Short-term in vivo exposure to murine lung neutrophils of differing oxidative capacity does not drive detectable genomic change in Aspergillus fumigatus

Neutrophil NADPH-oxidase (NOX)-derived reactive oxidative stress contributes to pulmonary clearance of Aspergillus fumigatus and is assumed to impose a strong selective pressure on inhaled conidia. It, however, remains unclear if repeated exposure to host neutrophil oxidative killing drives rapid genomic adaptation. We examined genomic and phenotypic outcomes of serial in vivo passages of neutrophil-associated conidia through wild type NOX2-sufficient and NOX2-deficient murine lungs. Whole genome sequence analysis compared four independent lineages from each host background with the parental Af293 population and did not identify any de novo single-nucleotide polymorphisms (SNPs) that met the applied filtering criteria. Four loci were initially identified to have undergone marked allele-frequency shifts, however, targeted amplicon sequencing revealed that the alternative alleles were equally present in both host groups, and these loci fell in regions of low sequencing coverage in the whole genome sequencing analysis. Mouse-passaged strains exhibited phenotypic change, including increased radial growth on standard and nutrient limiting conditions including carbon, iron, and zinc limited media. They also showed enhanced germination under glucose limiting conditions and lung homogenate. These phenotypic changes were recapitulated by in vitro passaged control strains indicating that they arise from repeated growth on minimal media rather than host immune selection. While host passaged strains did not exhibit enhanced resistance to neutrophil-mediated killing, all passaged strains showed reduced levels of thermotolerance, and NOX2-passaged lineages were more susceptible to hydrogen peroxide mediated oxidative stress. Together, these findings show that ten serial passages through oxidative and non-oxidative pulmonary environment were accompanied by phenotypic changes without detectable convergent SNP changes associated with host oxidative background or enhanced resistance to neutrophil killing. Early adaptation to the host appeared to proceed through non-genomic routes under pressures that are nutritional rather than oxidative.