Deletion of core septin gene aspB in Aspergillus fumigatus results in fungicidal activity of caspofungin

Abstract

Septins are a family of GTP-binding proteins found in many eukaryotic lineages. Although highly conserved throughout many eukaryotes, their functions vary across species. In Aspergillus fumigatus, the etiological agent of invasive aspergillosis, septins participate in a variety of processes, including conidiation, septation, and responses to cell wall stress. Previous studies determined that the {Delta}aspB strain had a greater sensitivity to anti-cell wall drugs, especially the echinocandins, yet mechanisms behind this augmented sensitivity are unknown. We performed cell viability staining of the deletion strains after caspofungin exposure and found that the {Delta}aspA, {Delta}aspB, and {Delta}aspC strains had significantly lower cell viability. Concomitant with the reduced viability, deletion strains are more susceptible to caspofungin on solid media. These results indicate that the septin cytoskeleton is important for A. fumigatus survival in the presence of caspofungin. Due to the potential of improved therapeutic outcome, we followed up using a neutropenic murine model of invasive aspergillosis. Animals infected with the {Delta}aspB strain and treated with caspofungin showed improved survival compared to the animals infected with akuBKU80 wild-type or complemented strains. Additionally, histological analysis showed reduced fungal burden and inflammation in the {Delta}aspB infected, caspofungin-treated group. Affinity purification coupled with quantitative proteomics identified proteins involved in the septin-dependent response to caspofungin, including four candidate interactors involved in cell wall stress response. Deletion of these candidate genes resulted in increased susceptibility to caspofungin and moderately reduced viability post-drug exposure. Taken together, these data suggest that septin AspB contributes to the fungistatic response to caspofungin. Author SummaryInvasive aspergillosis is a pulmonary disease caused by the fungus Aspergillus fumigatus that primarily occurs in immunocompromised patients. Invasive aspergillosis has a high mortality rate, ranging from 50-90%. Therapy options are limited due to few available drugs with fungicidal activity and growing global drug resistance. Treatment typically starts with triazoles, which target the fungal cell membrane. If unsuccessful, an echinocandin, which targets the cell wall, is given as a salvage therapy in the U.S. Echinocandins, including caspofungin, are fungistatic against A. fumigatus, slowing growth of the fungus rather than killing it. Due to this, echinocandins have a high therapeutic failure rate. Previous work suggests that deletion of the cytoskeletal septin genes increases sensitivity to caspofungin. Here we describe our finding that the septin genes aspA, aspB, and aspC are involved in the fungal response to caspofungin. Additionally, the deletion of aspB results in fungicidal activity of this otherwise fungistatic drug. These findings show promise for novel therapy options that block the septin-mediated response to caspofungin.