A reduced amount of 14-3-3 protein and the implications to Paracoccidioides brasiliensis infection.

Objectives:
Paracoccidioidomycosis (PCM), a systemic endemic mycosis with chronic and granulomatous evolution is caused by thermal dimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. The infection occurs through the inhalation of conidia derived from the mycelial form present in the natural environment. It can reach the lungs and converted into yeasts producing pulmonary lesions and subsequently spread to others organs. The down-expression of Pb14-3-3 (expression reduced by 55 %) previously obtained by antisense technology (aRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) conferred phenotype modifications to the transformant isolate, Pb14-3-3 aRNA, such as impairment in the dimorphism, reduced number of buds per yeast, decreased interaction with pneumocytes and attenuation of the virulence in Galleria mellonella model demonstrating the importance of this protein in P. brasiliensis and its interaction with the host. Our aim is to evaluate the pathogenic role of 14-3-3 protein of Paracoccidioides brasiliensis in a murine model of infection and the induction of pneumocytes apoptosis.

Methods:
Experimental paracoccidioidomycosis in mice with fungal transformant, Pb14-3-3 aRNA, PbWT (wild-type strain) and PbEV (empty-vector strain) assessing evidences as number of granulomas and fungi in the lung tissues and measurement of fungal burden after 15 days of infection. TUNEL assay was used to verify host cell apoptosis induction after 5 and 24 hours of interaction.

Results:
The lungs from mice infected with PbWT and PbEV showed a higher number of granulomas and these had a higher number of yeast cells, when compared with the mice group infected with Pb14-3-3 aRNA. Similarly, the number of CFU/g of organ recovered from the lungs of mice infected with Pb14-3-3 aRNA was approximately 87.7 % lower than the number recovered from the mice infected with PbWT and PbEV. When compared to PbWT and PbEV, the silenced strain Pb14-3-3 aRNA induces significantly less apoptosis for 5 and 24 hours.

Conclusion:
Pb14-3-3 aRNA displays attenuation of the virulence triggering the formation of fewer granulomas with decreased number of yeast cells in the lungs and reduced fungal burden, as well as, less ability to induce host cells apoptosis. Our results showed that 14-3-3 is an important virulence factor in pulmonary infection caused by P. brasiliensis.