Abstract
Improved diagnostics are needed to detect invasive pulmonary aspergillosis, a life-threatening infection caused by the pathogenic fungus Aspergillus fumigatus. We are investigating secreted fungal proteases as novel biomarkers for the diagnosis of this disease. Although the A. fumigatus genome encodes a multitude of secreted proteases, few have been experimentally characterized. Here, we employed an unbiased combinatorial library of internally quenched fluorogenic probes to detect infection-associated proteolysis in the lungs of guinea pigs experimentally infected with A. fumigatus. Comparative protease activity profiling revealed a prolyl endopeptidase activity that is reproducibly induced during infection but is not observed in healthy animals. This proteolytic activity was found in four independent animal experiments involving two A. fumigatus isolates. We synthesized a small, focused fluorogenic probe library to define the substrate specificity of the prolyl endopeptidase substrate motif and to identify optimal Probe sequences. These efforts resulted in the identification of a panel of six individual substrate-based fluorescent probes capable of detecting infection in guinea pigs with high statistical significance (P<0.005 in most cases). Receiver operating characteristic analyses demonstrated that this fluorogenic assay could detect A. fumigatus infection-associated proteolysis with comparable sensitivity and specificity as existing diagnostic procedures, suggesting that further optimization of the methodology may lead to improved diagnostics options for invasive pulmonary aspergillosis.
Acknowledgments
The authors wish to acknowledge the encouragement and guidance of Dr Joe Perrone, Director of Diagnostics at SRI Biosciences, and Dr Walter Moos, Vice President of SRI Biosciences. This work was supported by grant R21AI08502 and contract N01AI30041 and HHSN2722010000381 from the National Institute of Allergy and Infectious Diseases (NIAID). SRI International's Center for Advanced Drug Research was established through funding support from the Commonwealth of Virginia.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and the writing of the paper.