Histoplasma capsulatum 100-kilodalton antigen: recombinant production, characterization, and evaluation of its possible application in the diagnosis of histoplasmosis

CAPECE, PAULA; NUSBLAT, ALEJANDRO D.; MAGLIO, DANIEL GONZÁLEZ; IOVANNITTI, CRISTINA A.; TOSCANINI, MARÍA A.; POSSE, GLADYS; CUESTAS, MARÍA L.

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2020 vol. 104 p. 5861 - 5872

0175-7598

The goal of the present work was to develop a novel reagent with potential for histoplasmosis diagnosis. For this purpose, thegenetic sequence of the 100 kDa protein of Histoplasma capsulatum (Hcp100) was cloned and expressed as a secretory protein inPichia pastoris. After optimizing the culture conditions and purifying by immobilized metal ion affinity chromatography, thehighest yield of Hcp100 reached approximately 1.3mg/lwith > 90%purity in shake flasks using basal salt medium supplementedwith casamino acids after 72 h of methanol induction. To investigate its potential for diagnosis, its detection in urine samplesusing specific polyclonal antibodies as reagent was evaluated by dot blot in 6 patients with progressive disseminated histoplasmosis(PDH), of whom all had AIDS. Antigen was detected in urine from all 6 (100%) PDH patients. Urine samples from a poolof 20 healthy individuals did not react with the anti-Hcp100 antibodies. The dot blot assay performed in this study providespreliminary data of a simple technology that can be performed in medical institutions with limited resources to facilitate the rapiddiagnosis of histoplasmosis, particularly the disseminated forms. Hence, use of these assays may provide a rapid diagnostic toolof PDH in endemic areas for histoplasmosis where PDH-related mortality is high, hastening treatment and improving patientsurvival. Finally, this novel antigen and its specific antibodies may provide an alternative diagnostic reagent to the largelyunknown and poorly characterized polysaccharide antigens (HPA, galactomannan, histoplasmin) frequently used in the diagnostictests.