Immunoproteomics Reveals Pathogen's Antigens Involved in Homo sapiens- Histoplasma capsulatum Interaction and Specific Linear B-Cell Epitopes in Histoplasmosis

Abstract

Histoplasmosis is one of the most frequent systemic mycosis in HIV patients. In these patients, histoplasmosis has high rates of morbidity/mortality if diagnosis and treatment are delayed. Despite its relevance, there is a paucity of information concerning the interaction between Histoplasma capsulatum and the human host, especially regarding the B-cell response, which has a direct impact on the diagnosis. Culture-based "gold-standard" methods have limitations, making immunodiagnostic tests an attractive option for clinical decisions. Despite the continuous development of those tests, improving serological parameters is necessary to make these methods efficient tools for definitive diagnosis of histoplasmosis. This includes the determination of more specific and immunogenic antigens to improve specificity and sensitivity of assays. In this study, we performed a co-immunoprecipitation assay between a protein extract from the yeast form of H. capsulatum and pooled sera from patients with proven histoplasmosis, followed by shotgun mass spectrometry identification of antigenic targets. Sera from patients with other pulmonary infections or from healthy individuals living in endemic areas of histoplasmosis were also assayed to determine potentially cross-reactive proteins. The primary structures of H. capsulatum immunoprecipitated proteins were evaluated using the DNAStar Protean 7.0 software. In parallel, the online epitope prediction server, BCPREDS, was used to complement the B-epitope prediction analysis. Our approach detected 132 reactive proteins to antibodies present in histoplasmosis patients' sera. Among these antigens, 127 were recognized also by antibodies in heterologous patients' and/or normal healthy donors' sera. Therefore, the only three antigens specifically recognized by antibodies of histoplasmosis patients were mapped as potential antigenic targets: the M antigen, previously demonstrated in the diagnosis of histoplasmosis, and the catalase P and YPS-3 proteins, characterized as virulence factors of H. capsulatum, with antigenic properties still unclear. The other two proteins were fragments of the YPS-3 and M antigen. Overlapping results obtained from the two aforementioned bioinformatic tools, 16 regions from these three proteins are proposed as putative B-cell epitopes exclusive to H. capsulatum. These data reveal a new role for these proteins on H. capsulatum interactions with the immune system and indicate their possible use in new methods for the diagnosis of histoplasmosis.

Keywords: Histoplasma capsulatum; Homo sapiens; M antigen; YPS-3; catalase P; epitopes; immunoproteome; mass spectrometry.

Figure 1

Protein profile of Histoplasma capsulatum yeast cell extract. (A) Silver stained SDS-PAGE under reducing conditions. 1, Molecular mass standard; 2, Yeast cell extract of H. capsulatum. Numbers on the left correspond to the molecular mass (kDa) of the standards. (B) Colloidal Coomassie blue stained 2DE. The linear pH gradient range is indicated above the gel and the molecular mass standards (kDa) are on the left.

Figure 2

Venn diagrams depicting Histoplasma capsulatum proteins (potential antigens) recognized by the different pooled sera groups. (A) Number of proteins recognized by the pooled sera of patients with histoplasmosis (HPM) for the three biological replicates analyzed (HPM 1, HPM 2, and HPM 3); (B) The 132 proteins in common to all conditions in panel A were compared to the total list of proteins detected in at least one of the biological replicates for the other conditions: HET, pooled sera of patients with other fungal infections or tuberculosis; NHS, pooled sera of normal healthy donors; CON, mock control: phosphate buffered saline.

Figure 3

Functional classification and subcellular localization of immunoreactive proteins identified in Histoplasma capsulatum. (A) Functional classification of proteins based on UniProt database; (B) Subcellular localization of proteins based on the WoLF PSORT server.

Figure 4

In silico evaluation of putative B epitopes on candidate proteins for antigenic targets for the diagnosis of histoplasmosis. Results obtained from overlapping data from two bioinformatics tools: the software DNAStar Protean 7.0 and BCPREDS server. (A) M antigen; (B) Catalase P; (C) YPS-3. The flagged numbers refer to the peptides described in Table S7 .