MSACL 

15. Proteomic Identification of Coccidioidal Antigens from Lung Fluid of Infected Mice: Applications in Diagnostic Test Development
Tue 12:12 PM - PosterSplash Track 2
Forrest S.E. Helfrich
University of Arizona
Forrest S.E. Helfrich; Department of Chemistry and Biochemistry, University of Arizona
Erin Johnson; Department of Chemistry and Biochemistry, University of Arizona
Lisa F. Shubitz; Valley Fever Center for Excellence, Department of Veterinary Science and Microbiology, University of Arizona
Tao Peng; Valley Fever Center for Excellence, Bio5 institute, University of Arizona
Amy Hilderbrand; Department of Chemistry and Biochemistry, University of Arizona
John N. Galgiani; Valley Fever Center for Excellence, Bio5 Institute, University of Arizona
Vicki H. Wysocki; Department of Chemistry and Biochemistry, Bio5 Institute, University of Arizona
In the southwestern United States, coccidioidomycosis (Valley Fever) is an important cause of community acquired pneumonia (CAP), with more severe complications also occurring in both humans and pets. Early infections are indistinguishable from CAP of other causes and currently available serologic tests for Valley Fever are often insensitive to early infections. An alternative approach is to detect fungal proteins that present before host antibodies develop during infection. Bronchoalveolar lavage fluid (BALF) and lung homogenate (LH) samples were collected from mice infected with Coccidioides posadasii 14 d after intranasal infection. Multidimensional protein identification technology (MudPIT) and C-18 reverse phase liquid chromatography tandem mass spectrometry were utilized for the analysis of coccidioidal proteins in the BALF and LH samples. Data analyzed by SEQUEST, X! Tandem, and Scaffold confidently identified 21 coccidioidal proteins. By using the Basic Local Assignment Search Tool (BLAST), three of the proteins were found to have no mammalian homologs and show <45% amino acid similarity to all other fungal proteins in GenBank. These were selected as candidates for diagnostic targets to use in the development of an antigen capture diagnostic assay that will be amenable to point-of-care application.

All three of these coccidioidal proteins have been cloned, expressed in S. cerevisiae, and one was used to raise high affinity polyclonal antibodies in goats. The goat anti-serum identified the coccidioidal target protein by immunoblotting lung homogenates from infected mice. Furthermore, a competition ELISA assay sensitive to 1 ng/ml of antigen measured as much as 1000 ug per mouse lung 14 d after infection. There is a striking concordance in the antigen concentrations and the severity of infection. This is true over the duration of infection with respect to the relative susceptibilities of the three mouse strains studied.

Current studies are underway to identify oligopeptides through phage-display biopanning experiments that specifically bind to our biosignature protein targets. The oligopeptides will be used as capture agents for a fluorescence based diagnostic assay.

Funding
Arizona TRIF Grant to the Bio5 Institute
NIH AI065359
Email: [email protected]