Immunoproteomic Approach Identifies a Putative Virulence Chaperone DnaK Protein as a Candidate Diagnostic Marker and Therapeutic Target for Pythium insidiosum Infection

Pythiosis, a severe infectious disease caused by the oomycete Pythium insidiosum, continues to cause high levels of morbidity and mortality in humans and animals worldwide. However, there is a need to improve the method for diagnosing and treating the disease, and a better understanding of the causative agent is crucial for such need. In this study, we focused on identifying immunoreactive proteins of P. insidiosum, which could serve as promising candidates for diagnostic markers and therapeutic targets. The pathogen crude extract was separated using 2-dimensional gel electrophoresis, and the proteins were analyzed with Western blotting using pythiosis patient sera. Through LC-MS/MS analysis, we identified 55 immunoreactive spots corresponding to 42 unique proteins. Fifteen immunoreactive proteins were selected for in vitro synthesis, resulting in proteins S01-S21 in vitro generated directly from their PCR-amplified coding sequences. Only 4 synthesized proteins (S01, S11, S12, and S13) exhibited significant immunoreactivity against the pythiosis sera. Among them, S01 provided the highest protein yield and showed promise in differentiating the pythiosis group from the control. Additionally, S01 was annotated as a chaperone DnaK in P. insidiosum, part of a protein family involved in host immunity modulation, pathogenesis, and antifungal drug resistance of pathogenic fungi. In summary, we employed an immunoproteomic approach to successfully identify a chaperone DnaK in P. insidiosum, which could be a virulence protein of this pathogen. This protein holds potential as a diagnostic marker and therapeutic target for pythiosis and is worth exploring for its clinical application in the future.