Screening of chemical libraries for new antifungal drugs against Aspergillus fumigatus reveals sphingolipids are involved in the potential mechanism of action of miltefosine

<i>Aspergillus fumigatus</i> is an important fungal pathogen and the main etiological agent of aspergillosis, a disease characterized by a noninvasive process that can evolve to a more severe clinical manifestation called invasive pulmonary aspergillosis (IPA) in immunocompromised patients. The antifungal arsenal to threat aspergillosis is very restricted. Azoles are the main therapeutic approach to control IPA, but the emergence of azole-resistant <i>A. fumigatus </i>isolates has significantly increased over the last decades. Therefore, new strategies are necessary to combat aspergillosis and drug repurposing has emerged as an efficient and alternative approach for identifying new antifungal drugs. Here, we used a screening approach to analyze <i>A. fumigatus</i> in vitro susceptibility to 1,127 compounds. <i>A. fumigatus</i> was susceptible to 10 compounds, including miltefosine, a drug that displayed fungicidal activity against <i>A. fumigatus</i>. By screening an <i>A. fumigatus</i> transcription factor null library, we identified a single mutant, which has the <i>smiA</i> (<i>s</i>ensitivity to <i>m</i>iltefosine) gene deleted, conferring a phenotype of susceptibility to miltefosine. The transcriptional profiling (RNA-seq) of the wild-type and the Δ<i>smiA</i> strains and the Chromatin Immunoprecipitation coupled to next generation sequencing (ChIP-Seq) of a SmiA-tagged strain exposed to miltefosine revealed genes of the sphingolipids pathway that are directly or indirectly regulated by SmiA. Sphingolipids analysis demonstrated that the mutant has overall decreased levels of sphingolipids when growing in the presence of miltefosine. The identification of SmiA represents the first genetic element described and characterized which plays a direct role in miltefosine response in fungi.<b><br> </b>