Identification of a protective B-cell epitope of the <i>Staphylococcus aureus</i> GapC protein by screening a phage-displayed random peptide library

<div><p>The impact of epidemic <i>Staphylococcus aureus</i> (<i>S</i>. <i>aureus</i>) on public health is increasing. Because of the abuse of antibiotics, the antibiotic resistance of <i>S</i>. <i>aureus</i> is increasing. Thus, there is an urgent need to develop new immunotherapies and immunoprophylaxes. Previous studies showed that the GapC protein of <i>S</i>. <i>aureus</i>, which is a surface protein with high glyceraldehyde 3-phosphate dehydrogenase activity, transferrin binding activity, and other biological activities, is highly conserved. GapC induces an effective humoral immune response <i>in vivo</i>. However, the B-cell epitopes of <i>S</i>. <i>aureus</i> GapC have not been well identified. Here we used the bioinformatics tools to analyze the sequence of GapC, and we generated protective anti-GapC monoclonal antibodies (mAbs). A protective mAb (1F4) showed strong specificity to GapC and the ability to induce macrophages to phagocytose <i>S</i>. <i>aureus</i>. We screened the motif ²⁷²GYTEDEIVSSD²⁸², which was recognized by mAb 1F4, using a phage display system. Then, we used site-directed mutagenesis to identify key amino acids in the motif. Residues G272 D276 E277 I278 and V279 formed the core of the ²⁷²GYTEDEIVSSD²⁸² motif. In addition, we showed that this epitope peptide induced a protective humoral immune response against <i>S</i>. <i>aureus</i> infection in immunized mice. Our results will be useful for the further study of epitope-based vaccines against <i>S</i>. <i>aureus</i> infection.</p></div

anti-epitope serum-mediated phagocytosis of S. <i>aureus</i> Newman.

<p>PBS-washed Newman (2×10⁵ CFU) was incubated with 50 μl of the serum for 30 min. The negative control was incubated with the negative serum and the blank control comprised only phagocytes and bacteria. Differences in the internalization rates between anti-epitope peptide serum group and anti-negative serum group was statistically significant. The data represent the means ± SEM (<i>n</i> = 3). Statistical significance was measured using a Student’s t-test (**, <i>p</i> < 0.01).</p

Analysis of the ²⁷²GYTEDEIV²⁷⁹ epitope by confocal laser-scanning microscopy.

<p>The left panels show dark field fluorescence photos; the middle panels show bright field photos of the bacteria; and the right panels show the two fields combined. (<b>a–c</b>) The <i>S</i>. <i>aureus</i> (Newman) strain was observed. Primary antibodies were <i>S</i>. <i>aureus</i> anti-GapC serum, mAb 1F4, and the SP2/0 supernatant, respectively.</p

The ²⁷²GYTEDEIV²⁷⁹ epitope peptide confers protection against lethal challenges with <i>S</i>. <i>aureus</i> Newman.

<p>For the prophylaxis study, immunized and control mice (6 weeks old, <i>n</i> = 10) were challenged intraperitoneally with <i>S</i>. <i>aureus</i> (equivalent to 5 × 10⁸ CFU) two weeks after the last immunization. Mice were monitored for 15 days. The survival rates of GapC, GST-epitope were 70% and 50%, respectively. Statistical differences were determined by Student’s t-test. (***, <i>p</i> < 0.001).</p

Primers used in this study.

<p>Primers used in this study.</p

Alignment of the sequences surrounding the epitope-coding region of the GapC protein from different <i>S</i>. <i>aureus</i> strains.

<p>Alignment of the sequences surrounding the epitope-coding region of the GapC protein from different <i>S</i>. <i>aureus</i> strains.</p

Characterization of mAbs.

<p>(<b>a</b>) The purified 1F4 was analyzed by 12% SDS-PAGE. (<b>b</b>) GapC recognized by the mAb 1F4 was detected by western blotting. (<b>c</b>) The class of the mAb 1F4 was determined to be IgG1 and the κ chain. (<b>d</b>) The specificity of mAb 1F4 toward recombinant FnbpA,Trap, Mntc, IsdB of <i>S</i>. <i>aureus</i> and GapC from <i>S</i>. <i>dysgalactiae</i> was determined by indirect ELISA. (<b>e</b>) The reactivity of mAb 1F4 with the recombinant FnbpA, Trap, Mntc, IsdB proteins of <i>S</i>. <i>aureus</i> and GapC from <i>S</i>. <i>dysgalactiae</i> was determined by western blotting. (<b>f</b>) Effect of 1F4 on GapC GAPDH activity of <i>S</i>. <i>aureus</i>. The OD values were detected at 20 s (A1) and 5 min and 20 s (A2), the decrease of NADH reflected the level of GAPDH activity at <i>OD</i>₃₄₀. The data represent the means ± SEM (<i>n</i> = 3). (<b>g</b>) Passive immunization with mAb 1F4 protects against <i>S</i>. <i>aureus</i> infection. Mice were injected intraperitoneally with 5×10⁸ CFU of <i>S</i>. <i>aureus</i> (Newman) followed by the intravenous injection of anti-GapC serum, mAb 1F4, or SP2/0 cell supernatant (nagative control) after 24 h (<i>n</i> = 10).</p

Identification of strongly positive phage clones selected by 1F4 using a sandwich ELISA.

<p>The negative control was wild-type M13 phage and the blank control was <i>E</i>. <i>coli</i> ER2738. BSA-coated wells were used to exclude cross-activity.</p

Precisely defining the epitopes.

<p>The reactivity of the fusion proteins, in which each amino acid (²⁷¹FGYTEDEIVS²⁸⁰) was mutated to alanine, with mAb 1F4 was identified by western blotting. The G272A, D276A, E277A, I278A, and V279A mutations completely disrupted the reactivity of the epitope with mAb 1F4.</p

Structural analysis of the linear B-cell epitope.

<p>The PDB Protein Data Bank method was used to analyze the three-dimensional model of <i>S</i>. <i>aureus</i> GapC. The residues of the linear B-cell epitope that are recognized by mAb 1F4 are highlighted in red and shown in cartoon, stick, and dot formats, respectively. The core amino acids are shown in yellow.</p