Abstract The prevalence of Aspergillus fumigatus colonization in individuals with Cystic Fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low oxygen culture environment and conidia were more sensitive to oxidative stress inducing conditions compared to non-persistent isolates. Closely related persistent isolates harbor a unique allele of the high osmolarity glycerol (HOG) pathway mitogen activated protein kinase kinase, Pbs2 ( pbs2 C2 ). Data suggest this novel pbs2 C2 allele arose in vivo and is necessary for the fungal response to osmotic stress in a low oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through pbs2 C2 comes at the cost of decreased conidia stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent fungal CF isolates. Importance Aspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with Cystic Fibrosis (CF), Allergic Bronchopulmonary Aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates identified a persistent lineage that harbors a unique allele of the Pbs2 MAPKK necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased CF lung fitness at a cost of reduced in vitro growth in standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

Aspergillus fumigatusIn-Host HOG pathway mutation for Cystic Fibrosis Lung Microenvironment Persistence