The 74-kilodalton immunodominant antigen of the pathogenic oomycete Pythium insidiosum is a putative exo-1,3-beta-glucanase

Abstract

The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.

FIG. 1.

Peptide mass fingerprints of s74 and c74 immunodominant proteins of P. insidiosum after isolation from one-dimension SDS-polyacrylamide gels, in-gel tryptic digestion, and MS analysis. s74-1 and s74-2 were peptides (indicated by the names in the figure) of the s74 protein that matched a putative exo-1,3-ß-glucanase of Phytophthora infestans (EXO1).

FIG. 2.

Protein alignment and conserved domain of the putative glucanase of P. insidiosum. (A) Alignment of the deduced amino acid of the s74 partial protein of P. insidiosm (query; a 379-amino-acid long protein) and P. infestans EXO1 (subject; positions 278 to 653 of the 745-amino-acid long protein). Boxes at the beginning and at the end of the protein sequence indicate the locations of the s74-1 and s74-2 peptides of P. insidiosum that were predicted by MS analysis, respectively. Dotted lines at the beginning and at the end of the protein sequence indicate the positions of degenerate primers Pr1 and Pr4, respectively. In respect to the s74-2 peptide sequence of P. insidiosum, 3 deduced amino acids at positions 360, 364, and 369 (arrows) were different from the amino acids predicted by MS analysis. (B) Predicted conserved protein domains of the deduced amino acid sequence of the s74 partial protein gene of P. insidiosum.

FIG. 3.

Immunoreactivity of s74-1 (A) and s74-2 (B) peptides against pythiosis sera (samples T1 to T5) and control sera (samples C1 to C5) by ELISA. For the s74-1 peptide, the ELISA cutoff point (dashed line; the mean ELISA signal of the control sera plus 2 SDs) is 0.32, and the mean ELISA signals of the pythiosis and control sera are 0.48 (range, 0.26 to 1.17; SD, 0.39) and 0.15 (range, 0.07 to 0.27; SD, 0.09), respectively. For the s74-2 peptide, the ELISA cutoff point is 0.21 and the mean ELISA signal of the pythiosis and control sera are 0.57 (range, 0.33 to 1.22; SD, 0.39) and 0.09 (range, 0.05 to 0.18; SD, 0.06), respectively.

FIG. 4.

Phylogenetic analysis of 22 clinical isolates of P. insidiosum, based on partial putative exo-1,3-ß-glucanase DNA sequences, reveals that the organism can be divided into two clades, clade A (n = 16) and clade B (n = 6). P. infestans EXO1 is used as an outgroup for rooting.