Unlocking the cidal activity of echinocandins against Aspergillus fumigatus

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Invasive aspergillosis (IA), caused mainly by A. fumigatus, is the most prevalent invasive mold infection of immunocompromised individuals and is associated with mortality rates of 35-90%. Only three classes of anti- Aspergillus drugs exist. The global rise of resistance to the triazole class and the unacceptably high patient toxicity of the polyene class limits the use of two of these. The remaining class, the echinocandins, are generally considered effective therapeutic agents as their target is fungus-specific (cell wall biosynthesis) and they exhibit low toxicity. However, the echinocandins are not fungicidal for Aspergillus species and a paradoxical effect of treatment, characterized by decreased drug effectiveness with increasing drug concentrations, has been described both in vivo and in vitro. Likely due to these issues, high incidence breakthrough infections during echinocandin therapy have been reported. Therefore, echinocandin use for IA is also limited. The discovery of mechanisms essential for echinocandin stress adaptation and survival is expected to improve therapeutic efficacy with these important compounds by identifying fungal targets for future combination therapies. To delineate novel mechanisms orchestrating echinocandin stress responses, we recently completed the generation of a protein kinase disruption mutant library in a wild type genetic background. This library was constructed using CRISPR/Cas9 gene-editing to disrupt each of the 142 putative protein kinases encoded by the A. fumigatus genome. A total of 118 non-essential, unique gene disruptions were achieved and subsequently employed for in vitro echinocandin susceptibility assays. Our preliminary assays identified a total of 12 protein kinase disruption mutants displaying 4- to >32-fold decreased minimum effective concentrations (MEC) compared to the parental strain. We have discovered that two of these mutations, residing in the previously uncharacterized SepL and SidB kinases, impart fungicidal anti-Aspergillus activity to multiple echinocandins. These protein kinases are predicted to be core components of the Septation Initiation Network (SIN), a three- kinase cascade that is necessary for septation in fungi. Our exciting preliminary data show that blocking septation via loss of any single SIN kinase causes widespread hyphal damage and loss of viability in response to echinocandin treatment. In addition, employing the sepL disruption mutant, we have found that echinocandin therapy improves survival and eliminates residual tissue burden in a mouse model of IA. As septa are important for the limitation of mechanical injury to hyphae, and the A. fumigatus SIN is completely uncharacterized, exploration of this network is expected to reveal novel effectors of echinocandin stress survival. Our aims are to identify core SIN components required for unlocking echinocandin cidal activity (Aim 1), define temporal requirements for enhancement of echinocandin activity resulting from septation blockade (Aim 2), and to delineate the SIN pathway-dependent septum construction machinery (Aim 3). Our work will identify the SIN components with the highest potential for benefit when targeted in combination with echinocandin therapy.