From the development of three-dimensional human lung models of infection to the evolution of functional human lung models for studying the interaction with Histoplasma capsulatum biofilm

Abstract

Histoplasma capsulatum var capsulatum is the dimorphic fungus that causes histoplasmosis, a lung infection that can spread to other organs, causing severe forms of the disease. Histoplasmosis is the fourth mycosis that most affects people annually and is distributed worldwide, being considered an Invasive Fungal Infection (IFI) and classified as of high global relevance by the WHO in 2022. As it is an intracellular pathogen, several host cell genes have their expression induced by the fungus to make the macrophage more susceptible to survival and proliferation that can be directly related to the pathogen's virulence factors. We believe that the pathogen-host interaction of H. capsulatum biofilms triggers the activation of different genes that make macrophages more susceptible to infection by biofilms compared to infection by planktonic cells, and H. capsulatum biofilms are mechanisms of infection that trigger several virulence factors. In addition to the binding of communities to the substrate, we believe that the cell-cell interactions that characterize a biofilm affect the aggressiveness of the infection. Thus, this research project aims to develop and characterize models of three-dimensional cell cultures more reliable to what happens in vivo of spheroids mono and co-culture of human lung using MRC-5 and THP-1 macrophage-like cells, and Functional human lung tissue model using co-culture of hSAEC cells differentiated by Air-Liquid Interface (ALI), MRC-5 and THP-1 macrophage-like through confocal laser scanning microscopy (MCLV), scanning electron microscopy (SEM) ), transmission electron microscopy (TEM) and RT-PCR to evaluate the biofilm-host interaction of H. capsulatum; characterize live infection by H. capsulatum biofilms with adhesins blocked by monoclonal antibodies (Hsp60, 14-3-3, enolase, GAPDH and Dectin-1) in three-dimensional models using the Cell ASIC Onix microfluidic system (MilliPore), characterizing the infective process by RT-qPCR of the genes Hsp60, CatB, DDR48, GAPDH and CBP1 related to the fungus and which are differentially expressed in biofilms, and the host cell genes C3ar1, Gnb2 and Pdcl (from the GPCR signaling pathway), Ithb2 and Fermt3 (from the alternative pathway of integrin activation), and Actb,Nckap1l (from the actin cytoskeleton), and confocal microscopy; to determine the mechanisms of pathogen-host interaction important in the infection by H. capsulatum biofilms through whole-genome screening by CRISPR/Cas9 of THP-1 CRISPRKO cells transformed and inserted into three-dimensional models; and finally, to evaluate the influence of host cell genes important in the survival of H. capsulatum biofilms after treatment with nitrofuran derivatives using total genome screening by CRISPR/Cas9 in models produced with transformed THP-1 CRISPRKO. (AU)