Staphylococcus epidermidis Esp Degrades Specific Proteins Associatedwith Staphylococcus aureus Biofilm Formation and Host-PathogenInteraction

Shinya Sugimoto,a Takeo Iwamoto,b Koji Takada,c Ken-ichi Okuda,a Akiko Tajima,a Tadayuki Iwase,a Yoshimitsu Mizunoea

Department of Bacteriology,a Division of Biochemistry, Core Research Facilities,b and Department of Biochemistry,c The Jikei University School of Medicine, Tokyo, Japan

Staphylococcus aureus exhibits a strong capacity to attach to abiotic or biotic surfaces and form biofilms, which lead to chronicinfections. We have recently shown that Esp, a serine protease secreted by commensal Staphylococcus epidermidis, disassemblespreformed biofilms of S. aureus and inhibits its colonization. Esp was expected to degrade protein determinants of the adhesiveand cohesive strength of S. aureus biofilms. The aim of this study was to elucidate the substrate specificity and target proteins ofEsp and thereby determine the mechanism by which Esp disassembles S. aureus biofilms. We used a mutant Esp protein(EspS235A) with defective proteolytic activity; this protein did not disassemble the biofilm formed by a clinically isolated methicil-lin-resistant S. aureus (MRSA) strain, thereby indicating that the proteolytic activity of Esp is essential for biofilm disassembly.Esp degraded specific proteins in the biofilm matrix and cell wall fractions, in contrast to proteinase K, which is frequently usedfor testing biofilm robustness and showed no preference for proteolysis. Proteomic and immunological analyses showed thatEsp degrades at least 75 proteins, including 11 biofilm formation- and colonization-associated proteins, such as the extracellularadherence protein, the extracellular matrix protein-binding protein, fibronectin-binding protein A, and protein A. In addition,Esp selectively degraded several human receptor proteins of S. aureus (e.g., fibronectin, fibrinogen, and vitronectin) that are in-volved in its colonization or infection. These results suggest that Esp inhibits S. aureus colonization and biofilm formation bydegrading specific proteins that are crucial for biofilm construction and host-pathogen interaction.

Staphylococcus aureus is a major cause of community- and hos-pital-associated infectious diseases in humans, ranging from

minor skin infections, such as furuncles and boils, to severe infec-tions, such as pneumonia, osteomyelitis, endocarditis, and sepsis(1). Colonization of the nose by S. aureus represents a major riskfactor for these infections (2). In recent years, the rapid emergenceof virulent strains resistant to many antibiotics, such as methicil-lin-resistant S. aureus (MRSA), has become a major problemworldwide (3). S. aureus exhibits a strong capacity to attach to thesurface of implanted medical devices and forms multilayeredcommunities of bacteria called biofilms (4).

Biofilm formation proceeds in two distinct phases: primaryattachment and proliferation (5–7). In the human body, the at-tachment directly to the surface of implanted medical devices orhuman extracellular matrix (ECM) and plasma proteins, such asfibronectin (Fn), fibrinogen (Fg), and vitronectin (Vn), is the firststep of S. aureus biofilm formation (8). Then, proliferation pro-ceeds through the production of biofilm matrices that contributeto bacterial accumulation in multiple layers. In staphylococcalbiofilms, cells are embedded in extracellular matrices composed ofproteins, polysaccharides (polysaccharide intercellular adhesin[PIA] or poly-N-acetylglucosamine [PNAG]), extracellular DNA(eDNA), and, presumably, host factors. Some surface proteins,such as major autolysin (Atl) (9, 10), biofilm-associated protein(Bap) (11), clumping factors (ClfA and ClfB) (12), ECM protein-binding protein (Emp) (13), Fn-binding proteins (FnBPA andFnBPB) (14, 15), S. aureus surface proteins (SasC and SasG) (16,17), and staphylococcal protein A (Spa) (18), have been identifiedin S. aureus as being important in cell-to-cell and cell-to-surfaceinteractions. Recently, �-toxin, a neutral sphingomyelinase, wasfound to have a structural role in S. aureus biofilm matrix, incooperation with eDNA from lysed cells (19). Staphylococcal bio-

films display extraordinary resistance to antimicrobial killing,limiting the efficacy of antibiotic therapy, and surgical interven-tion is often required to remove infected tissues or medical de-vices, such as catheters and orthopedic implants. S. aureus is there-fore a frequent cause of biofilm-associated infections, which are atremendous burden on our health care system. The prevention ofbiofilm-associated infections of S. aureus is a major goal of hospi-tal infection control.

Recently, we have reported that Esp, a serine protease pro-duced by a Gram-positive commensal bacterium, Staphylococcusepidermidis, inhibits S. aureus nasal colonization and biofilm for-mation and disassembles preformed S. aureus biofilms (20). Thisbiofilm-degrading enzyme is effective against biofilms formed byMRSA and vancomycin-intermediate S. aureus (VISA). Thesefindings attracted us to develop an innovative strategy for control-ling the colonization and infection of the pathogen by using Esp orits derivatives. However, how Esp inhibits nasal colonization andbiofilm formation of S. aureus and disassembles preformed bio-films has largely been unknown. Since Esp is an extracellular ser-ine protease (20–23), we hypothesized that Esp degrades variousproteins, including determinants for the adhesive (adhesion to

Received 9 September 2012 Accepted 7 January 2013

Published ahead of print 11 January 2013

Address correspondence to Shinya Sugimoto, ssugimoto@jikei.ac.jp, orYoshimitsu Mizunoe, mizunoe@jikei.ac.jp.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.01672-12.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.01672-12

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abiotic or biotic surfaces) and cohesive (bacterium to bacterium)strength of S. aureus biofilms. The goal of the present study was toelucidate target proteins of Esp, including S. aureus surface pro-teins and host ECM proteins which are crucial for S. aureus colo-nization and biofilm formation.

MATERIALS AND METHODSProteins. Fn and hemoglobin (Hg) from human, papain from Caricapapaya, proteinase K from Tritirachium album, thermolysin from Bacillusthermoproteolyticus, Vn from human plasma, and protein A from S. au-reus were purchased from Sigma. Fg from human plasma was from Wako.Recombinant S. epidermidis Esp was purified as previously described (23).Dispersin B from Aggregatibacter actinomycetemcomitans was gifted fromKaren LoVetri (Kane Biotech Inc.).

Bacterial strains. Bacterial strains used in this study are listed in TableS2 in the supplemental material. E. coli strains were grown at 37°C in LBmedium containing appropriate antibiotics (100 �g/ml ampicillin). S.aureus strains were grown at 37°C in brain heart infusion (BHI) medium(Difco) or BHI medium supplemented with 1% glucose (BHIG).

Plasmid construction. The efb gene was amplified from S. aureusMR23 genomic DNA using a primer set, Efb-BamHI-F and Efb-His-HindIII-R (see Table S3 in the supplemental material), and cloned intopNCMO2 (TaKaRa) using BamHI and HindIII restriction sites. The re-sulting plasmid was termed pEfb-His (see Table S2 in the supplementalmaterial). The eap gene was amplified from S. aureus MR23 genomic DNAusing a primer set, Eap-PstI-brevi-F and Eap-His-XbaI-R (see Table S3 inthe supplemental material), and cloned into pNCMO2 using PstI andXbaI restriction sites. The resulting plasmid was named pEap-His (seeTable S2 in the supplemental material). The S. aureus MR23 eap genecontaining the 5= untranslated region (UTR) was ligated with the gfpuv

gene by splicing by overlap extension PCR (SOE-PCR) (24). In the firstPCR, the eap and gfpuv genes were amplified using primer sets Eap-5=UTR-PstI-F/Eap-GFP-R and Eap-GFP-F/GFP-XbaI-R, respectively.The second PCR was performed to amplify the gfp-fused eap gene usingthese amplified fragments as the templates and a primer set: Eap-5=UTR-PstI-F/GFP-XbaI-R. The resulting fragment was digested by PstI and XbaIand was then cloned into pNCMO2 using PstI and XbaI restriction sites.The gfpuv gene was amplified from pGFPuv (Clontech). The DNA frag-ment containing sarA promoter P1 of S. aureus SH1000 was also ligatedwith the gfpuv gene by SOE-PCR using primers PsarA-PstI-F, PsarA-GFP-R, PsarA-GFP-F, and GFP-HindIII-R, and the resulting fragmentwas cloned into pNCMO2 using PstI and HindIII restriction sites. Theconstructed plasmids were named pEap-GFP and pP1GFP, respectively(see Table S2 in the supplemental material).

Purification of recombinant proteins. Recombinant S. aureus pro-teins, including the extracellular adherence protein (Eap) fused with aC-terminal His6 tag and extracellular fibrinogen-binding protein (Efb)fused with a C-terminal His6 tag, were purified using a Brevibacillus ex-pression-secretion system as previously described (23), with a minormodification for Eap. Eap was associated on the surface of Brevibacilluschoshinensis cells and was therefore extracted with 1 M NaCl. The extractwas subjected to Ni-affinity column chromatography. Purified recombi-nant proteins were dialyzed against phosphate-buffered saline (PBS) buf-fer containing 10% glycerol and stored at �80°C. Protein concentrationswere determined using a protein assay kit (Bio-Rad).

Biofilm formation. Single colonies of S. aureus strains were grown in5 ml BHI medium at 37°C by shaking overnight. The overnight cultureswere 1,000-fold diluted in 5 ml BHIG medium and then placed in 35-mm-diameter polystyrene dishes (Nunc). The cultures were incubated at 37°Cfor 24 h under static conditions. After the incubation, the dishes weregently shaken and the culture media were discarded. The dishes were thenwashed twice with PBS and dried under atmosphere.

Cell aggregation and biofilm formation of B. choshinensis cells weretested as follows: B. choshinensis cells harboring pNCMO2 (an empty vec-tor), pEap-His, or pEfb-His (see Table S2 in the supplemental material)

were grown in MT medium (1% glucose, 1% peptone, 0.5% meat extract,0.2% yeast extract, 0.001% FeSO4 · 7H2O, 0.001% MnSO4 · 4H2O,0.0001%, ZnSO4, 20 mM MgCl2) supplemented with 10 �g/ml neomycin(MTNm) at 30°C under shaking conditions at 150 rpm for 4 days. Thecultures were incubated in standing glass tubes at room temperature for 3h, and cell aggregations were observed. For the biofilm formation assay,these strains were grown in MTNm at 30°C under shaking conditions at150 rpm for 2 days. The cultures were then diluted 100-fold in freshMTNm, and the diluted cultures (200 �l) were incubated at 30°C understatic conditions in 96-well polystyrene plates (Corning) for 2 days. Afterthe incubation, the plates were gently shaken to remove the depositedbacteria and the culture media were discarded. After twice washing withPBS, biofilms were stained with 0.25% safranin and eluted with 70% eth-anol (200 �l). The absorbance at 492 nm of the ethanol extract was mea-sured using a microplate reader (Tecan). Assays were performed in trip-licate.

Biofilm destruction assay. Biofilms of various S. aureus strainsformed in 35-mm-diameter polystyrene dishes were incubated with theindicated proteases, 5 �g/ml Esp, 5 �g/ml thermolysin, 5 �g/ml protei-nase K, and 5 �g/ml papain, in 100 mM Tris-HCl (pH 8.0) at 37°C for theindicated periods. After the incubation, the dishes were gently shaken andthe supernatants were discarded. The dishes were then washed twice with10 mM Tris-HCl (pH 8.0) and dried under atmosphere. The pictures ofthe dishes were taken by digital camera. Profiles of the fractionated pro-teins were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis (PAGE) with Coomassie brilliant blue (CBB) staining.

Isolation of biofilm matrix proteins, cell wall-anchoring proteins,and cytoplasmic proteins. S. aureus proteins were fractionated into bio-film matrix, cell wall, and cytoplasmic fractions. Biofilms of various S.aureus strains formed in the test dish were detached by mixing with wash-ing buffer composed of 10 mM Tris-HCl (pH 8.0) and protease inhibitorcocktail (Nacalai) and transferred into a test tube. If required, the biofilmwas mechanically peeled away from the surface of the test dish. Theyielded biofilm was shaken by vortexing and centrifuged at 5,000 � g for10 min (washing fraction). The supernatant was transferred into a newtest tube. The pellet was dissolved in a matrix extraction buffer composedof 10 mM Tris-HCl (pH 8.0), 1 M NaCl, and protease inhibitor cocktail,and the mixture was incubated at 25°C for 30 min with gentle rotation(biofilm matrix fraction). After the incubation, the mixture was centri-fuged at 5,000 � g for 10 min and the supernatant was transferred into anew test tube. The fractionated proteins were analyzed by SDS-PAGE. Ifrequired, dispersin B (0.2 mg/ml) was added to the biofilm matrix fractionto check for components of the insoluble materials in the fraction. Thepellet was dissolved in a cell wall-anchored protein extraction buffer com-posed of 10 mM Tris-HCl (pH 8.0), 25% sucrose, 0.2 mg/ml lysostaphin,and protease inhibitor cocktail and incubated at 37°C for 30 min understatic conditions. After the incubation, the mixture was centrifuged at15,000 � g for 10 min and the supernatant was transferred into a new testtube (cell wall fraction). For the analysis of cytoplasmic proteins, the pelletwas dissolved in 10 mM Tris-HCl (pH 8.0) and protease inhibitor cocktailand sonicated. After centrifugation at 15,000 � g for 10 min, cytoplasmicsoluble proteins were recovered (cytoplasmic fraction).

Cell surface proteins, which were degraded by various proteases, werealso fractionated from S. aureus biofilms and analyzed by SDS-PAGE asdescribed above.

Scanning electron microscopy. Structures of S. aureus SH1000 bio-films with or without 1 M NaCl treatment and the NaCl-extracted biofilmmatrix were observed by scanning electron microscopy according to themethod previously reported (25).

Cell aggregation and biofilm promotion by addition of the NaClextract from S. aureus biofilms. S. aureus MS4 was grown in BHI me-dium at 37°C overnight. The overnight culture was diluted 50-fold in BHImedium supplemented with or without 1 M NaCl extract (100 �l) fromSH1000 biofilms as described above. NaCl (1 M, 100 �l) was also usedinstead of the NaCl extract. These diluted cultures were incubated at 37°C

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for 1 h in 35-mm-diameter polystyrene dishes. After the incubation, thedishes were gently shaken and the culture media were discarded. Theplates were then washed twice with PBS buffer and dried under atmo-sphere. The remaining biofilms were stained with 0.1% crystal violet.

Identification of proteins. Cell surface proteins (biofilm matrix andcell wall fractions) isolated from S. aureus MR23 biofilms were separatedby one-dimensional (1D) SDS-PAGE. The major protein bands with ap-parent molecular masses of 60 kDa and 15 kDa were identified by N-ter-minal amino acid sequence analyses as described previously (20). Top-down proteomic analysis was also performed to identify proteins that aredegraded by Esp (details are described in the supplemental material).

Promotion of S. aureus biofilm formation by exogenously supple-mented recombinant proteins. Single colonies of S. aureus MS4 weregrown in 5 ml BHI medium at 37°C overnight. The overnight cultureswere 1,000-fold diluted in 5 ml BHIG medium supplemented with theindicated concentrations (0.05 to 1 �M) of purified Eap and Efb, andthe diluted cultures were then placed into 96-well polystyrene plates. Theplates were incubated at 37°C for 24 h under static conditions. After theincubation, the plates were gently shaken and the culture media werediscarded. The plates were then washed twice with PBS buffer and driedunder atmosphere. The remaining biofilms were stained with 0.1% crystalviolet, and the absorbance at 595 nm was monitored using a microplatereader (Tecan). Assays were performed in triplicate.

Western blotting. Eap in the biofilm matrix fraction of S. aureusMR23 was detected by Western blotting. Anti-Eap polyclonal antibody(primary antibody; Abcam) and goat anti-rabbit IgG-horseradish perox-idase (HRP; secondary antibody; Bio-Rad) were diluted 1,000- and200,000-fold, respectively, in Tris-buffered saline (TBS)–Tween (TBS-T;20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.1% [vol/vol] Tween 20). Sam-ples were separated by SDS-PAGE and transferred to an Immobilon-Pmembrane (Millipore Corp.). To block unspecific interactions with IgG-binding proteins, the membrane was incubated for 1 h with 5% (vol/vol)goat serum (Sigma) dissolved in TBS buffer containing 0.3% (wt/vol)bovine serum albumin (BSA; Sigma) as previously described (26). Spa inS. aureus MR23 biofilm matrix and cell wall fractions was detected usingrabbit IgG (Sigma) and goat anti-rabbit IgG-HRP (Bio-Rad). The primaryantibody and secondary antibody were diluted 20,000-fold in TBS-T.Samples were separated and transferred as mentioned above. The mem-branes were incubated for 1 h with 3% BSA (Sigma) in TBS-T. The mem-brane was treated with the primary antibody at 25°C for 1 h and thenwashed twice with TBS-T. Subsequently, the membrane was incubated inTBS-T containing the secondary antibody. After washing three times withTBS-T, the immunoreactive signals were detected with an ECL-plus de-tection kit (GE Healthcare).

Degradation of purified proteins by Esp in vitro. Purified staphylo-coccal proteins, including Eap and Spa, and human proteins, includingFg, Fn, Vn, and Hg (1 �M each), were incubated with or without 1 �MEsp in PBS buffer at 37°C. At the indicated time points, aliquots (10 �l) ofthe solutions were collected and mixed with 10 �l of 2� SDS samplebuffer (150 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 10% 2-mer-captoethanol). Proteins were analyzed by SDS-PAGE (12% gel) and visu-alized with CBB staining. If required, the band intensity on the gel wasestimated using an ImageQuant system (GE Healthcare).

Fluorescence microscopy. S. aureus RN4220 (27) was used for mod-ification of recombinant plasmids generated in E. coli. S. aureus MR23 wastransformed with pEap-GFP or pP1GFP, both of which were extractedand purified from RN4220. These transformants were inoculated intoBHI medium containing 50 �g/ml neomycin and cultured at 37°C over-night. The overnight cultures were 1,000-fold diluted in BHIG mediumsupplemented with 50 �g/ml neomycin and further incubated at 37°Cunder static conditions. At the indicated times, cell morphology and lo-calization of Eap-GFP were observed by fluorescence microscopy with agreen fluorescent protein (GFP) filter.

RESULTSA high concentration of sodium chloride triggers the detach-ment of biofilm matrix from S. aureus cells. Fractionation of cellsurface proteins in S. aureus biofilms is necessary to determinewhich proteins are degraded by Esp and thereby to figure out howEsp destructs S. aureus biofilms. The cell surface proteins are com-posed of cell surface-associated proteins, including biofilm matrixproteins and cell wall-anchored proteins harboring an LPXTGmotif. To date, cell wall-anchored proteins of S. aureus biofilmshave been extensively examined by proteomic approaches (28–30), but biofilm matrix proteins are largely unknown. It has beenreported that a high concentration of salt (1 M NaCl) induces therelease of TasA, a biofilm matrix protein, from Bacillus subtiliscells (31). Here, we also used 1 M NaCl to detach biofilm matrixcomponents from S. aureus cells. First, we prepared biofilmsformed by S. aureus SH1000 (32), a laboratory strain frequentlyused for biofilm assays, and compared the surface structure ofintact biofilms and that of 1 M NaCl-treated biofilms by scanningelectron microscopy. As shown in Fig. 1A, SH1000 cells were em-

FIG 1 Isolation of biofilm matrix proteins and cell wall-anchored proteinsfrom S. aureus biofilms. The surface structures of biofilms produced by S.aureus SH1000 without (A) and with (B) 1 M NaCl treatment were observed byscanning electron microscopy. (C) The structure of the NaCl-extracted bio-film matrices from the SH1000 biofilm was also analyzed. (D) The culturesupernatant fraction (lane 1), 10 mM Tris-HCl (pH 8.0) washing fraction (lane2), 1 M NaCl-extracted fraction (lane 3), cell wall fraction obtained by lyso-staphin treatment (lane 4), and cytoplasmic fraction yielded after sonication(lane 5) were dissolved by SDS-PAGE and stained with CBB. An arrow indi-cates insoluble materials stacked on the well. (E) A 1 M NaCl extract from theSH1000 biofilm was treated without (lane 1) or with (lane 2) 0.2 mg/ml dis-persin B and then subjected to SDS-PAGE. Dispersin B was also subjected toSDS-PAGE as a control (lane 3). The arrow indicates insoluble materialsstacked on the well, and the arrowhead represents dispersin B. In panels D andE, molecular sizes are given at the left. (F) The effect of 1 M NaCl extracts frombiofilms on cell aggregation and attachment to an abiotic surface was exam-ined. S. aureus MS4, which forms less biofilm, was incubated at 37°C for 1 h inthe absence or presence of a 1 M NaCl extract from SH1000 biofilms. Cellsattached to polystyrene dishes were stained with crystal violet. NaCl solution(1 M) instead of 1 M NaCl extract was added as a negative control.

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bedded in adherent materials in the biofilm. After gently mixingwith 1 M NaCl, surface-adherent materials were removed and thecell surface became more obvious (Fig. 1B). In addition, networksof fibrillar structure were confirmed in a 1 M NaCl-extracted sam-ple by scanning electron microscopy (Fig. 1C). Notably, severalproteins and insoluble materials that were stacked in the well of anSDS-polyacrylamide gel were observed in the biofilm matrix frac-tion (Fig. 1D, lane 3). The insoluble materials were degraded bydispersin B (Fig. 1E), a polysaccharide-degrading enzyme (33),but not proteinase K and DNase I, indicating that the biofilmmatrix of SH1000 contains polysaccharides. This is consistentwith the susceptibility of the SH1000 biofilm to dispersin B. Next,we examined whether 1 M NaCl extracts from S. aureus SH1000are responsible for biofilm formation and cell aggregation. Theextracts were added into the culture of S. aureus MS4, a clinicallyisolated methicillin-sensitive S. aureus (MSSA) strain. MS4 aloneformed less biofilm, and buffer containing 1 M NaCl did not affectits biofilm formation (Fig. 1F). The addition of 1 M NaCl extractfrom SH1000 biofilms triggered the cell aggregation and partialbiofilm formation of MS4 (Fig. 1F), suggesting that the treatmentwith a high concentration of NaCl enabled us to detach and yieldS. aureus biofilm matrices. In addition, as described below, biofilmmatrix proteins were identified by this technique combined withmass spectrometry. This is the first time that biofilm matrix pro-teins have been identified using a systems biology approach.

Profiles of biofilm matrix proteins and cell wall-anchoredproteins of S. aureus clinical isolates. We examined the profilesof biofilm matrix proteins and cell wall-anchored proteins from alaboratory MSSA strain (SR6, a derivative of JCM 2874), clinicallyisolated MRSA strains (MR10 and MR23), and MSSA strains(MS4 and MS10) by 1D SDS-PAGE analyses. Interestingly, theprofiles of proteins in the biofilm matrix (Fig. 2A) and cell wall

fractions (Fig. 2B) were distinct among the strains tested. Thebiofilm matrices of SR6 and MR10, both of which form a dispersinB-sensitive biofilm (F. Sato et al. unpublished data), containedinsoluble materials (Fig. 2A), as seen in the SH1000 biofilm(Fig. 1D, lane 3). Remarkably, the biofilm matrix of MR23, whichforms proteinase K-susceptible and dispersin B- and DNase I-re-sistant biofilms (Sato et al. unpublished), contained two abundantproteins with molecular masses of approximately 60 kDa and 15kDa (Fig. 2A). The CBB-stained protein bands were then dissectedand subjected to N-terminal amino acid sequence analysis. Ho-mology searches based on the retrieved amino acid sequences (60-kDa band, A-A-K-P-L-D-K-S-S-S-X-L; 15-kDa band, S-E-G-Y-G-P-X/P-E-K-/E-K-P/K-V/P-V; X represents any amino acid)revealed that the predominant 60-kDa and 15-kDa proteins re-covered were the extracellular adhesion protein (Eap), also knownas the major histocompatibility complex (MHC) class II analogprotein (Map), and the extracellular fibrinogen-binding protein(Efb), respectively. In contrast to the biofilm matrices fromSH1000, SR6, and MR10, the biofilm matrix of MR23 lacked in-soluble materials stacked on the top of an SDS-polyacrylamide gel.In addition, MS4 and MS10, which form proteinase K- and DNaseI-sensitive and dispersin B-resistant biofilms, also did not containthem. Therefore, the existence of the insoluble materials stackedon the top of the gel could be one of the hallmarks of biofilmmatrices composed of polysaccharides. To identify Esp target pro-teins, strain MR23 was used as described below, since this strainforms the most robust biofilm among the proteinaceous biofilmproducers examined.

Degradation of specific proteins is required for Esp-depen-dent S. aureus biofilm disassembly. Previously, we showed usingamidinophenylmethanesulfonyl fluoride (APMSF), a serine pro-tease inhibitor, that proteolytic activity is involved in biofilm dis-assembly by Esp purified from S. epidermidis culture supernatant(20). To more carefully examine whether proteolytic activity isinvolved in biofilm disassembly by Esp, we used a mutant Espprotein (EspS235A), which lacks proteolytic activity (34) (see Fig.S1A in the supplemental material). As shown in Fig. 3A and Fig.S1B in the supplemental material, wild-type Esp (EspWT) disas-sembled biofilms formed by MR23, while EspS235A did not,thereby indicating that the proteolytic activity of Esp is essentialfor biofilm disassembly. Next, we treated preformed MR23 bio-films using various proteases, including thermolysin (metallopro-tease), proteinase K (serine protease), and papain (cysteine pro-tease), in addition to Esp (serine protease). MR23 biofilms weredisassembled by the addition of all proteases tested (Fig. 3A). Cellsurface proteins (biofilm matrix and cell wall fractions) degradedby these proteases were examined according to the procedure de-scribed above (Fig. 1 and 2). Interestingly, proteinase K and pa-pain degraded proteins in the biofilm matrix fraction almost com-pletely, whereas EspWT and thermolysin degraded certain specificproteins (Fig. 3B). These differences were not due to the level ofproteolytic activity (see Fig. S2 in the supplemental material). Ofnote, EspWT degraded certain proteins in the biofilm matrix frac-tion (200 kDa, 85 kDa, and 70 kDa) (Fig. 3C). Degradation of cellwall-anchored proteins by these proteases showed similar pat-terns; EspWT and thermolysin degraded several specific proteins,while proteinase K and papain hydrolyzed many more proteins(Fig. 3D; see Fig. S3 in the supplemental material). EspWT signif-icantly degraded a protein with a high molecular mass (200 kDa)in the cell wall fraction (Fig. 3D).

FIG 2 Profiles of biofilm matrix and cell wall-anchored proteins isolated fromS. aureus biofilms. (A) Biofilm matrices were extracted from the indicated S.aureus strains by 1 M NaCl treatment and analyzed by SDS-PAGE. Arrows,insoluble materials stacked on the well; arrowheads, Eap (60 kDa) and Efb (15kDa), which were determined by N-terminal amino acid sequencing. (B) Cellwall-anchored proteins were isolated from the indicated S. aureus strains afterlysostaphin treatment and centrifugation. The supernatants were analyzed bySDS-PAGE. Molecular sizes are given at the left.

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Esp degrades biofilm-associated proteins in the biofilm ma-trix fraction of S. aureus. Proteins, including ones specificallydegraded by EspWT in the MR23 biofilm, were identified by aproteomic analysis (see Fig. S4 in the supplemental material). Pro-teins in the biofilm matrix fractions before and after treatmentwith EspWT were subjected to one-dimensional SDS-PAGE. Allvisible protein bands in the sample without the treatment withEspWT were cut out, and the proteins were extracted and identifiedby nano-liquid chromatography/tandem mass spectrometry(nano-LC-MS/MS) (see Fig. S4A in the supplemental material).Reference gels for the sample treated with Esp were also analyzed(see Fig. S4B in the supplemental material). Identified proteinswith similar migration in SDS-polyacrylamide gels were com-pared between these two samples (e.g., see band 1A versus 1B and

band 2A versus 2B in Fig. S4 in the supplemental material), andproteins that were identified only in the sample without the treat-ment with EspWT were determined to be target proteins of Esp. Assummarized in Table S1 in the supplemental material, EspWT de-graded at least 74 proteins in the biofilm matrix, including 10secreted proteins harboring a secretion signal, 8 membrane pro-teins, 3 cell wall-anchoring proteins containing an LPXTG motif,45 cytoplasmic proteins, and 8 proteins whose localization has notbeen determined. Among the proteins that were identified as tar-gets of Esp in the biofilm matrix fraction, Atl (9, 10), �-toxin (19),Emp (13), FnBPA (14, 15), and Spa (18) have been found to beresponsible for S. aureus biofilm formation (Table 1). Spa andFnBPA, cell wall-anchoring proteins harboring an LPXTG motif,are thought to be released from the cell wall by cell lysis, proteo-

FIG 3 Biofilm destruction triggered by various proteases. (A) Biofilms produced by MR23 were destructed by wild type Esp (EspWT), protease-defective mutantEsp (EspS235A), and the indicated proteases (5 �g/ml) at 37°C for 24 h. Biofilms without protease treatment (control) are also shown. (B) Proteins in the biofilmmatrix fractions with or without treatment with the indicated proteases were isolated and analyzed by SDS-PAGE, as shown in Fig. 1. (C) The time-dependentdegradation of proteins in the biofilm matrix fraction by EspWT was analyzed. As a control, PBS was added instead of EspWT, and the biofilm was incubated at37°C for 8 h. Molecular sizes are given at the left. (D) The time-dependent degradation of cell wall-anchored proteins by EspWT was also analyzed as describedfor panel C. Arrowheads in panels C and D, proteins that were significantly degraded by EspWT.

TABLE 1 Biofilm- or infection-associated proteins that are degraded by Esp in S. aureus MR23 biofilm

Proteinname Description

LPXTGmotifa

Length(no. ofaminoacids)

Molecularmass (Da) Extracellular function

Reference(s) orsource

SdrD Serine-aspartate repeat-containing protein D � 1,381 149,448 Adhesion, abscess formation 35, 36Atl Bifunctional autolysin � 1,248 136,751 Biofilm formation, adhesion 9, 10, 37FnBPA Fibronectin-binding protein A � 1,018 111,707 Biofilm formation, adhesion 10, 14, 15, 38Eap (Map) Extracellular adherence protein (MHC class

II analog protein)� 689 77,047 Biofilm formation, adhesion, anti-inflammatory,

antiangiogenic, abscess formationThis study, 36, 39–42

Spa Immunoglobulin G-binding protein A � 520 56,833 Biofilm formation, immune evasion, abscessformation

18, 35, 43

Sbi Immunoglobulin-binding protein � 436 50,012 Complement evasion 44IsdA Iron (Fe2�) ABC superfamily ATP binding

cassette transporter� 350 38,793 Adhesion, abscess formation 35, 36, 45

Emp Extracellular matrix protein-binding protein � 340 38,495 Biofilm formation, adhesion, abscess formation 13, 36, 46Hlb �-Hemolysin/�-toxin � 340 33,036 Biofilm formation 19SceD Probable transglycosylase � 231 24,077 Nasal colonization 47Efb Fibrinogen-binding protein � 165 18,764 Adhesion, immune evasion 48

a �, presence; �, absence.

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lytic processing, or unknown mechanisms and attach to the cellsurface by noncovalent bonds during biofilm development. Thesebiofilm-associated proteins play important roles in biofilm devel-opment as the glue in cell-to-cell and cell-to-surface interactions.It is therefore reasonable to consider that a primary mechanism ofS. aureus biofilm disassembly triggered by Esp is proteolytic deg-radation.

Eap forms a biofilm matrix architecture and contributes tobiofilm development. Eap was the most abundant protein in thebiofilm matrix fraction of MR23, which was identified by N-ter-minal amino acid sequencing (Fig. 2A). Eap was also determinedto be a target of Esp, since it was identified in the sample of thebiofilm matrix fraction without EspWT treatment (see bands 1A,2A, 6A, 7A, 12A, and 13A in Fig. S4 in the supplemental material)but not in that with the treatment (see bands 1B, 2B, 6B, 7B, 12B,and 13B in Fig. S4 in the supplemental material; summarized inTable S1 in the supplemental material). However, the band inten-sity of Eap stained with CBB was changed slightly from before toafter the EspWT treatment (compare band 4A to band 4B in Fig. S4in the supplemental material). To assess more precisely whetherEspWT degrades Eap, we examined its degradation in vitro usingpurified recombinant Eap (Fig. 4A and B) and in the biofilm usingan anti-Eap polyclonal antibody (Fig. 4C and D). Eap was gradu-

ally degraded within 24 h in vitro and in the biofilm, indicatingthat Eap is a target of Esp.

To address the role of Eap in biofilm formation, we used aBrevibacillus choshinensis expression-secretion system which en-ables us to produce a particular protein in the extracellular envi-ronment (23). As shown in Fig. 5, extracellular production ofrecombinant Eap fused to a C-terminal His6 tag triggered cellaggregation and biofilm formation of B. choshinensis cells,whereas Efb (48), the other abundant protein in the biofilm ma-trix fraction of MR23 (Fig. 2A), did not. A large amount of recom-binant Eap was purified from the cell-associated fraction(Fig. 5C), indicating that Eap was predominantly associated withthe surface of B. choshinensis cells. In addition, the purified recom-binant Eap was supplemented into medium at the beginning ofbiofilm formation of S. aureus MS4, which forms less biofilm andalso shows less extracellular proteolytic activity. As shown inFig. 5D, Eap stimulated the biofilm formation of S. aureus MS4 ina dose-dependent manner, while recombinant Efb only slightlyaffected the biofilm formation. These results clearly indicate thatEap potentially contributes to biofilm formation not only by S.aureus but also by B. choshinensis.

In order to visualize the cellular localization of Eap in S. aureusbiofilms, C-terminal green fluorescent protein-fused Eap (Eap-

FIG 4 Esp-dependent degradation of Eap in vitro and in S. aureus biofilm. (A) Purified recombinant Eap (1 �M) was incubated in the presence of EspWT at 37°Cfor 24 h. As a control, PBS was added instead of EspWT. (B) Band intensities on the gel (A) were estimated using the ImageQuant system. Data points representthe means and standard deviations of results from three independent experiments. The standard deviation is less than the size of the symbol if no error bars areseen. (C) The time-dependent degradation of Eap by EspWT in the biofilm was analyzed by SDS-PAGE with CBB staining and Western blotting using an anti-Eappolyclonal antibody. As a control, PBS was added instead of Esp. Molecular sizes are given at the left. (D) Band intensities on the X-ray film (C) were estimatedusing ImageQuant. The means and standard deviations of triplicate determinations are represented.

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GFP) was expressed in MR23 biofilms. As a control, GFPuv wasalso expressed using the same plasmid. GFPuv spread inside thecell (Fig. 6A), whereas Eap-GFP localized on the cell surface after1 day (Fig. 6B). A similar cellular localization pattern of Eap wasobserved by immunofluorescence microscopy (see Fig. S5 in thesupplemental material). Interestingly, an increased amount ofEap-GFP localized on the cell surface and formed biofilm matrixstructures after 2 days. To our knowledge, this is the first time thatbiofilm matrix architectures composed of Eap have been visual-ized during S. aureus biofilm formation.

Esp degrades specific proteins in the cell wall fraction of S.aureus. Proteins degraded by EspWT in the cell wall fraction ofMR23 biofilms were also identified by a similar procedure for thebiofilm matrix fraction. As shown in Fig. S6 in the supplementalmaterial, FnBPA and the Ser-Asp repeat-containing protein D(SdrD) (35) were identified as target proteins of Esp. Spa wasdetected as a major cell wall-anchored protein, but no significantdegradation was observed after the EspWT treatment (see Fig. S6 inthe supplemental material). In contrast, non-cell wall-anchoredSpa (in the biofilm matrix fraction) was degraded by EspWT, asmentioned above (see Table S1 in the supplemental material).This discrepancy suggested that cell wall-anchored Spa is not de-graded by Esp in the biofilm. To address this possibility moreprecisely, Western blotting was performed using cell wall and bio-film matrix fractions from MR23 biofilms treated with EspWT. TheSpa in the biofilm matrix fraction was degraded immediately afterthe addition of EspWT, while that in the cell wall fraction was not(Fig. 7A). Purified Spa was also degraded by EspWT in vitro (Fig. 7Band C), suggesting that cell wall-anchored Spa is protected in thebiofilm from the proteolytic action of Esp, presumably by a mech-anism in which its processing sites are embedded in cell wall orbiofilm matrix materials.

Esp degrades human ECM and plasma proteins importantfor host-pathogen interaction. S. aureus is a pathogen of medical

FIG 5 Stimulation of biofilm formation by heterologous expression or supple-mentation of recombinant Eap. (A) Aggregation of Brevibacillus choshinensis cellsharboring pEap-His or pEfb-His was observed. B. choshinensis cells harboringpNCMO2 (an empty vector) were used as a control. (B) The biofilm formation ofB. choshinensis cells harboring pEap-His was compared with that of B. choshinensiscells harboring pNCMO2 or pEfb-His. Biofilms were stained with safranin andestimated by measuring the absorbance at 492 nm (ABS492). The means and stan-dard deviations of triplicate determinations are represented. (C) C-terminally His-tagged Eap was purified from culture supernatant and cell-associated fractionsusing a nickel-affinity resin. Arrow, recombinant Eap. Molecular sizes are given atthe left. (D) The indicated concentrations of purified Eap and Efb were supple-mented into BHIG medium at the initial stage of biofilm formation by S. aureusMS4, which forms less biofilm under the tested conditions. After incubation at37°C for 24 h, biofilms were stained with crystal violet and estimated by measuringthe absorbance at 595 nm. As a control, PBS was used instead of Eap (defined as100%). The means and standard deviations of triplicate determinations arerepresented.

FIG 6 Eap is a component of the S. aureus biofilm matrix. The cell morphol-ogy and GFP fluorescence of S. aureus MR23 cells harboring pP1GFP (A) andpEap-GFP1 (B) were observed during biofilm formation (day 1 and day 2) byphase-contrast and fluorescence microscopy. Fourfold-magnified imagestaken under a GFP filter are also shown on the right. Bars, 1 �m.

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device-associated infection. Host ECM and plasma proteins coverthe devices soon after insertion, and thus, the specific interactionbetween these proteins and S. aureus surface proteins is importantfor colonization and infection. We have reported that Esp not onlydisassembles S. aureus biofilms but also inhibits its nasal coloni-zation (20). To elucidate how S. aureus colonization is inhibitedand how Esp affects the host-pathogen interaction, we testedwhether several human proteins are degraded by Esp. Recently, ithas been reported that the presence of Hg in nasal secretions con-tributes to S. aureus nasal colonization via prevention of the agrquorum-sensing system (49). We first asked whether Hg is de-graded by EspWT in vitro. As shown in Fig. 8A, no degradation ofHg was observed. We next tested whether major human ECM andplasma proteins, such as Fn, Fg, and Vn, are degraded by EspWT invitro, since they are key receptors for S. aureus colonization on

human epithelial and endothelial cells (50–52). Fn was degradedinto various fragments (Fig. 8B), while Vn was partially processedto a 40-kDa fragment (Fig. 8C). In the case of Fg, which is com-posed of 2 sets of 3 chains designated the �, �, and � subunits (53),only � chains were digested by EspWT (Fig. 8D).

Several biofilm-associated proteins (Atl, Emp, FnBPA, Eap,and Spa) and adhesins (Efb, IsdA, and SdrD), all of which interactwith host ECM and plasma proteins (18, 35, 37–40, 45, 46, 48),were degraded by Esp. In addition, SceD, a protein essential forcolonization of S. aureus in a cotton rat model (47), was also de-graded by Esp. These results together suggest that this proteasecould inhibit nasal colonization of S. aureus presumably via deg-radation of S. aureus surface proteins and host receptors that arecrucial for host-pathogen interactions.

DISCUSSION

Once a biofilm is fully developed, it can be disassembled to singlecells or larger clusters through both mechanical and active pro-cesses (54). Biofilm disassembly is crucial for the dissemination ofbacteria to other colonization sites and triggered by several fac-tors, such as enzymes (proteases, glycosidases, and DNases) andsurfactants (phenol-soluble modulins) that degrade or solubilizeadhesive molecules in the biofilm matrix (55–57). An in-depthunderstanding of biofilm disassembly mechanisms could not onlyprovide fundamental insights into bacterial lifestyles associatedwith infectious diseases but also lead to treatment options. Todate, several approaches to disassemble and eliminate biofilmsformed by various bacterial species have been reported. These

FIG 7 Degradation of Spa by Esp. (A) Degradation of Spa in the biofilmmatrix and cell wall-anchored fractions from EspWT-treated biofilms of S.aureus MR23 were examined by Western blotting. (B) Purified Spa was de-graded by EspWT in vitro. Molecular sizes are given at the left. (C) Band inten-sities on the gel (B) were estimated using ImageQuant. Data points representthe means and standard deviations of results from three independent experi-ments. The standard deviation is less than the size of the symbol if no error barsare seen.

FIG 8 Degradation of staphylococcal receptor proteins of humans by EspWT.Human ECM and plasma proteins, including Hg (A), Fn (B), Vn (C), and Fg(D), were incubated with or without EspWT in PBS buffer at 37°C. At theindicated time points, proteins were analyzed by SDS-PAGE. Molecular sizesare given at the left.

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approaches use biofilm matrix-degrading enzymes, such as bovineDNase I (58), dispersin B (59), and lysostaphin (60), and smallmolecules, such as D-amino acids (61) and norspermidine (62).

In the present study, we focused on extracellular serine pro-tease Esp produced by commensal S. epidermidis and molecularmechanisms of Esp-dependent disassembly of pathogenic S. au-reus biofilms (20). We identified 75 cell surface-associated pro-teins (in the biofilm matrix and cell wall fractions) as targets of Espin the biofilm of MRSA (see Table S1 in the supplemental mate-rial). They include several known biofilm-associated proteins (Ta-ble 1), such as Atl (9, 10), �-toxin (19), Emp (13), FnBPA (14, 15),and Spa (18). They play important roles in biofilm development asthe glue in cell-to-cell or cell-to-surface interactions. It has previ-ously been shown that Eap contributes to the biofilm developmentof S. aureus Newman under certain conditions (e.g., iron-depletedconditions and serum-containing conditions) (13, 40). In thesestudies, an S. aureus eap-deletion mutant was used to evaluate therole of Eap in biofilm development. Considering the fact that bio-film formation is a multifactorial event, a single-gene deletioncould be compensated for by other factors, and thus, no pheno-type may be observed. In the case of extracellular proteins, sup-plementation of the purified protein into the culture of biofilm-negative strains is an efficacious approach. In this study, we foundthat supplementation of recombinant Eap increased the biofilmformation of S. aureus MS4, a biofilm-negative strain (Fig. 5D). Inaddition, heterologous expression of specific genes in biofilm-negative bacterial species can be an alternative strategy. Using aBrevibacillus expression-secretion system, we also proved that Eapstimulates cell aggregation and biofilm formation and it stronglybound to the cell surface (Fig. 5A to C). Furthermore, Eap formeda structural framework in the biofilm matrix (Fig. 6B; see Fig. S5 inthe supplemental material). Our proteomic and immunologicalanalyses revealed that Esp degrades Eap (Fig. 4; see Table S1 in thesupplemental material), in addition to the other biofilm-associ-ated proteins mentioned above. It is therefore reasonable to con-sider that a primary mechanism of S. aureus biofilm disassemblytriggered by Esp is proteolytic degradation of these biofilm-asso-ciated proteins.

Several biofilm-associated proteins, such as Atl (37), Emp (46),FnBPA (38), Eap (39), and Spa (18), as well as adhesins, such asEfb (48), IsdA (45), and SdrD (35), interact with host ECM andplasma proteins. They therefore play an important role in theinfection of host cells by S. aureus. Our results indicate that Espdegrades these surface proteins of S. aureus (Table 1) and humanECM and plasma proteins, such as Fn, Fg, and Vn (Fig. 8). Inaddition, SceD, a protein essential for nasal colonization of S.aureus in cotton rats (47), is also degraded by Esp (Table 1; seeTable S1 in the supplemental material). These results togethersuggest that Esp inhibits nasal colonization of S. aureus presum-ably via degradation of S. aureus surface proteins and host recep-tors that are crucial for the pathogen-host interaction.

Surface proteins of S. aureus are known to be important forcausing infectious diseases in humans, ranging from minor skininfections to severe infections (1). SdrD, Spa, IsdA, and Emp arenecessary for abscess formation (36). Of note, Eap has drawn theattention of researchers because of its importance in various as-pects of infection (e.g., anti-inflammatory and antiangiogenicproperties and abscess formation) (41, 42). Immunoglobulin-binding proteins Spa and Sbi contribute to immune evasion byforming complexes with VH3-type IgM on the surface of B cells,

pathogen-specific antibodies, or complement factor 3d (43, 44).Efb is also associated with immune evasion through the binding tothe � chain of complement factor 3 (63). Our data clearly indicatethat Esp degrades these surface proteins and toxins, such as�-toxin and coagulase (Table 1; see Table S1 in the supplementalmaterial), suggesting that Esp might be applicable for the preven-tion or treatment of infectious diseases caused by this pathogen.However, we should pay attention to the use of Esp for therapeuticpurposes, since the safety of this protease has not yet been estab-lished. Development of a vaccine targeting proteins that are de-graded by Esp and also contribute to S. aureus biofilm formationand colonization of human skin and the human nasal cavity couldbe an alternative approach.

ACKNOWLEDGMENTS

This work was supported by a Grant-in-Aid for Research Activity Start-upto S.S. from the Japan Society for the Promotion of Science (JSPS), by aGrant-in-Aid for Young Scientists (B) to S.S. from JSPS, by a grant to Y.M.from the Uehara Memorial Foundation, by a grant to Y.M. from TheScience Research Promotion Fund, by a grant to Y.M. from MEXT-Sup-ported Program for the Strategic Research Foundation at Private Univer-sities, 2012–2016, and by a grant to S.S. from the Joint Usage/ResearchCenter for Developmental Medicine, IMEG, Kumamoto University.

We acknowledge Yasuaki Hiromasa (Department of Biochemistry, Kan-sas State University) for mass spectrometry analyses, Teru Ogura (Depart-ment of Molecular Cell Biology, Kumamoto University, Japan) for sending E.coli strains and plasmids, Karen LoVetri (Kane Biotech Inc.) for gifting dis-persin B, and Fumiya Sato (Division of Infectious Disease and Control, TheJikei University School of Medicine, Japan) for providing clinically isolated S.aureus strains. We also thank Hitomi Shinji for discussion, Satomi Yamada,Takuya Kitamura, Rintarou Shigemori, and Kensuke Sekiguchi for experi-mental support, and Yumi Tokoro for secretary work.

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