Phenotypic and Proteomic Analysis of the Aspergillus fumigatus ΔPrtT, ΔXprG and ΔXprG/ΔPrtT Protease-Deficient Mutants

Abstract

<i>Aspergillus fumigatus</i> is the most common mold species to cause disease in immunocompromised patients. Infection usually begins when its spores (conidia) are inhaled into the airways, where they germinate, forming hyphae that penetrate and destroy the lungs and disseminate to other organs, leading to high mortality. The ability of hyphae to penetrate the pulmonary epithelium is a key step in the infectious process. <i>A. fumigatus</i> produces extracellular proteases that are thought to enhance penetration by degrading host structural barriers. This study explores the role of the <i>A. fumigatus</i> transcription factor XprG in controlling secreted proteolytic activity and fungal virulence. We deleted <i>xprG</i>, alone and in combination with <i>prtT</i>, a transcription factor previously shown to regulate extracellular proteolysis. <i>xprG</i> deletion resulted in abnormal conidiogenesis and formation of lighter colored, more fragile conidia and a moderate reduction in the ability of culture filtrates (CFs) to degrade substrate proteins. Deletion of both <i>xprG</i> and <i>prtT</i> resulted in an additive reduction, generating a mutant strain producing CF with almost no ability to degrade substrate proteins. Detailed proteomic analysis identified numerous secreted proteases regulated by XprG and PrtT, alone and in combination. Interestingly, proteomics also identified reduced levels of secreted cell wall modifying enzymes (glucanases, chitinases) and allergens following deletion of these genes, suggesting they target additional cellular processes. Surprisingly, despite the major alteration in the secretome of the <i>xprG/prtT</i> null mutant, including two to fivefold reductions in the level of 24 proteases, 18 glucanases, 6 chitinases, and 19 allergens, it retained wild-type virulence in murine systemic and pulmonary models of infection. This study highlights the extreme adaptability of <i>A. fumigatus</i> during infection based on extensive gene redundancy.