Heterologous Expression, Purification, and Immunological Reactivity of a Recombinant HSP60 from Paracoccidioides brasiliensis

The complete coding cDNA of HSP60 from Paracoccidioides brasiliensis was overexpressed in an Escherichia coli host to produce high levels of recombinant protein. The protein was purified by affinity chromatography. A total of 169 human serum samples were tested for reactivity by Western blot analysis with the purified HSP60 recombinant protein. Immunoblots indicated that the recombinant P. brasiliensis HSP60 was recognized by antibodies in 72 of 75 sera from paracoccidioidomycosis patients. No cross-reactivity was detected with individual sera from patients with aspergillosis, sporotrichosis, cryptococcosis, and tuberculosis. Reactivity to HSP60 was observed in sera from 9.52% of control healthy individuals and 11.5% of patients with histoplasmosis. The high sensitivity and specificity (97.3 and 92.5%, respectively) for HSP60 suggested that the recombinant protein can be used singly or in association with other recombinant antigens to detect antibody responses in P. brasiliensis-infected patients. Paracoccidioidomycosis is a fungal disease caused by Paracoccidioides brasiliensis, a thermal dimorphic fungus which is geographically confined to Latin America (13). The significance of paracoccidioidomycosis results from its high prevalence in areas of endemicity and from the severity of its clinical forms (7). It is estimated that 10 million people may be infected by P. brasiliensis in those areas, and up to 2% of them might develop the infection (15). Acute and subacute forms are found predominantly in children and young adults, and chronic forms predominate in infected adult men (21). The fungus grows as yeast at body temperature and as mycelium at 22 to 26°C. The mycelia produce conidia that differentiate into yeast cells when inhaled by the host, thus establishing the infection (12). Members of the heat shock protein (HSP) family participate in several cellular processes, including acting as molecular chaperones (6, 11). In addition to their central role in transferring peptides through cells, HSPs are recognized as important molecules in the modulation of the immune system. Of the HSP family members, HSP60 has been shown to be a major immunodominant antigen in parasites and a target of the cellmediated and humoral immune responses to infections (9). In fact, immune responses to HSPs have been reported in infectious diseases caused by bacteria, protozoa, and fungi and in models of experimental infection (5, 24, 25, 29). Vaccination using a Histoplasma capsulatum recombinant HSP60 induces a protective cellular immune response in experimental mice against intranasally administrated sublethal doses of fungal cells (10). HSP60 from the human-pathogenic fungus Coccid