A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus

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Abstract
Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy. The pathogenic mold Aspergillus fumigatus is the leading cause of airborne fungal infections in immunocompromised patients. The fungus normally resides in compost, an environment that challenges the organism to obtain nutrients by degrading complex organic polymers. This is accomplished by secreted enzymes, some of which may also contribute to nutrient acquisition during infection. Extracellular enzymes are folded in the endoplasmic reticulum (ER) prior to secretion. If the folding capacity of the ER is overwhelmed by increased secretory demand, the resulting ER stress triggers an adaptive response termed the unfolded protein response (UPR). In this study, we uncover a previously unknown function for the master transcriptional regulator of the UPR, HacA, in fungal virulence. In the absence of HacA, A. fumigatus was unable to secrete high levels of proteins and had reduced virulence in mice. In addition, loss of HacA caused a cell wall defect and increased susceptibility to two major classes of antifungal drugs used for the treatment of aspergillosis. These findings demonstrate that A. fumigatus relies on HacA for growth in the host environment and suggest that therapeutic targeting of the UPR could have merit against A. fumigatus, as well as other eukaryotic pathogens with highly developed secretory systems.