Browsing by Author "Renshaw, Hilary"

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    Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus.
    (PLoS One, 2015) Falloon, Katie; Juvvadi, Praveen R; Richards, Amber D; Vargas-Muñiz, José M; Renshaw, Hilary; Steinbach, William J
    Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn't show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.
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    Myosin V Function and Regulation in the Morphogenesis of Aspergillus Fumigatus
    (2018) Renshaw, Hilary

    Over the last decade, a growing number of fungal infections of animals and plants has risen and persisted. The lack of diversity in antifungal drugs as well as rising antifungal resistance in many pathogens has exacerbated the problem. Thus there is a critical need for basic molecular understanding of fungal morphogenesis and pathogenesis to design new ways to combat these diseases.

    One of the fungal infections in need of new treatments is Aspergillus fumigatus, the etiological agent of invasive aspergillosis. A. fumigatus is an obligate filamentous fungus that is commonly found in the soil and air. It generally does not cause invasive disease in immunocompetent hosts; however immunocompromised people are at risk for invasive aspergillosis. To better understand the morphogenesis and pathogenesis of this fungus, I decided to study myosins, a group of actin-based motor proteins that are involved in myriad of integral processes in other organisms.

    Through gene deletion, I revealed the importance of the class II myosin, MyoB, in septal formation and conidiation. The class V myosin, MyoE, was required for hyphal polarity, radial extension, septal frequency and conidiation. Importantly, MyoE was required for full virulence in a murine model of invasive aspergillosis. Given the importance of MyoE in critical processes such as hyphal growth and pathogenesis, I aimed to understand the molecular requirements of MyoE. Through iterative truncations of MyoE’s N-terminal tail domain, I revealed the importance of the tail domain in hyphal growth, polarity, and MyoE localization. I identified several phosphorylated residues on the MyoE protein, but mutational analysis did not reveal that any one residue was required for MyoE function. In the absence of the serine/threonine phosphatase, calcineurin, MyoE was phosphorylated at an additional residue. Mutational analysis of a residue in the tail domain revealed it was required for septal localization but not hyphal tip localization, growth, or septation.

    Because MyoE is a cargo binding protein, it likely participates in several pathways that are required for growth and septation of the fungus. To identify novel roles of class V myosins, I identified the MyoE interactome using LC-MS/MS analysis. This analysis revealed several components of the COPII pathway for ER to Golgi transport, suggesting that MyoE may play a role in this protein transport system. My future work aims to understand this role.