Using bar-seq as a proxy to study population dynamics of Aspergillus fumigatus deletion libraries during infection of mammalian cells

<ns7:p> Background Pulmonary infections caused by <ns7:italic>Aspergillus fumigatus</ns7:italic> are a global health concern. In the lungs, <ns7:italic>A. fumigatus</ns7:italic> employs a variety of virulence and adaptation mechanisms that allows it to survive and evade local immune responses. <ns7:italic>A. fumigatus</ns7:italic> protein kinases co-ordinately regulate adaptation to the lung environment and antifungal agents. However, to improve our understanding of the role of these fungal protein kinases in subverting the antifungal responses exhibited by specific host cells, <ns7:italic>in vitro</ns7:italic> infection models should be developed. Here, we describe a functional genomics approach to evaluate the role of <ns7:italic>A. fumigatus</ns7:italic> protein kinases upon infection of airway epithelial cells, leveraging the use of our existing library of <ns7:italic>A. fumigatus</ns7:italic> deletion mutants for kinase functions and bar-seq. Methods We infected A549 alveolar epithelial cells with a pool of 111 <ns7:italic>A. fumigatus</ns7:italic> barcoded null mutant strains in genes encoding protein kinases and used bar-seq to identify which of these are involved in fungal survival upon A549 infection. To assess the optimal method for understanding host-pathogen interactions, three approaches aimed at removing extracellular conidia were evaluated: pharmacological treatment with the antifungal nystatin, washes of unbound conidia, and differential fluorescent staining of extracellular and intracellular conidia followed by cell sorting. The identified candidates were further validated using targeted approaches. Results Our data revealed new roles for <ns7:italic>A. fumigatus</ns7:italic> protein kinases during infection of airway epithelial cells. Modification of the experimental setup allowed us to differentiate between the genes associated with uptake and those required to withstand other stresses imposed by exposure to mammalian cells. Conclusions This protocol provides a new platform to ascribe pathogenicity-associated functions to <ns7:italic>A. fumigatus</ns7:italic> genes and allows prioritization for downstream experiments in mice, potentially reducing the number of studies needed in live animals. This is particularly pertinent, as genome-wide barcoded collections with thousands of strains have become available. </ns7:p>