Invasive aspergillosis, caused mainly by A. fumigatus, is the most prevalent invasive mold infection of immunocompromised individuals and is associated with mortality rates of 35-90%. Therapy options are extremely limited for invasive aspergillosis and resistance to the triazole class of antifungals is on the rise. Significant knowledge gaps concerning how A. fumigatus adapts to drug-induced stress (i.e., drug tolerance) impair our ability to improve antifungal therapy and halt the rise in resistance. Our central hypothesis is that protein phosphorylation events orchestrate cellular processes crucial for A. fumigatus triazole antifungal tolerance. To delineate novel roles for the A. fumigatus kinome in triazole tolerance, we have completed a preliminary phospho-proteomics analysis of the response to voriconazole treatment. Our preliminary data reveal significant changes in the phosphorylation status of ~1400 proteins, implying extensive kinase-mediated adaptation to drug-induced stress. Filtering this dataset for proteins known to mediate triazole tolerance, we identified voriconazole-induced phosphorylation state changes in HapB. The HapB protein is a subunit of the heterotrimeric CCAAT-binding complex (CBC), a crucial transcriptional repressor of ergosterol biosynthesis genes. Interestingly, clinical triazole resistance in select A. fumigatus isolates has been ascribed to mutations that alter functionality of the CBC. Therefore, delineation of CBC regulatory mechanisms could lead to novel interventions targeting antifungal tolerance and/or resistance.
In Aim 1, we will test the working hypothesis that CBC-mediated regulation of triazole tolerance is controlled by differential phosphorylation of the HapB subunit.
In Aim 2, we will identify novel protein kinases supporting antifungal fitness in A. fumigatus using our novel CRISPR/Cas9 mutational approach. Through these aims, we expected to uncover multiple, novel contributions of protein kinases to A. fumigatus antifungal fitness. In addition, we will potentially delineate a phospho- regulatory mechanism controlling the CBC, a crucial transcriptional regulator of the triazole stress response. As they are considered the second largest class of proteins currently functioning as drug targets, identification of the protein kinases crucial to triazole tolerance could reveal novel targets for use in new stand-alone or combination therapies. Therefore, the potential impact of this work is the improvement of antifungal therapy and significant advance towards the sustained clinical utility of triazole antifungals against Aspergillus.
Invasive aspergillosis is associated with high mortality rates in patients with suppressed or ineffective immune systems. Successful treatment of Aspergillus fumigatus infections is becoming more difficult, as resistance to currently available antifungal drugs is on the rise. The proposed research will significantly increase our understanding of how A. fumigatus survives in the presence of antifungal therapy and will yield important information needed to improve current drug therapy.