resonance spectrometry was used. FAFwas immobilized to the surface of a chip, and MK, BRAK/CXCL14, or hBD-3 were injected and left to interact to satura-tion. Binding to FAF was obtained with MK (KD = 13 nM) andBRAK/CXCL14 (KD = 8.9 nM), but not hBD-3. However, when,instead, hBD-3 was immobilized and FAF injected over the sur-face, a stable interaction was observed (KD = 0.7 nM) (Fig. 5A).Due to the relatively low and sometimes inconsistent bactericidalactivity of hBD-2 in viable counts experiments, blocking experi-ments were not performed with this peptide. In addition, surfaceplasmon resonance experiments showed no or low binding of FAFto hBD-2 (data not shown).Then we analyzed whether protein SIC, which is produced by

some strains of S. pyogenes including the highly virulent M1 se-rotype (21), is also able to inhibit the bactericidal activity of MKand BRAK/CXCL14. SIC blocked the killing of S. pyogenes withthese peptides, although not as efficiently as protein FAF (Fig.5B). Furthermore, using surface plasmon resonance spectrometry,SIC was found to bind to both MK and BRAK/CXCL14, but witha more transient kinetic compared with the interaction of FAF(Fig. 5B). SIC has previously been shown to bind and interferewith the activity of human b-defensins and was therefore notanalyzed (19, 39).

Colocalization of MK and BRAK/CXCL14 with SufA and FAFof F. magna and SpeB and SIC of S. pyogenes in infested skin

Healthy skin infested ex vivo with F. magna and biopsies fromS. pyogenes-infected skin (erysipelas) were investigated usingelectron microscopy (Fig. 6A, upper panels). In both cases, bac-teria were seen in the vicinity of keratinocytes, with characteristicmorphological features (i.e., being coccoid and, in the case ofS. pyogenes, an extracellular rim of M protein).Using immunoelectron microscopy on healthy skin biopsies

infested with F. magna ex vivo, MK and BRAK/CXCL14 werecolocalized with SufA and FAF in close proximity of F. magnabacteria (Fig. 6A). In skin biopsies from erysipelas (caused byS. pyogenes), MK and BRAK/CXCL14 colocalized with SpeBand SIC, respectively, close to the bacterial surface (Fig. 6A). Todetermine the concentrations of the AMPs and the bacterialproteins in the skin samples, the gold probe density on thespecific primary Abs and on normal rabbit Ig controls wasquantified as described in Materials and Methods. The valuesobtained (Fig. 6B) confirmed increased production of MK anda decreased production of BRAK/CXCL14 during infection.Skin infected ex vivo with F. magna may resemble the situationduring an opportunistic infection causing inflammation ratherthan colonization as a commensal. The findings are summarizedin Fig. 7.

DiscussionIn this study, we show that F. magna, a member of the normalmicrobiota, and the virulent pathogen S. pyogenes differentlymodulate the activity of constitutively and induced AMPs, re-spectively. Although F. magna possess the means (i.e., SufA andprotein FAF) to circumvent the constant pressure of the consti-tutively expressed AMPs MK and BRAK/CXCL14, S. pyogenespossess a broader activity (via SpeB and protein SIC) includingthe important AMPs hBD-2 and hBD-3 that are expressed duringinflammation (summarized in Figure 7).During healthy, noninflamed conditions, F. magna colonize

the basal parts of epidermis and are likely to encounter the

FIGURE 4. Antibacterial activity of SufA-gener-

ated MK and BRAK/CXCL14fragments against F.

magna and S. pyogenes, respectively. A and B, Bac-

teria, grown to midlogarithmic phase, were incubated

for 1 h at 37˚C with intact peptides (MK and BRAK/

CXCL14, respectively, at 1 mM) or with fragments

thereof generated by SufA cleavage at 1 and 18 h,

respectively. To quantify the bactericidal activity, the

number of CFUs was determined and compared with

that obtained after incubation of bacteria in buffer

alone. The data shown represent mean 6 SEM of

three separate experiments. Student t test for paired

observations was applied to investigate statistical dif-

ferences. C, Electron micrographs showing the mor-

phology of F. magna and S. pyogenes, respectively,

after negative staining. Bacteria were incubated in

buffer alone, MK or BRAK/CXCL14 (1 mM), and

MK or BRAK/CXCL14 that had been subjected to

degradation by SufA for 1 h. Membrane disruption

and leakage of intracellular contents are seen in

the presence of intact MK and BRAK/CXCL14 and

proteolytic fragment, in the latter case to a lesser

degree for F. magna. Scale bar, 100 nm.

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constitutively expressed MK and BRAK/CXCL14, as demon-strated in the current study. However, during inflammation,BRAK/CXCL14 production is downregulated, as seen in atopicdermatitis and psoriasis (3, 40). Epidermal growth factor is up-regulated during inflammation and wound healing and has a stronginhibitory activity on BRAK/CXCL14 gene expression (41). MKis upregulated during inflammation in some tissues of the adultand can be detected in plasma of healthy individuals (42, 43).Binding of many proinflammatory cytokines to their correspond-ing receptors and activation of TLRs by pathogen-associatedmolecular patterns result in activation of NF-kB–dependent genetranscription (44). MK has an NF-kB–responsive element in itspromoter region and is thus likely to be upregulated during in-flammation (45). Another property of MK is that it recruits andactivates neutrophils to sites of inflammation, suggesting severalimportant roles for this growth factor (46).

In a previous study, it was shown that BRAK/CXCL14 hasantibacterial activity against fungi and both Gram-positive and-negative aerobic bacteria (3). The finding in the present work thatBRAK/CXCL14 had higher activity against F. magna than S.pyogenes might reflect this chemokine’s role in controlling thehost burden of commensal bacteria. During inflammation causedby more virulent bacteria, additional AMPs such as hBD-2 andhBD-3 are upregulated to restrict the spread of the infection (47).Thus, in such an event, it is necessary for commensal bacteria tocope with another set of host defense molecules to survive. It istherefore not surprising that F. magna is less sensitive to MK.Interestingly, hBD-2 showed relatively weak antibacterial activityagainst both F. magna and S. pyogenes compared with the otherAMPs investigated. During invasion, S. pyogenes, being a virulentpathogen, operates during a limited timeframe. This may be oneexplanation for SpeB being more efficient and less specific than

FIGURE 5. A, FAF of F. magna inhibits the antibacterial activity of MK, BRAK/CXCL14, and hBD-3. MK, BRAK/CXCL14, and hBD-3 were pre-

incubated with FAF at indicated ratios for 20 min before incubation with bacteria, grown to midlogarithmic phase, for 1 h. To quantify the bactericidal

activity, the number of CFUs was determined and compared with that obtained after incubation of bacteria in buffer alone. The data shown represent mean6SEM of three separate experiments. To investigate the binding kinetics and affinity, surface plasmon resonance was applied. MK and BRAK/CXCL14

were injected over a chip with immobilized FAF. In the case of hBD-3, the setup had to be reversed to achieve binding, and FAF was injected over

immobilized hBD-3. Binding to FAF was obtained with MK (KD = 13 nM), BRAK/CXCL14 (KD = 8.9 nM), but not hBD-3. However, when hBD-3 was

instead immobilized and FAF injected over the surface, a stable interaction was observed (KD = 0.7 nM). B, Protein SIC of S. pyogenes binds and inhibits

the antibacterial activity of MK and BRAK/CXCL14. MK and BRAK/CXCL14 were preincubated with protein SIC at the ratios indicated for 20 min

before incubation with bacteria, grown to midlogarithmic phase, for 1 h. To quantify bactericidal activity, the number of CFUs was determined and

compared with that obtained after incubation of bacteria in buffer alone. The data shown represent mean 6 SEM of three separate experiments. To in-

vestigate binding kinetics using surface plasmon resonance, protein SIC was immobilized on a BIAcore chip (BIAcore) followed by injection of MK and

BRAK/CXCL14.

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SufA, enhancing successful invasion and dissemination, over-whelming the host. In contrast, it is not in the interest of F. magna,being a colonizing commensal, to disturb homeostasis.Common characteristics for AMPs are a high content of the

positively charged amino acids arginine and lysine. Interestingly,SufA preferentially cleaves both MK and BRAK/CXCL14 at thesepositions and thereby reduces the antibacterial activity of thesemolecules. For a commensal bacterium, this is an importantmechanism that will lead to improved survival, unarming consti-tutively produced AMPs, at the site of the colonization. The

preference of SufA to cleave at positions of basic amino acidshas been described for other subtilases of human and plant origin(48). In contrast, SufA cannot cope with the b-defensins, possiblybecause they are more compact molecules. However, F. magnastrains that express FAF have an advantage through the efficientneutralizing capacity of this molecule against hBD-3. The peptidehBD-2 has a relatively low bactericidal activity and may notpresent a similar threat to the bacterium.In the case of a pathogen, such as S. pyogenes, it is important

for the bacterium to modulate a broad spectrum of AMPs to

FIGURE 6. Colocalization of MK and BRAK/

CXCL14 with SufA and FAF of F. magna and

SpeB and SIC of S. pyogenes in infested skin. A,

Healthy skin biopsies, ex vivo infested with F.

magna (left panels) and biopsies from S. pyo-

genes-infected skin (erysipelas; right panels),

were investigated using electron microscopy.

Bacteria are seen in the vicinity of keratinocytes

(top left panel, arrows). Scale bars, 2.5 mm. At

higher magnification, individual bacteria are vi-

sualized. In the case of S. pyogenes, a typical rim

of M protein is seen (upper right panel). Scale

bars, 0.5 mm. Immunoelectron microscopy shows

colocalization of MK (as detected by 10-nm

colloidal gold particles conjugated with second-

ary Abs) and BRAK/CXCL14 (10-nm colloidal

gold particles) with SufA (5-nm colloidal gold

particles) and FAF (5-nm colloidal gold particles),

respectively, in the close proximity of F. magna

bacteria (bottom left panels). Scale bar, 0.1 mm.

In a skin biopsy from erysipelas (caused by S.

pyogenes), MK (as visualized by Abs labeled

with 10-nm colloidal gold particles) and BRAK/

CXCL14 (10-nm colloidal gold particles) coloc-

alize with SpeB (5-nm colloidal gold particles)

and SIC (5-nm colloidal gold particles), re-

spectively, close to the bacterial surface (bottom

right panels). Scale bar, 0.1 mm. B, Morphometric

evaluation of AMPs and bacterial protein con-

centrations in skin. The probe density of gold-

labeled Abs was quantified on electron micro-

graphs of individual skin samples. Molar Ag

concentrations were calculated based on the

number of gold particles per mm2.

FIGURE 7. Schematic illustration of bacterial sur-

vival strategies during noninflamed and inflamed con-

ditions, respectively. F. magna resides in the basal parts

of epidermis, where it adheres to BM-40 of the BM.

Keratinocytes constitutively produce MK and BRAK/

CXCL14, for which bactericidal activity F. magna can

attain by the release of SufA and FAF. Upon invasion

of the virulent pathogen S. pyogenes, the host responds

with inflammation, resulting in downregulation of

BRAK/CXCL14 expression and a concomitant upreg-

ulation of MK, hBD-2, and hBD-3. S. pyogenes can

neutralize these AMPs by release of the cysteine pro-

tease SpeB and the AMP-binding protein SIC, whereas

the countermeasures executed by F. magna are less ef-

ficient in the inflamed context, because SufA has no

effect on hBD-2 and hBD-3.

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efficiently invade host tissues. In this study, we find that SpeBrapidly degrades all investigated AMPs (MK, BRAK/CXCL14,hBD-2, and hBD-3). This is in line with previous studies show-ing a broad capacity for SpeB to cleave AMPs (38).In the case of F. magna, we are not aware of other enzymes or

proteins interacting with AMPs, whereas S. pyogenes produceseveral additional enzymes counteracting innate and adaptive im-mune mechanisms (e.g., SpyCep [degrading IL-8/CXCL8], C5a-peptidase [degrading C5a], IdeS [degrading IgG], and EndoS [gly-canase activity against IgG]). However, the latter enzymes havea very selective mode of action (49).The release of protein FAF and protein SIC by F. magna and S.

pyogenes, respectively, equips the bacteria with means to inacti-vate AMPs at a distance. Interestingly, FAF was proven to bindand neutralize the antibacterial activity of both MK and BRAK/CXCL14 more efficiently than SIC. The survival of F. magnabacteria is critically dependent on balancing the pressure fromconstitutive host-defense activities, limiting its proliferative activ-ity and virulence, whereas virulent pathogens such as S. pyogenesbacteria do not have a biology that needs such considerations.Taken together, the current study demonstrates how F. magna cancounteract the activities of constitutively produced AMPs, thuscreating a protected habitat within the epidermis.

AcknowledgmentsWe thank Pia Andersson and Ulla Johannesson for excellent technical as-

sistance and Dr. Bjorn Walse for molecular models.

DisclosuresThe authors have no financial conflicts of interest.

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