An epigenetic mechanism of azole tolerance facilitates acquired antifungal resistance in Aspergillus fumigatus

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, and particularly in filamentous fungi, remain elusive. Here, we identified an In hibitor of G rowth domain-containing protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus . The loss of ingB promotes supra-MIC growth on agar surfaces despite susceptible MICs in standardized assays. Moreover, established Δ ingB biofilms are also less susceptible to azoles in vitro . In a murine model of invasive pulmonary aspergillosis, loss of ingB results in higher pulmonary fungal levels when animals are treated with voriconazole compared to the wild-type control. Subsequent exposure of the Δ ingB -tolerant strain to high azole concentrations in vitro resulted in rapid acquired resistance, most notably driven by a frameshift mutation in a putative 20S proteasome maturation protein-encoding gene, ump A, while the susceptible wild-type strain failed to acquire adaptive mutations. The data suggest that loss of IngB provides an epistatic background for the emergence of azole resistance. Our work shows that drug tolerance in a critical fungal pathogen can facilitate azole resistance emergence. IMPORTANCE While antimicrobial drug resistance causes adverse effects on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remain elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus . In a murine model of invasive pulmonary aspergillosis treated with voriconazole, loss of ingB facilitated higher fungal burden levels than the wild-type control, suggesting the observed in vitro tolerance translates to the murine pulmonary environment. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure in vitro and led to the discovery of a new gene associated with azole resistance, umpA . Our work identifies a novel regulator of antifungal tolerance in a critical human fungal pathogen and suggests that drug tolerance can pave the way for resistance emergence.