Functional characterization of genes related to the biofilm formation in the fungal pathogens Histoplasma capsulatum and Paracoccidioides brasiliensis

Abstract

Fungal infections are neglected by the social and political point of view. However, these affect more than one billion people, resulting in approximately 11.5 million deadly infections and more than 1 to 5 million deaths per year. Despite advances in several public health areas, many pathogens remain important because of their pathogenicity and represent an important source of infection for humans and animals. Systemic mycoses emerge worldwide as an increasingly important problem and the ability of fungi to form biofilms is associated with increased drug tolerance. Histoplasmosis and paracoccidioidomycosis are considered important endemic and systemic mycoses in the world and Latin America, respectively. These mycoses are caused by the dimorphic fungi Histoplasma capsulatum and Paracoccidioides brasiliensis. It is noteworthy that the most affected population are immunocompromised individuals, mainly those with HIV/AIDS or who are receiving therapies that include corticosteroids or biological compounds such as monoclonal antibodies against tumor necrosis factor alpha (TNF-a), and associated with exposure to the rural environment and to agricultural activities, among others. The incidence of these diseases can be altered by climate phenomena and mainly by human migration and occupation of poorly explored territories. These fungal pathogens exhibit several virulence factors such as thermal dimorphism, adhesins, and expression of genes involved in the detoxification of reactive oxygen species produced by host phagocytic cells. In addition, it has been demonstrated that H. capsulatum and P. brasiliensis have the capability to form biofilm, considered as an important virulence factor. However, it is not known what genes are involved in the biofilm formation by H. capsulatum and P. brasiliensis.Herein, the present join proposal aims to characterize and evaluate genes involved in the biofilm formation by H. capsulatum and P. brasiliensis in order to elucidate the role of these molecules as virulence factors and to search for possible therapeutic targets. This proposal is framed and support by the FAPESP research project, process 2018/02785-9: "Dermatophytes and Dermatophytosis: biofilm formation and development of control strategies".To achieve this, a search for orthologous genes associated with the formation of biofilms in Candida albicans and other importantly medical fungi will be performed. Once the genes have been selected, a search of these orthologous genes in the annotated genome of H. capsulatum and P. brasiliensis will be done, and their expression will be evaluated using a model of Histoplasma and P. brasiliensis biofilm formation on lung epithelial cells (A549) and, finally, the CRISPR/Cas9 technique will be used to obtain mutants for the selected genes and determine their role in the biofilm formation process. In addition, the inflammatory response induced by planktonic and biofilm cells of H. capsulatum and P. brasiliensis on epithelial cells through expression of genes coding for proinflammatory cytokines (IL-1, IL-6, IL8 and TNF-a), as well as apoptosis will be determined. This proposal is framed in the area of Health, specifically in the area of Pharmacy, Microbiology and Immunology. (AU)