Study of the effects of mutations in the genes of Histoplasma capsulatum, generated by CRISPR-Cas9, on the survival and intracellular replication of yeasts

Abstract

Histoplasmosis is an infection caused by the dimorphic fungus Histoplasma capsulatum, being one of the most important systemic and endemic mycoses with a wide worldwide distribution, and areas of predominance in the Americas. Histoplasma is found mainly in soils contaminated with the excrement of certain species of birds or bat guano; consequently, infection with this pathogen usually occurs after exposure to these contaminated environments. Histoplasma spp. is an intracellular pathogen, yeasts are highly adapted to the host, using macrophages as a proliferative niche while avoiding microbicidal mechanisms inside these cells. As a result, innate immune cells are unable to control H. capsulatum on their own. During infection of H. capsulatum in murine macrophages, the expression of a gene encoding a co-activating protein, 100 KDa protein (Hc100p), was observed; this protein seems to play an important role in the adaptation and survival mechanisms of this fungus in hostile conditions inside macrophages. However, so far the characterization and function of this protein are unknown. Also, within the intracellular compartment, the replication of Histoplasma yeasts requires the acquisition of several essential nutrients, including metal ions. Studies show that although iron and zinc are sufficiently abundant in macrophages at rest, activation of cytokines from cellular hosts causes restriction of these metals to yeast as a form of nutritional immunity. Thus, the objective of this study is to elucidate and characterize, genes and proteins involved in the mechanisms of virulence, survival and intracellular replication of the fungal pathogen Histoplasma capsulatum. Methods: Histoplasma capsulatum strain G217B will be cultured to obtain mutant strains using the CRISPR-Cas9 editing system, for the gene coding for the coactivating protein (Hcp100), as well as for those coding for proteins involved in the acquisition of micronutrients iron and zinc. The survival and replication of the mutant and wild strains will be evaluated in an in vitro model using macrophages activated by cytokines. Subsequently, a proteomic analysis of the wild yeasts and mutants of H. capsulatum, from activated macrophages, will be performed to characterize the proteins and molecular pathways involved in the virulence, survival and replication of this pathogen. Together with these analyzes, the survival and replication of mutant strains obtained from H. capsulatum in an in vivo model of Pulmonary Histoplasmosis will be evaluated, as well as the analysis of pro and anti-inflammatory cytokine production in the lungs of infected mice. (AU)