Hypoxia morphology of Aspergillus fumigatus impacts diagnostics - Aspergillus fumigatus (Af) is a common environmental mould that can cause high-mortality mycoses, such as Invasive Pulmonary Aspergillosis (IPA). Disease caused by A. fumigatus has remained significantly difficult to diagnose due to physical limitations of sample collection and limited diagnostic methods, meaning Af-mycoses are often not definitively, if ever, diagnosed until disease has progressed to a severe state. Contemporary clinical diagnosis of A. fumigatus disease relies on non-culture-based methods of organism detection including Platelia and FungiTell microplate assays to detect fungal galactomannan antigen and beta-glucans respectively. However, the extent to which heterogeneity in A. fumigatus clinical isolates impacts disease diagnosis has not been studied. Numerous clinical observations over the years strongly suggest significant heterogeneity in A. fumigatus morphologies that I hypothesize impacts diagnostic results. Our lab has previously identified that increased expression of a subtelomeric gene hrmA via SNP D304G (strain: HrmAREV) is sufficient to induce a clinically observed hypoxia-fit morphologies (H-MORPH) of A. fumigatus. H-MORPH is clinically relevant as strains exhibiting this morphology can be isolated from patients, however it remains unknown how this morphology impacts the ability to detect and diagnose IPA. My preliminary data suggests that the lab-generated H-MORPH HrmAREV exhibits increased levels of both beta-glucan and galactomannan antigens compared to isogenic N-MORPH strains in vitro. My central hypothesis is that H-MORPH isolates will result in the increased ability to detect and diagnose IPA both in vitro and in vivo. To address this central hypothesis, I will pursue the following two aims: 1.) I will test the hypothesis that the H-MORPH associated gene, hrmA, regulates antigenic extracellular polysaccharide composition, and 2.) I will test the hypothesis that HrmA induction increases the ability to diagnose in vivo infection using contemporary clinical diagnostic markers. As we have previously identified that HrmA has a weakly predicted RNA recognition motif (RRM) I hypothesize HrmA mediates the alteration of the extracellular polysaccharide profile through post- transcriptional RNA modulation, which I will probe with targeted RNA immunoprecipitation. After determining the extracellular polysaccharide composition, I will use N- and H-MORPH strains in vitro and in vivo to determine how morphology impacts detection using clinical diagnostic Fungitell and Platelia microplate assays. In my approach, I will combine genetics, biochemical assays, and clinical microbiology diagnostic assays to determine the role of HrmA in A. fumigatus clinically relevant diagnostic antigen production. Overall, the experiments outlined in this proposal will identify a role for the fungal specific gene, hrmA, in modulating fungal biology through translational implications of disease as it relates to a clinically relevant morphotype of A. fumigatus, while providing me with important pre-doctoral training in fungal genetics, biochemistry, bioinformatics, and clinical microbiology.
