Production and characterization of a novel antigen of Histoplasma capsulatum for its potential use in diagnosis and therapeutics

TOSCANINI MA; PARK E; LOPEZ RIZZOLO A; IOVANNITTI C; NUSBLAT A; CUESTAS ML

Congreso; 16TH INFOCUS; 2018

Introduction: Histoplasmosis is a systemic and endemic mycosis widely distributed in the Americas caused by the dimorphic fungus Histoplasma capsulatum. The infection is usually asymptomatic in immunocompetent individuals. However, immunocompromised patients may contract the disseminated form of the disease, which has a bad prognosis and requires rapid diagnosis and treatment. The definitive diagnosis involves the isolation of H. capsulatum by culture from clinical specimens, which may take up to 4 weeks. In addition, molecular methods are expensive and have low sensitivity whilst immunoassays present many false-positive results. Liposomal amphotericin B, which is the treatment of choice for severe cases, is highly expensive. Unfortunately, there is a lack of other therapeutic alternatives for this severe clinical form of histoplasmosis as initial intensive induction treatment. Thus, there is a need to look for new antigens candidates for diagnosis as well as for new therapeutic targets. In this regard, a 100kDa protein of H. capsulatum, Hcp100, was proposed as a novel target for histoplasmosis therapy and diagnosis due to its essential role in the fungal adaptation and survival inside the macrophages during infection. Hence, the aim of this work was to express and purify Hcp100 to develop novel diagnostics tools and to perform characterization studies of this protein as a first approach for the potential development of novel therapeutic strategies.Materials and Methods: The gene that encodes for Hcp100 was constructed with a secretory signal and a polyhistidine-tag and expressed in the Pichia pastoris X-33 strain. Cell culture supernatants from different induction times were analyzed by SDS-PAGE, Western blot and mass spectrometry. Purification of Hcp100 from a 24h cell culture supernatant was carried out using a Ni-NTA affinity chromatography column and flow-throws and eluates were analyzed by SDS-PAGE and Western blot. Due to its homology with the human transcription coactivator p100, DNA binding ability of Hcp100 was evaluated by chromatography using DNA-agarose and heparin columns. Hcp100 immunoreactivity was analyzed by dotblotting using rabbit polyclonal antibodies against H. capsulatum, Paracoccidioides brasiliensis and Coccidiodes sp. Similar sequences of Hcp100 retrieved from the National Center of Biotechnology Information using tblastn tool and GBID CAA06786.1 as query were compared to infer orthology ralations using the OrthoMCL and OMA orthology databases. Results: Hcp100 was successfully expressed and purified (90% purity) from the cell culture supernatant and its identity was confirmed by Western blot and mass spectrometry. No DNA or heparin binding ability was observed despite the presence of the Oligonucleotide-Oligosaccharide binding fold in this protein. Hcp100 proved to be immunoreactive against anti-H. capsulatum antibodies and no cross-reactions were observed with anti-P. brasiliensis and anti-Cocciciodes sp. antibodies. Conclusions: P. pastoris proved to be an excellent system for recombinant Hcp100 expression with potential applications in diagnosis, prognosis and molecular targeted therapy. Further characterization of the different Hcp100 domains are needed so as to elucidate its role in fungal pathogenesis.