Loss Of Septation Initiation Network (Sin) Kinases Blocks Tissue Invasion And Unlocks Echinocandin Cidal Activity Against Aspergillus Fumigatus

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.Author summary Aspergillus fumigatus is a ubiquitous fungal pathogen and the major causative agent of a life-threatening infection known as invasive aspergillosis (IA). IA is a disease in which, typically immune compromised, patients experience unfettered growth of this pathogenic mold in the lungs with dissemination to other organs being common. Therapeutic options are limited by multiple factors and only three major antifungal drug classes are currently in use. Although effective as treatment for other fungal diseases, the echinocandin class of antifungals have limited usefulness against IA. Our overall goal is to identify novel fungal proteins that, if targeted for inhibition as part of a co-therapeutic approach, could improve the anti-Aspergillus activity of echinocandins and therefore lead to better patient outcomes. Here, we sought to identify A. fumigatus genes required for fungal survival under echinocandin stress by generating and screening an A. fumigatus mutant library composed of disruptions in genes encoding putative protein kinases. We found that protein kinases required for hyphal septation are essential for survival in the presence of echinocandins and, surprisingly, for the ability of A. fumigatus to invade lung tissue. Our results suggest that novel septation inhibitors could enhance echinocandin activity while simultaneously limiting A. fumigatus virulence.