Expression, purification, and characterization of the recombinant exo-1,3-β-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum

Tiwa Rotchanapreeda; Yothin Kumsang; Pattarana Sae-Chew; Thidarat Rujirawat; Tassanee Lohnoo; Wanta Yingyong; Penpan Payattikul; Onrapak Reamtong; Theerapong Krajaejun

doi:10.1016/j.heliyon.2020.e04237

Expression, purification, and characterization of the recombinant exo-1,3-β-glucanase (Exo1) of the pathogenic oomycete Pythium insidiosum

Heliyon. 2020 Jun 19;6(6):e04237. doi: 10.1016/j.heliyon.2020.e04237. eCollection 2020 Jun.

Authors

Tiwa Rotchanapreeda¹, Yothin Kumsang², Pattarana Sae-Chew², Thidarat Rujirawat², Tassanee Lohnoo², Wanta Yingyong², Penpan Payattikul², Onrapak Reamtong³, Theerapong Krajaejun¹

Affiliations

¹ Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
² Research Center, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
³ Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.

Abstract

Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-β-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall β-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codon-optimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS- and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.

Keywords: Exo-1,3-β-glucanase; Exo1; Immunoreactive protein; Medical microbiology; Microbiology; Mycology; Protein engineering; Pythiosis; Pythium insidiosum; Recombinant protein; Vaccines.