Coccidioidomycosis (Valley fever), caused by inhalation of Coccidioides arthroconidia, 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.
Antibiotic resistance genes (ARGs) are a new type of pollutant in the environment, and insects serve as the medium for their spread. Maggots, as an important organism in polluted environments, can come into contact with, acquire, and transfer ARGs; however, the patterns indicating the spread of ARGs remain unclear. Here, we showed that tetA, sul2, amp, and km were widely distributed in maggots in chicken farm, pond, trash can, and hospital. Soil and food were important sources of ARGs in maggots. Sul2 was found in the head, chest, and abdomen. Both maggots and adult houseflies have ARGs. Feeding experiments firstly showed that the conjugative transfer of ARGs mediated by plasmids occurred in maggots. Providencia rustigianii and Providencia alcalifaciens in Proteobacteria were the main receptor bacteria of ARGs in maggots. Maggots can serve as new indicators of ARG contamination, and it is of great significance for preventing and controlling the spread of ARGs.
