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Fine-mapping of immunodominant linear B-cell epitopes of the Staphylococcus aureus SEB antigen using short overlapping peptides.

ABSTRACT: Staphylococcal enterotoxin B (SEB) is one of the most potent Staphylococcus aureus exotoxins (SEs). Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB) and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97-114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257) were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97-112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97-112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC3943954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics, and by its fast evolution into new lineages that rapidly spread within the healthy human population. This calls for the development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available as yet, the present studies were aimed at identifying new leads for their development. For this purpose, we thoroughly profiled the cell surface-exposed staphylococcal proteome by combining two surface shaving approaches. In parallel, non-covalently cell wall-bound proteins were extracted with KSCN and analyzed by gel-free proteomics, and also the exoproteome was analyzed through gel-free proteomics. Lastly, we screened a selection of the identified cell wall-attached proteins for binding of immunoglobulin G from patients that have been challenged with different types of S. aureus over extended periods of time due to chronic wound colonization. The combined results of these analyses highlight particular cell surface-exposed S. aureus proteins with highly immunogenic epitopes as potentially powerful targets for the development of protective anti-staphylococcal immunization strategies. Bioinformatics pipeline: Reduction and alkylation, desalting of the samples, mass spectrometric (MS) analysis and database searches were performed as previously described (PMID: 20662103). The strain-specific uniprot databases were used for the /S. aureus/ strains Newman and USA300, respectively including concatenated reversed databases with 5250 and 5298 entries. Validation of MS/MS-based peptide and protein identifications was performed with Scaffold (version Scaffold_2_04_00, Proteome Software Inc., Portland, OR). Peptide identifications were accepted if they exceeded the specific database search engine thresholds. Sequest identifications required at least deltaCn scores of greater than 0.10 and XCorr scores of greater than 1.9, 2.2, 3.8 and 3.8 for singly, doubly, triply and quadruply charged peptides, respectively. All experiments were conducted in independent triplicates. Peptides were only accepted as identified if they were detected in at least two out of the three replicates per sample set. With these filter parameters no false positive hits were obtained.

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Fine-mapping of immunodominant linear B-cell epitopes of the Staphylococcus aureus SEB antigen using short overlapping peptides.

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