Identification of a novel 74-kiloDalton immunodominant antigen of Pythium insidiosum recognized by sera from human patients with pythiosis

Abstract

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the etiologic agent of pythiosis, a life-threatening infectious disease of humans and animals that has been increasingly reported from tropical, subtropical, and temperate countries. Human pythiosis is endemic in Thailand, and most patients present with arteritis, leading to limb amputation and/or death, or cornea ulcer, leading to enucleation. Diagnosis of pythiosis is time-consuming and difficult. Radical surgery is the main treatment for pythiosis because conventional antifungal drugs are ineffective. The aims of this study were to evaluate the use of Western blotting for diagnosis of human pythiosis, to identify specific immunodominant antigens of P. insidiosum, and to increase understanding of humoral immune responses against the pathogen. We performed Western blot analysis on 16 P. insidiosum isolates using 12 pythiosis serum samples. These specimens were derived from human patients with pythiosis who had different forms of infection and lived in different geographic areas throughout Thailand. We have identified a 74-kDa immunodominant antigen in all P. insidiosum isolates tested. The 74-kDa antigen was also recognized by sera from all patients with pythiosis but not by control sera from healthy individuals, patients with thalassemia, and patients with various infectious diseases, indicating that Western blot analysis could facilitate diagnosis of pythiosis. Therefore, the 74-kDa antigen is a potential target for developing rapid serodiagnostic tests as well as a therapeutic vaccine for pythiosis. These advances could lead to early diagnosis and effective treatment, crucial factors for better prognosis for patients with pythiosis.

FIG. 1.

Map of Thailand showing geographic distribution of patients with pythiosis as sources of Pythium insidiosum isolates (P) and sera (S).

FIG. 2.

Coomassie brilliant blue-stained SDS-PAGE gel (A), India ink-stained blotted membrane (B), and Western blot analysis (C, D) of Pythium insidiosum P1 to P16 SABH (lane 1 to 16, respectively) probed with S2 (C) and S12 (D) pythiosis serum. Molecular markers (A, B) or predicted sizes (C, D) are shown in kDa on the left.

FIG. 3.

Coomassie brilliant blue-stained SDS-PAGE gel (A) and Western blot analysis (B) of Pythium insidiosum CFAs P1 (lane 1), P15 (lane 2), P10 (lane 3), P6 (lane 4), P8 (lane 5), and P7 (lane 6) probed with S12 pythiosis serum. Molecular markers (A) or predicted sizes (B) are shown in kDa on the left.

FIG. 4.

Western blot analysis of Pythium insidiosum P1 CFA probed with S1 to S12 pythiosis sera (lane 1 to 12, respectively) at 1:1,000 dilution. Lanes 13 and 14 represent S12 serum at 1:10,000 and 1:100,000 dilutions, respectively. Predicted sizes are shown in kDa on the left.

FIG. 5.

Western blot analysis of Pythium insidiosum P1 SABH (lanes 1 and 3) and Pythium insidiosum P1 CFA (lanes 2 and 4) probed with S12 pythiosis serum. Proteins were separated in 12% (lanes 1 and 2) and 8% (lanes 3 and 4) SDS-PAGE gels. Predicted sizes (kDa) are shown at both sides.

FIG. 6.

Coomassie brilliant blue-stained SDS-PAGE gel (A) and Western blot analysis (B) of C. coronatus CAM (lane 1), Rhizopus sp. CAM (lane 2), Pythium deliense CAM (lane 3), Pythium aphanidermatum CAM (lane 4), Pythium insidiosum P1 SABH (lane 5), Pythium insidiosum P1 CFA (lane 6), and concentrated Sabouraud dextrose broth (lane 7) probed with S12 pythiosis serum. Molecular markers (A) and predicted sizes (B) are shown in kDa on the left.