Polarized hyphal growth and septation facilitate invasion of host tissue by A. fumigatus. To better understand these processes, we are studying myosins, a group of motor proteins categorized into classes based on their structure. A. fumigatus contains three myosins encompassing three classes: class I (myoA), class II (myoB), and class V (myoE). These proteins are involved in growth, morphology and septation in other fungi; however, the role of myosins in the growth and virulence of a human pathogen has never been explored. Because of myosins’ involvement in critical cellular processes, an understanding of their precise functions and regulation in A. fumigatus will lead to fundamental knowledge of pathogenesis which may help identify novel drug targets.
We generated two myosin single deletion strains (ΔmyoB and ΔmyoE) and a double deletion strain (ΔmyoB ΔmyoE). Attempts to delete the myoA gene were unsuccessful. Radial growth was assessed on solid media daily for five days and conidiation quantified. Transmission electron microscopy was used to visualize septa and hyphal morphology. Conidia viability was assessed using bis-(1,3-Dibarbituric acid)-trimethine oxanol (DiBAC). Virulence was determined using a persistently immunosuppressed murine model of invasive aspergillosis. Phosphorylated resides were determined by GFP-Trap protein purification of MyoE-GFP, TiO2 phosphopeptide enrichment, and LC/MS-MS.
While the ΔmyoB strain showed no significant difference in radial extension, the ΔmyoE and ΔmyoB ΔmyoE strains resulted in a significant defect (p<0.001 at day 5). Both MyoB and MyoE are required for full conidiation (p<0.0001). Deletion of myoE resulted in hyperbranching and loss of polarity. TEM revealed that the cell walls of both the ΔmyoB and the ΔmyoE strains appear normal but the ΔmyoB strain contains incomplete, thicker septa. Septa in the ΔmyoE strain appear wild-type; however staining revealed that deletion of myoE resulted in hyperseptation (p<0.001). In our murine model, both the ΔmyoB and ΔmyoE strains were hypovirulent (p<0.001 and p<0.01, respectively). Deletion of myoB resulted in a 2-fold increase in inviable conidia (p<0.05), and deletion of myoB or myoE resulted in significantly delayed germination. Because of the significant radial extension and hypovirulence in the ΔmyoE strain, we became interested in MyoE as a drug target and wanted to understand its regulation. We determined that MyoE is phosphorylated at eight residues, encompassing each of its four domains.
We demonstrated that myosins have distinct roles in hyphal morphology and virulence and that MyoA is likely essential, as in other fungi. MyoB and MyoE are required for conidiation. MyoE has a role in preserving hyphal polarity and/or suppressing new growth foci. MyoB is important for proper septa formation, while MyoE may have a role in septa frequency. MyoE is required for virulence in a murine model. The reduction of virulence in the ΔmyoB strain may be due to lack of conidia viability. We are currently exploring the importance of phosphorylation as a regulation mechanism of MyoE to better understand its role in virulence.