Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection

Abstract

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

Fig 1. Gp43 gene cloning and expression of the gp43 protein by the transformed S. cerevisiae.

(A) Representative scheme of recombinant gp43 cloned into the vector pBG1805 by Gateway system. The recombinant gp43 is fused to 6X His, HA, a cleavage site of protease 3C and ZZ domain of protein A in C-terminal site, besides being on promoter induction of GAL1. B1 and B2 are recombinant sites of the pBG1805. (B) Detection of recombinant gp43 protein by Western blotting. Overexpression of the gp43 fusion protein in S. cerevisiae yeast cells was induced by adding galactose to the YPD media. The cells grew for 4 hours (lanes 2 and 5) or overnight (3 and 6). Non-induced samples were used as controls (lanes 1 and 4). Total protein extracts were separated by SDS-PAGE, transferred to nitrocellulose membranes and reacted with anti-HA or anti-gp43 antibodies. A polypeptide of approximately 66 kDa, which is the predicted molecular weight of the gp43-tagged fusion protein, was detected either by an anti-HA (Fig. 1B, left) or anti-gp43 polyclonal antibodies (Fig. 1B, right). The bands of molecular weight lower than 66 kDa may correspond to degradation products.

Fig 2. Pulmonary histopathology and morphometric analysis of the granulomas.

The pulmonary histopathology of immunized mice infected with P. brasiliensis were analyzed thirty day post infection. Lungs of mice immunized with yMAgp43 (Vaccine Group) presented discrete granulomatous infiltrates (HE, 10X, G) if compared to controls (Vector and PBS Groups, D and A). Silver methenamine staining revealed that the lungs in the Vaccine Group (H and I) presented less yeast cells and granulomas when compared to controls (B, C, E and F). Panel J: The number of granulomas/mm² in the tissue was obtained using an optical microscopy with the aid of an integrator lens (Carl Zeiss Jena, Germany). Each bar represents the average ± SD of lung sections from 4 animals. Panel K: the granuloma area (mm²) was determined by means of KS-100 program (Carl Zeiss Jenamed 2, USA). The data represent the average ± SD of lung sections from 4 animals, where: *P < 0.05 in relation to the PBS group.

Fig 3. Quantification of fungal burden in the lung and spleen of recombinant yeast-immunized mice, after infection with 1x10⁶ yeasts of P. brasiliensis.

Bars represent CFU levels in lungs (A and C) and spleens (B and D). BALB/c mice were intraperitoneally inoculated with PBS (PBS group), or yMA (Vector Group), or yMAgp43 (Vaccine group) and intravenously infected with P. brasiliensis. One and two months after infection, all mice were sacrificed. The Vaccine group mice presented reduced levels of colony forming units (CFU) when compared with the PBS groups in both organs and both time (A-D). In addition, sixty days post infection, the CFU number in the Vector Group mice was significantly lower than PBS group (C and D). Data are representative of a typical experiment independently repeated three times, where: * P<0.05, **P<0.01, and ***P<0.001 in relation to the PBS Group.

Fig 4. Quantification of cytokines in the lung (A and B) and spleen (C and D) of mice inoculated (i.p) with PBS, or yMA, or yMAgp43, and infected (i.v.) with 1x10⁶ yeasts P. brasiliensis.

Bars represent levels of IL-12p40 (A and C) and IFN-γ (B and D) detected in the organs of mice from different experimental groups at 30 days post-infection. All immunized mice (Vaccine group) showed higher levels of IL-12 and IFN-γ, in both organs, when compared to controls (PBS). Data are representative of a typical experiment independently repeated three times, where: * P<0.05, and **P<0.01 in relation to the PBS Group.