Table 1.
List of primers used in this study.
Fig 1.
Identification and annotation of full length exo-1,3-β-glucanase gene (exo1) from P. insidiosum:
(A) known 924-bp partial sequence of the exo1 gene (accession number: GU994093); (B) Adaptor and (C) RACE PCR used to identify upstream and downstream regions of the partial exo1 gene from genomic DNA (gDNA) and complementary DNA (cDNA), respectively; (D) and (E) Full length exo1 coding sequence amplified from gDNA and cDNA, respectively [forward and reverse primers for PCR and sequencing (Table 1) are depicted above and below the sequence structure, respectively]; (F) Promoter [including the core-promoter sequence and the transcriptional start site (1+)], 5’-untranslated, open reading frame, and 3’-untranslated regions of the exo1 gene; (G) Exo1 protein structure showing signal peptide (SP), BglC domain, X8 domain, transmembrane region (TM), and positions and sequences of Peptide-A, -B, and -C. Numbers indicate amino acid position.
Table 2.
List of oomycete and fungal microorganisms whose top BLAST-hit DNA sequences vs. the P. insidiosum’s exo1 gene were used for phylogenetic analysis.
All sequences were retrieved from the FungiDB database [27], and BLAST searched against the NCBI nucleotide database.
Table 3.
P. insidiosum’s transcriptome-derived Exo1 homologous proteins that share the Peptide-A, -B, or -C sequences.
Predicted structures of these proteins are shown in Fig 2. NCBI accession number, protein length, calculated protein molecular weight (MW), number of 454-derived transcript reads (when P. insidiosum grew at 37°C), and sequence alignment analysis against Exo1 (including: query sequence coverage, E-value, and sequence identity), corresponding to each transcriptome-derived protein, are summarized in the table.
Fig 2.
Protein structure of Exo1 and six transcriptome-derived homologous proteins.
Protein domains of Exo1 and homologous proteins (UN05080, UN00475, UN03240, UN01457, UN24957 and UN22794; Table 3) were predicted by SignalP, TMHMM, and NCBI’s conserved domain search programs (Methods). The DOG program was used to draw protein structures. Numbers indicate the first and last amino acid positions of each protein. Detailed characteristics of the homologous proteins are shown in Table 3. (Symbols: A, Peptide-A; B, Peptide-B; C/1, Peptide-C (the first half portion); C/2, Peptide-C (the second half portion); SP, signal peptide; BglC, BglC domain; X8, X8 domain; and TM, transmembrane region; The grey regions are sequences that do not match any protein domain defined by the NCBI’s conserved domain search program).
Fig 3.
Phylogenetic analysis of glucanase genes from oomycetes and fungi.
exo1 gene sequences from 6 strains of P. insidiosum (accession number: LC033486 to LC033491), and glucanase-encoding genes (top exo1-BLAST hit sequences) from 9 other oomycetes and 26 fungi (Table 2) were included for phylogenetic analysis. Phylogenetic reconstruction was performed using the PhyML program (Methods). Reliability for internal branch was analyzed using the aLRT test (Methods).
Fig 4.
Expression of exo1 in response to temperature, culture duration, and dextrose availability.
Real-time PCR was used to measure exo1 mRNA levels in P. insidiosum (strain Pi-S) at 5 culture conditions: (i) 28°C for 7 days (28c-7d-1x); (ii) 37°C for 7 days (37c-7d-1x); (iii) 28°C for 14 days (28c-14d-1x); and 28°C for 7 days with (iv) 2 mg/ml dextrose (28c-7d-0.1x), or (v) no dextrose (28c-7d-0x) supplement. exo1 expression in each condition was normalized to the reference actin gene (act1). Asterisk indicates significant up- or down-regulation, relative to 28c-7d-1x.
Fig 5.
Immunoreactivity of Exo1 peptides against rabbit anti-Exo1 peptide sera.
ELISA result of rabbit pre-immune or anti-Exo1 peptide serum (raised against the combination of Peptide-A, -B, and -C) with the individual peptides (used to coat an ELISA plate).
Fig 6.
Western blot analysis of P. insidiosum’s crude protein extracts using rabbit anti- Exo1 peptide serum.
Crude proteins (SABH and CFA) extracted from P. insidiosum were separated in a SDS-PAGE gel, transferred to a Western blot membrane, and probed with the rabbit pre-immune or post-immune serum. The arrow and arrow head indicate the 82- and 78-kDa band, respectively. Protein molecular weight markers (7–175) are shown in kDa. (SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; CFA, culture filtrate antigen; SABH, soluble antigen from broken hyphae; Pre-immune, rabbit pre-immune serum; Post-immune, rabbit anti-Exo1 peptide serum).
Fig 7.
Peptide pre-absorption of the rabbit anti-Exo1 sera for Western blot analysis of P. insidiosum crude protein extracts.
Separated crude proteins (SABH) of P. insidiosum were probed with rabbit pre-immune serum (Lane 1), rabbit post-immune serum (Lane 2), and rabbit post-immune serum pre-absorbed with Peptide-A, -B, and -C (Lane 3), Peptide-A and -B (Lane 4), Peptide-A and -C (Lane 5), and Peptide-B and -C (Lane 6). The arrow and arrow head indicate the 82- and 78-kDa band, respectively. [Abbreviations: SABH, soluble antigen from broken hyphae; Pre-immune, rabbit pre-immune serum; Post-immune, rabbit anti-Exo1 peptide serum; ‘+’, used as probe (pre- and post-immune sera) or used for pre-absorption (Peptide-A, -B, or -C); ‘-’, not used as probe nor used for pre-absorption]
Fig 8.
Immunoreactivity of Exo1 peptides against pythiosis patient sera by ELISA.
ELISA results of serum samples from pythiosis patients (n = 3; PS1-3) and healthy blood donors (n = 3; CS1-3; control) and (A) Peptide-A, (B) Peptide-B, (C) Peptide-C, and (D) a mixture of the peptides (used to coat an ELISA plate). Number in the parenthesis is the mean ELISA signal.
Fig 9.
Measurement of hydrolytic activity of Exo1 by agar plate enzymatic assay:
The pPinsEXO1-harboring E. coli cell suspension [1 x 107 (A) and 1 x 109 (B) cells/ml] and the positive controls [T. harzianum’s lysing enzyme (100 mg/ml; C) and T. reesei’s cellulase (100 mg/ml; D)] gave hydrolytic/clear zones in LB agar supplemented with laminarin. The negative controls [bacteria with the pRSET-C empty plasmid (1 x 109 cells/ml; E) and plain LB broth (F)] did not produce a hydrolytic/clear zone in the laminarin plate. * indicates location where the material (bacteria, enzyme, or broth) was spotted.
Fig 10.
Linear correlation of clear zone diameters and amounts of hydrolytic enzyme used in the agar plate enzymatic assay.
Laminarin-supplemented LB agar was hydrolyzed by various concentrations of cellulase enzyme (X-axis). Resulting clear zones (Y-axis) were visualized by staining with iodine. There is a linear relationship between the amount of enzyme and the diameter of clearing when displayed on a semi-log plot. Clear zone diameters generated by various pPinsEXO1-harboring E. coli cell suspensions (horizontal dash lines) were correlated with amounts of enzyme (vertical dash lines).