Global Profiling of Secreted Protease Activities in Coccidioides posadasii

Coccidioidomycosis (Valley fever), caused by inhalation of Coccidioides spores, remains a diagnostic challenge due to its clinical similarity to other respiratory illnesses and the absence of a rapid, definitive diagnostic test. Current approaches, including serology and imaging, often result in delayed or inconclusive diagnosis. Here, we profiled secreted protease activity in cell-free conditioned media from a single strain of Coccidioides posadasii . This activity could potentially form the basis of a protease-targeted diagnostic assay for clinical specimens. Using multiplex substrate profiling by mass spectrometry (MSP-MS) applied to cell-free conditioned media, we characterized global proteolytic activity at acidic (pH 2.5) and neutral (pH 7.4) conditions. At acidic pH, we detected dominant aspartyl protease activity that was inhibited by pepstatin and exhibited substrate preferences similar to human cathepsin D. Consistent with these findings, a commercial cathepsin D fluorogenic substrate was efficiently cleaved under acidic conditions. At neutral pH, we observed distinct proteolytic activity consistent with contributions from both exopeptidases and endopeptidases. Inhibition profiling and fluorogenic substrate assays further suggested involvement of serine and threonine proteases. These findings provide an activity-based characterization of secreted proteases from a single C. posadasii strain and establish a resource for future studies evaluating their biological and diagnostic relevance.