Invasive aspergillosis (IA) caused by the fungus Aspergillus fumigatus has surpassed invasive candidiasis as the most frequent fungal cause of death in immunocompromised patients. The Infectious Diseases Society of America highlighted A. fumigatus as one of only six pathogens for which a substantive treatment breakthrough is urgently needed. Despite an increasing incidence of disease and mortality, the basic determinants underlying A. fumigatus pathogenesis are not well understood. Our long-term goal is to understand the molecular control of A. fumigatus growth and disease. The septum is a fundamental biologic structure important for the growth of many fungi and an ideal therapeutic target. A. fumigatus hyphae produce compartmentalizing septa to advance and invade host tissue. We have shown that calcineurin stably localizes to the A. fumigatus septum and genetic or pharmacologic inhibition of calcineurin blocks hyphal growth and disrupts septation. Despite its pivotal role in growth, no studies have focused on the hyphal septum to understand pathogenesis, and calcineurin-mediated control of septum formation is not understood. Our central hypothesis is that calcineurin complexes with other proteins at the A. fumigatus septum to regulate septum formation and hyphal extension. The objective of this application is to define those proteins that bind to calcineurin at the hyphal septum. Our approach will be divided into two specific aims: 1) Identify calcineurin- binding proteins in A. fumigatus through calcineurin A and B pulldown assays followed by LC/MS/MS analysis. Calcineurin A and B are highly concentrated at the septum, arguing for a strong interaction, a role different than calcineurin's known function as a phosphatase. We will identify what calcineurin binds at the septum to begin to understand the mechanism of growth regulation. 2) Validate septal localization and calcineurin-dependency of putative calcineurin-binding protein. We will prioritize and localize those newly identified proteins in wild-type A. fumigatus to verify their cellular location and function. To characterize the calcineurin-dependency of the identified calcineurin-binding septal proteins, we will utilize our unique set of calcineurin deletion strains. These results will have a significant impact in understanding underlying growth mechanisms, focusing on the novel theory that calcineurin directly regulates septum formation to orchestrate hyphal growth and disease. Our approach to define the proteins interacting with calcineurin at the septum will begin to unravel the larger molecular basis of A. fumigatus pathogenesis. This strategy will move us toward our long- term goal of developing novel IA therapeutics by identifying exploitable fungal-specific targets to block septum formation and growth, leading to a substantial impact on IA mortality.

R21AI097541

2012-07-01

2014-06-30