Antigenic specificity of the MAbs.

<p>The antigenic specificities of the MAbs at 1 ng/mL and at higher concentrations (10 ng/mL, 100 ng/mL) against proteins in supernatants of lysostaphin-treated <i>S. epidermidis</i> RP62A (“RP62A”) and ATCC 12228 (“12228”) were analyzed using Western blot. The multiple proteins (between 250 kDa–300 kDa) in <i>S. epidermidis</i> RP62A, or the single protein (180 kDa) in <i>S. epidermidis</i> ATCC 12228, probed by 1 ng/mL MAbs, corresponded to full-length or proteolytically processed Aap, based on an analysis of these bands using a 4700 MALDI-TOF/TOF proteomics analyzer (Applied Biosystems, <a href="http://www.appliedbiosystems.com" target="_blank">http://www.appliedbiosystems.com</a>).</p

Aap expression in biofilms of <i>S. epidermidis</i>.

<p>Aap in the biofilms of <i>S. epidermidis</i> RP62A was probed with MAb_25C11 (10 ng/mL) and Cy3-conjugated secondary antibody (1∶100 diluted, red fluorescence), and the bacteria were further stained with SYTO9 (1 µM, green fluorescence). Aap expression was observed under a Leica TCS SP5 CLSM. Confocal microscopy Z-series of the biofilms were acquired in 0.5-µm increments. “PC”: positive control (antigens contained in the biofilm were probed using mouse anti-<i>S. epidermidis</i> serum (1∶400 diluted) and Cy3-conjugated secondary antibody, showing that antibodies could diffuse to the inner side of the biofilm), “NC”: negative control (the biofilm formed in the presence of MAb_25C11 was probed with Cy3-conjugated secondary antibody alone to establish that the MAb-treated biofilms no longer contained initially added MAb (10 µg/mL), that could cause false-positive immunofluorescence, after 14 h culture), “RP62A”: untreated, “Mock”: normal mouse IgG-treated, the red arrow indicates the crater-like micropores.</p

Fluorescence quantities of the Live/Dead stained biofilms.^a

<p><b>a</b>. The biofilm of <i>S. epidermidis</i> RP62A formed in the presence of each MAb (10 µg/mL) was visualized using Live/Dead viability staining (SYTO9/PI). After obtaining the 3-D structure of the biofilms under a CLSM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020918#pone-0020918-g003" target="_blank">Figure 3</a>), the Z-stack composite confocal photomicrographs were further generated, and the stacks of viable cells, dead cells, and both cells (viable & dead) were generated separately. The fluorescence quantities of SYTO9-stained viable cells and PI-stained dead cells were determined using ImageJ program (<a href="http://rsbweb.nih.gov/ij" target="_blank">http://rsbweb.nih.gov/ij</a>).</p><p><b>b</b>. “SYTO9” stands for the fluorescence quantities of the viable cell stacks.</p><p><b>c</b>. “PI” stands for the fluorescence quantities of the dead cell stacks.</p><p><b>d</b>. “Total” represents the fluorescence quantities of both cell stacks.</p><p><b>e</b>. “PI/Total” represents the proportion of the dead cell in the biofilms.</p

Oligonucleotide primers used in the present study.

<p><b>a</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> ATCC 12228 (GenBank NC_004461).</p><p><b>b</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> RP62A (GenBank NC_002976).</p><p><b>c</b>. Primers were designed according to the gene sequence of the 56-residue B1 immunoglobulin binding domain (GB1) of immunoglobulin G-binding protein from <i>Streptococcus dysgalactiae</i> subsp. <i>equisimilis</i> GGS_124 (amino acids 303–357, GenBank YP_002997067).</p

Aap expression in <i>S. epidermidis</i>.

<p>(A) Protein expression level. Aap expression in <i>S. epidermidis</i> RP62A co-cultured with each MAb (10 µg/mL) was detected using Western blot with MAb_25C11 (1 ng/mL). After separation of the proteins using 7% SDS-PAGE, the gel pieces carrying high molecular-weight proteins (>130 kDa) were excised for Western blot assay, and the remaining gel was stained using Coomassie brilliant blue as the endogenous control. (B) Relative transcriptional level. The transcriptional level of <i>aap</i> was detected by Q-RT-PCR using RNA sample extracted from <i>S. epidermidis</i> RP62A co-cultured with each MAb (10 µg/mL). The housekeeping gene <i>gyrB</i> was used as an endogenous control, and all samples were analyzed in triplicate and normalized against <i>gyrB</i> transcription (means ± SD). “RP62A”: untreated, “Mock”: normal mouse IgG-treated.</p

The substitution of V₇₅H₇₆ by T₇₅E₇₆ in TF_1–102 reduced the interaction between the fragment and MAb_25C11 and MAb_20B9.

<p>The AapBrpt1.5 fragment, TF_{1<b>–</b>102}, with the substitution of V₇₅H₇₆ by T₇₅E₇₆ was established using site-directed mutagenesis. The interactions between the mutated fragment and MAb_25C11 and MAb_20B9 were detected using immunoprecipitation. “TF_1–102V₇₅H₇₆”: wild type, “TF_1–102T₇₅E₇₆”: mutant, “IgG H”: IgG heave chain.</p

Epitope mapping of anti-AapBrpt1.5 MAbs.

<p>AapBrpt1.5 N-terminally fused with a GB1-tagged six-histidine (GB1-His) tag was truncated into a series of fragments as shown in the schematic diagrams, and the binding ability between the truncated fragments and MAbs was analyzed using immunoprecipitation. (A) Preliminary epitope mapping. (B) Precise recognition site mapping of MAb_25C11 and MAb_20B9. (C) Precise recognition site mapping of MAb_18B6. (D) Domain structures of Aap from <i>S. epidermidis</i> RP62A (“RP62A”) and ATCC 12228 (“12228”). The A-repeat region, the putative globular domain (α/β), the B-repeat region containing 6 or 12 tandem Brpt constructs, the collagen-like proline/glycine-rich region, the domain boundary of AapBrpt1.5, and the MAb epitopes are illustrated. (E) Amino acid sequence alignment of AapBrpt constructs. The AapBrpt construct in AapBrpt1.5 (GenBank NP_763730) and twelve distinct AapBrpt constructs from <i>S. epidermidis</i> RP62A (RP62A 01-12, GenBank YP_189945) were aligned using the ClustalW2 program (<a href="http://www.ebi.ac.uk/Tools/clustalw2" target="_blank">http://www.ebi.ac.uk/Tools/clustalw2</a>). The identified epitopes of the MAbs are shown in boxes, and the identical residues are marked with asterisks. The conserved substitutions are represented by “:”, and semi-conserved substitutions are represented by “.”. Two conserved His residues in AapBrpt constructs are marked with triangles.</p

EPS biosynthesis in biofilm of <i>S. epidermidis</i>.

<p>(A) Extracellular DNA quantification. Extracellular DNA was isolated from the biofilm matrices of <i>S. epidermidis</i> RP62A, and Q-PCRs of four chromosomal loci (<i>gyrA</i> (gyrase A), <i>serp0306</i> (ferrichrome transport ATP-binding protein A), <i>lysA</i> (diaminopimelate decarboxylase A), and <i>leuA</i> (2-isopropylmalate synthase)) were performed for eDNA quantification in each biofilm. The biomass that represented the biofilm density was quantified at A₆₀₀, and the eDNA measurement was normalized to biofilm biomass as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020918#pone.0020918-Mann1" target="_blank">[27]</a>. Data are depicted as averages of three Q-PCR detections with the standard deviation, and the results represent one of three independent experiments. (B, C) Biofilm stability against DNase treatment. When exposed to DNase I (0.14 U/µL), the biofilm was more severely disintegrated in the presence of each MAb compared with that formed in the absence of the MAbs. The data are means ± SD of three independent experiments. The biofilm density ratios of the untreated biofilms and biofilms treated with DNase I were plotted (C). (D) PIA synthesis in biofilm of <i>S. epidermidis</i>. PIA synthesis in biofilm of <i>S. epidermidis</i> RP62A was detected using the WGA-HRP dot blot assay. Serial dilutions of the PIA extractions from biofilm bacteria were spotted onto nitrocellulose transfer membranes, and the HRP activity was visualized using chromogenic detection. The data represent one of three independent experiments. “RP62A”: untreated; “Mock”: normal mouse IgG-treated.</p

Effects of anti-AapBrpt1.5 MAbs on different <i>S. epidermidis</i> strains.

<p><b>a.</b> “Relative biofilm formation” of the strains cultured in the presence of each MAb (10 µg/mL) in polystyrene plates was calculated by dividing the mean density of MAb-treated biofilms by that of untreated biofilms. “-” means no biofilm formation was observed.</p><p><b>b.</b> Planktonic aggregation of the strains statically cultured in the presence of each MAb (10 µg/mL) was described using “+++”, “++”, “+”, or “−”, which means that very many, many, a few, or no bacterial clusters were observed, respectively.</p><p><b>c.</b> Biofilm formation by the strains without treatment was described using “++”, “+”, “+/−”, or “−”, which means that strong, weak, slight, or no biofilm formation was observed in polystyrene plates, respectively.</p><p><b>d.</b> Cell aggregation of the strains without treatment was described using “+” or “−”, which means that a few or no bacterial clusters were observed, respectively.</p><p><b>e.</b> The clinical strains of <i>S. epidermidis</i> were obtained from Zhongshan Hospital and Ruijin Hospital, Shanghai, China.</p

Cell aggregation mediated by the MAbs.

<p><i>S. epidermidis</i> RP62A was statically cultured in TSB medium containing each MAb (10 µg/mL, 0.07 µM) alone or both MAb (10 µg/mL, 0.07 µM) and AapBrpt1.5 (untagged, 3.2 µg/mL, 0.14 µM). The photomicrographs were obtained by a Nikon TE2000-U inverted microscopy using a 40x objective lens (Nikon, <a href="http://www.nikoninstruments.com/" target="_blank">http://www.nikoninstruments.com/</a>). “RP62A”: untreated; “Mock”: normal mouse IgG-treated.</p