# Immunoproteomic approach identifies a putative virulence chaperone DnaK protein as a candidate diagnostic marker and therapeutic target for Pythium insidiosum infection

## TL;DR

This study identifies a protein called DnaK in Pythium insidiosum that could help diagnose and treat pythiosis, a severe infectious disease.

## Contribution

The study introduces a chaperone DnaK protein as a novel diagnostic marker and therapeutic target for pythiosis.

## Key findings

- Fifteen synthesized proteins from P. insidiosum were tested, with only four showing significant immunoreactivity.
- S01, identified as a chaperone DnaK protein, showed the highest diagnostic potential.
- DnaK is linked to host immunity modulation, pathogenesis, and drug resistance in fungi.

## Abstract

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 of these proteins were selected for in vitro synthesis, resulting in proteins S01-S21 being 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.