Identification and evaluation of Histoplasma capsulatum virulence factors involved in the biofilm formation to identify new therapeutics targets

Abstract

Histoplasma capsulatum is the dimorphic fungus that causes histoplasmosis, respiratory and systemic mycosis, which affects even immunocompetent individuals. Several strategies, such as intracellular permanence, thermodymorphism, expression of adhesins, and, more recently, biofilm formation, have already been described in the literature as important to permanence and multiplication in the various conditions imposed by the host. Thus, the present study intends to verify the expression kinetics of virulence factors genes of Histoplasma capsulatum in its planktonic form and during the biofilms formation on abiotic surface, as well as the importance of them in the process of adhesion and formation of biofilms. It is also proposed to investigate the importance of biofilm in virulence by recovering yeasts from this condition and evaluating the capacity of infection in in vivo and in vitro models. The expression kinetics of the genes related to the virulence factors GAPDH, Enolase, Hsp60, CBP1, alpha-1,3 glucan and Yps3 will be evaluated by real-time PCR (q-PCR). In order to identify the impact of these virulence factors on biofilm formation, biofilms will be made in the presence and absence of polyclonal antibodies related to virulence factors, blocking them, and thus will be analyzed for metabolic activity, extracellular matrix production and total biomass, in addition to physical analysis by scanning electron microscopy. The in vitro infection rate in pneumocytes (A549) and macrophages (RAW 264.7) will be analyzed by flow cytometry, and the virulence and infectivity of planktonic and biofilm yeasts will be evaluated in the alternative model Galleria mellonella, through the establishment of survival curve and fungal load. It is hoped that the results obtained will clarify the role of the various virulence factors of the fungus in the adhesion and consolidation of H. capsulatum biofilms, in order to find possible therapeutic targets related to this condition, as well as to elucidate if the fungus virulence is exacerbated when in biofilm condition.