Virulence factors of Histoplasma capsulatum involved in biofilm formation

Author(s)

Nathália Ferreira Fregonezi

• Document type: Master's Dissertation
• Press: Araraquara. 2020-11-10.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2020-09-22
• Advisor: Ana Marisa Fusco Almeida; Haroldo Cesar de Oliveira

Abstract

The dimorphic fungus Histoplasma capsulatum is the etiological agent of histoplasmosis, a respiratory and systemic mycosis with worldwide distribution. The pathogenesis of histoplasmosis occurs as a result of microconidia inhalation, which primarily affects the lungs, where they turn into the yeast phase and can spread to other organs. Several mechanisms could be involved in the pathogenesis process, allowing growth in the face of host defense. Among these, the mechanisms for intracellular survival stand out, and, more recently described, the ability to form biofilms could contribute to virulence. Thus, this study aimed to evaluate the impact of the biofilms on the adhesion and virulence of the H. capsulatum EH-315 and G186A strains. Also, we evaluate the role of several proteins in the establishment of these biofilms and in the interaction with the host using in vitro and in vivo models of infection. To assess the virulence profile of biofilms during infection, cell lines A549 (human alveolar basal epithelial cells), J774 and RAW 264.7 (murine macrophages), and the alternative animal model Galleria mellonella were used. In addition, biofilms were evaluated for total biomass, matrix production, and metabolic activity after blocking the proteins Enolase, 14-3-3, PL02996, and GAPDH, described as adhesins and virulence factors for Paracoccidioides spp., and the protein Hsp60, an important binding molecule of H. capsulatum to host macrophages, using specific antibodies. The blockage of proteins with their respective antibodies was also used as a strategy to evaluate the influence of these proteins on the interaction with the G. mellonella model. According to the results, the EH-315 strain demonstrated a higher ability to interact with pulmonary epithelial cells (A549) than the G186A strain, in both planktonic (p <0.0001) and biofilm (p<0.05) cultures. Besides, the passage through biofilm increased the interaction capacity by 32.26% for the G186A strain, and 20.89% for the EH-315 strain. The interaction with macrophages was also influenced after passing through the biofilm, with an increase of 12.75% for the G186A strain and 13.94% for the EH-315 strain in RAW 264.7 cells, as well as in the intracellular proliferation capacity in J774 cells, with a 76.77% increase in proliferation after passage through biofilm in the EH-315 strain. Despite being able to establish the infection and reduce survival in the G. mellonella model, there was no significant difference between the survival curves of the infections with the fungus in planktonic and recovered from the biofilm. However, the passage through biofilm induced earlier death, in addition to inducing a significant reduction (p<0.05) in the number of hemocytes when compared to larvae infected with planktonic cultivation in the EH-315 strain. Regarding the influence of proteins on biofilms, blocking Enolase, 14-3-3, PL02996, Hsp60 and GAPDH caused a significant reduction in biomass, polysaccharide content, and metabolic activity, demonstrating that they are important for the process of consolidation of biofilms. Except for the 14-3-3 protein for the EH-315 strain, these proteins also appear to play an important role in establishing infection in the alternative model G. mellonella, since the blockade led to increased larval survival (p<0.05). Further studies still need to be done to prove the role of these proteins as new virulence factors for H. capsulatum. However, the studied proteins proved to be important in the adhesion to the substrate and formation of H. capsulatum biofilms, and also for interaction with the host. Considering the clinical aspects of the biofilms, and the importance of the different ligands and their receptors in the host for the intracellular fate of H. capsulatum, the knowledge of the repertoire of surface molecules involved in the biofilm formation, as well as interacting with host cells, can contribute to a better understanding of the cell biology and virulence of H. capsulatum, in addition to providing new targets to be explored with antifungal approaches. Also, the results demonstrate the correlation that may exist between the virulence factors of Paracoccidioides spp. and H. capsulatum. We also highlight the use of the G.mellonella model for further study of the pathogenesis and profile of the immune response triggered by this fungus. (AU)

• FAPESP's process: 19/04882-4 - Identification and evaluation of Histoplasma capsulatum virulence factors involved in the biofilm formation to identify new therapeutics targets
• Grantee: Nathália Ferreira Fregonezi
• Support Opportunities: Scholarships in Brazil - Master