W170 - Identification of an Immunoreactive Protein Candidate for Developing a Diagnostic and Controlling Measure for Pythium Insidiosum Infection Using Proteome-based Mass Spectrometric Analysis and Cell-free Protein Synthesis

Chalisa Jaturapaktrarak – Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Pattarana Sae-Chew – Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Thidarat Rujirawat – Faculty of Medicine Ramathibodi Hospital, Mahidol Universityy, Bangkok, Thailand; Onrapak Reamtong – Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand

Abstract Text: Pythiosis is a devastating infectious disease caused by a fungus-like pathogen, Pythium insidiosum. The disease processes high morbidity and mortality in affected humans and animals worldwide. Effective diagnostic and therapeutic modalities still need to be improved. Besides, a basic understanding of the disease and the causative agent is trivial. In this study, we explored and characterized immunoreactive proteins of P. insidiosum, which are excellent candidates for being a diagnostic marker and drug or vaccine target. Crude extract of the pathogen was subjected to protein separation using 2-dimensional gel electrophoresis for Western analyses against three representative sera from patients with active pythiosis. A total of 55 protein spots were recognized by all tested sera, but not a control serum, and thus subjected to isolation and liquid chromatographic-mass spectrometric (LC-MS/MS) analysis against our in-house P. insidiosum genome-derived proteomic library. As a result, eight spots generated a peptide profile that can match 15 proteins in the library. By employing the cell-free protein synthesis (CFPS) workflow, we can simultaneously express these proteins in vitro to validate their immunoreactive properties using Western blot analysis against anti-HisTag antibody and three pythiosis sera. Only the CFPS-based recombinant protein S01, annotated as a chaperone DnaK, demonstrated immunoreactivity. S01-coated ELISA showed a significant signal of an array of pythiosis sera (n = 23) compared with control sera (n = 24). In conclusion, we successfully identified and produced an immunoreactive chaperone DnaK protein of P. insidiosum, a potential target for developing a diagnostic and control measure for pythiosis.