Rho2-dependent cell wall remodeling boosts the fungistatic activity of manogepix against Aspergillus fumigatus

Fosmanogepix is a new antifungal agent currently undergoing phase 3 clinical trials. Its active moiety, manogepix, inhibits Gwt1 which is an enzyme essential for the assembly and attachment of glycosylphosphatidylinositol anchors to cell wall proteins. Manogepix has a strong fungistatic activity against most human pathogenic fungal species. Here we characterized the activity of manogepix against the major human pathogenic mold Aspergillus fumigatus. We show that manogepix susceptibility is linked to the expression of gwt1, the gene encoding Gwt1, thereby demonstrating that overexpression of gwt1 can confer resistance. In agreement with a previous study conducted with a different fungal pathogen, we observed an increase of cell well chitin after manogepix treatment. Using a luciferase-based reporter assay, we show that manogepix activates the cell wall integrity stress response pathway. However, manogepix only occasionally causes cell lysis. Mutants that lack the cell wall stress sensor Wsc1 or the Rho GTPase Rho4, which is important for septum formation, are slightly more susceptible to manogepix. In contrast, mutants that lack the Rho GTPase Rho2 or the cell wall stress sensor MidA, both of which are key for A. fumigatus to survive cell wall stress caused by granulocytes, heat and other cell wall perturbing chemical compounds, grow better than wild-type when exposed to manogepix. We show that both mutants, especially {Delta}rho2 but also {Delta}midA, fail to upregulate cell wall chitin to wild-type-like levels in response to manogepix. These results implicate a role for the Rho2-dependent stress response in the fungistatic activity of manogepix against A. fumigatus.