The Elicitin-Like Glycoprotein, ELI025, Is Secreted by the Pathogenic Oomycete Pythium insidiosum and Evades Host Antibody Responses

Pythium insidiosum is a unique oomycete that can infect humans and animals. Patients with a P. insidiosum infection (pythiosis) have high rates of morbidity and mortality. The pathogen resists conventional antifungal drugs. Information on the biology and pathogenesis of P. insidiosum is limited. Many pathogens secrete proteins, known as effectors, which can affect the host response and promote the infection process. Elicitins are secretory proteins and are found only in the oomycetes, primarily in Phytophthora and Pythium species. In plant-pathogenic oomycetes, elicitins function as pathogen-associated molecular pattern molecules, sterol carriers, and plant defense stimulators. Recently, we reported a number of elicitin-encoding genes from the P. insidiosum transcriptome. The function of elicitins during human infections is unknown. One of the P. insidiosum elicitin-encoding genes, ELI025, is highly expressed and up-regulated at body temperature. This study aims to characterize the biochemical, immunological, and genetic properties of the elicitin protein, ELI025. A 12.4-kDa recombinant ELI025 protein (rELI025) was expressed in Escherichia coli. Rabbit anti-rELI025 antibodies reacted strongly with the native ELI025 in P. insidiosum’s culture medium. The detected ELI025 had two isoforms: glycosylated and non-glycosylated. ELI025 was not immunoreactive with sera from pythiosis patients. The region near the transcriptional start site of ELI025 contained conserved oomycete core promoter elements. In conclusion, ELI025 is a small, abundant, secreted glycoprotein that evades host antibody responses. ELI025 is a promising candidate for development of diagnostic and therapeutic targets for pythiosis.


Introduction
All strains were maintained on Sabouraud dextrose agar at room temperature and sub-cultured once a month.

Serum samples
Three serum samples were obtained from pythiosis patients diagnosed by culture identification or serological tests [36][37][38][39][40][41]. To serve as controls, three serum samples were obtained from healthy blood donors who came to the Blood Bank Division, Department of Pathology, Ramathibodi Hospital. Rabbit anti-rELI025 sera were purchased from the Biomedical Technology Research Laboratory, Faculty of Associated Medicine, Chiang Mai University, Thailand. To block the rabbit anti-rELI025 antibodies from the rabbit serum, 20 μl of rELI025 (2.4 mg-/ml) and 1.5 ml of diluted rabbit serum [1:2,000 in 5% skim milk in TBS (pH 7.5)] were co-incubated with gentle agitation at 4°C overnight. All sera were kept at -20°C until use.

Protein preparation
Crude protein extracts of P. insidiosum, including soluble antigen from broken hyphae (SABH; containing intracellular proteins) and culture filtrate antigen (CFA; containing secreted proteins), were prepared according to the methods described by Chareonsirisuthigul et al [41]. Briefly, 100 ml Sabouraud dextrose broth was inoculated from an actively growing P. insidiosum colony and incubated, with shaking (~150 rpm), at 37°C for 10 days. The organism was killed with 0.02% Thimerosol (Sigma). Hyphae were collected by filtration on a 0.22-μm-pore-size membrane (Durapore, Merck Millipore), and ground in a mortar with precooled distilled water (1.5 g hyphae per 30 ml water). Supernatant, following centrifugation (10,000 x g) of the cell lysate at 4°C for 30 min, was filtered through a 0.22-μm-pore-size membrane (Durapore, Merck Millipore). Both filtered supernatant (SABH) and cell-free broth (CFA) were 100-fold concentrated by ultrafiltration (10,000 molecular weight cut-off membrane; Amicon Ultra 15M, Merck Millipore). Protein concentration was measured by Bradford's assay [42]. SABH and CFA were stored at -20°C until use.
Genomic DNA extraction P. insidiosum genomic DNA (gDNA) was extracted using the modified method of Lohnoo et al [43]. Briefly, hyphal mat (~500 mg) was transferred to a 2-ml tube containing glass beads (~1,000-μm diameter; Sigma) and 400 μl of homogenizing buffer [0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0)]. The tube was shaken at 30 Hz for 2 min, in a tissue homogenizer (TissueLyzer, QIAGEN), before adding 20% sodium dodecyl sulfate (final concentration, 2%) and proteinase K (final concentration, 400 μg/ml). The cell lysate was incubated, with gentle inversion, at 55°C, overnight. The sample was then mixed with 300 μl of 6 M NaCl, vigorously vortexed for 30 s, and centrifuged (10,000 x g) at room temperature for 30 min. The supernatant was then mixed with an equal volume of isopropanol, incubated at -20°C for 1 hr, and centrifuged (12,000 x g) at 4°C for 20 min. The gDNA pellet was collected and washed with 70% ethanol, air dried, and resuspended in sterile water. All extracted gDNAs were kept at -20°C until use.

SDS-PAGE and Western blot
SABH, CFA, and rELI025 were separated by SDS-PAGE (4% stacking and 12% separating gel) at 150 V, for 65 min, using the Mini-PROTEAN II apparatus (Biorad). Proteins were stained with either Coomassie blue R-250 or Silver staining kit (Thermo Scientific). The Image Lab 3.0 program (Biorad) was used to estimate protein molecular weight (kilo Dalton; kDa) based on migration of pre-stained broad range protein markers (Biorad). For Western blot analysis, the separated proteins were transferred and immobilized onto a 0.2-μm-pore-size PVDF membrane (Merck Millipore), using the Biorad Mini Trans-Blot cell (setting: 100 V for 60 min). The blotted membrane was blocked with 5% skim milk (Sigma) in blocking buffer [TBS; 150 mM NaCl, 10 mM Tris-HCl (pH 7.5)] at 4°C, overnight, or room temperature for an hour. The membrane was washed 3 times with the washing buffer [TTBS; 500 mM NaCl, 20 mM Tris-Cl, and 0.1% Tween-20 (pH 7.5)]. The membranes were incubated with the primary antibodies diluted in the blocking buffer [1:1,000 for mouse anti-6x histidine antibody (Merck Millipore); 1:2,000 for rabbit anti-rELI025 serum; 1:1,000 for patient serum] for 2 hr at room temperature (anti-6x histidine and anti-rELI025) or overnight at 4°C (patient serum), and washed 3 times with TTBS. The secondary antibodies, diluted in the blocking buffer [1:2,000 for goat antimouse IgG conjugated with horseradish peroxidase (Merck Millipore); 1:5000 for goat antirabbit IgG conjugated with alkaline phosphatase (Southern Biotech); 1:3000 for goat antihuman IgG conjugated with horseradish peroxidase (Biorad)], were added to the membrane and incubated for 2-3 hr at room temperature. After washing the membrane 3 times with TTBS, substrate and chromogen (4CN and H 2 O 2 for horseradish peroxidase; NBT and BCIP for alkaline phosphatase) were added to develop Western blot signals. Intensities of Western blot bands were quantitated by the GelQuant.NET software (http://biochemlabsolutions. com/GelQuantNET.html).

Mass spectrometric analysis
The 10-and 15-kDa bands present on SDS-PAGE gel and Western blot were excised from gel and PVDF membrane, respectively. Proteins were extracted and trypsin digested, using the method described by Shevchenko et al. [45]. The digested proteins were analyzed by an Ultimate 3000 LC System (Dionex, USA) coupled to an HCTultra PTM Discovery System (Bruker Daltonics Ltd., U.K.) at the Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology, Thailand. The Bruker Daltonics Data Analysis version 4.0 (Bruker Daltonics Ltd., U.K.) was used to analyze raw mass spectrometric data. The MASCOT software (Matrix Science, UK) was used to search the obtained MS and MS/MS data against~15,000 genome-derived predicted proteins of P. insidiosum (unpublished data).

Deglycosylation of glycoprotein
A glycoprotein deglycosylation kit (Calbiochem) was used to remove sugar moieties (N-and O-linked glycosylation) from the native ELI025 in CFA. Briefly, 50 μg of CFA, 10 μl of 5x kit deglycosylation buffer, 2.5 μl of the kit denaturation solution, and distilled water were mixed to the final volume of 50 μl. The mixture was heated at 100°C for 5 min and cooled down to room temperature, before adding 2.5 μl of 15% TRITON X-100. To remove N-glycosyl groups, 1 μl of N-glycosidase F was added to the reaction. To remove O-glycosyl groups, 1 μl of the enzyme mixture, including α-2-3,6,8,9-neuraminidase, endo-α-N-acetylgalactosaminidase, β1,4-galactosidase, and β-N-acetylglucosaminidase, was added to the reaction. The protein-enzyme mixture was incubated at 37°C for 3 hr.

Immunohistochemical staining assay
Immunohistochemical staining assay was performed, using the method described by Keeratijarut et al [46], with some modifications. A paraffin-embedded tissue section (4-μm thickness) from a patient with vascular pythiosis was pretreated with xylene and absolute ethanol (Merck), before washing with phosphate buffered saline (PBS; pH 7.4). The tissue section was incubated with Tris-EDTA buffer (TE buffer; pH 9.0) at 95°C for 40 min, treated with 10% H 2 O 2 in PBS for 10 min, and washed with PBS. Then, the tissue section was incubated with 200 μl of either rabbit pre-immune serum or rabbit anti-rELI025 serum (1:16,000 in PBS) at 4°C overnight, washed with PBS (5 min each), and incubated with 200 μl of mouse anti-rabbit IgG antibody conjugated with horseradish-peroxidase (Thermo Scientific, USA) for 30 min. To develop color, the substrate 3,3'-diaminobenzidine tetrahydrochloride (DAKO, USA) was added to the tissue section and incubated at room temperature for 5 min. The tissue section was counterstained with hematoxylin before examination with a light microscope (ECLIPSE Ci, Nikon, Japan).

Polymerase chain reaction and DNA sequencing
A draft genome sequence of the P. insidiosum strain Pi-S (unpublished data) was used to design primers for PCR amplification of the rELI025-encoding sequence and its promoter region (ELI025_promoter_F2, 5'-CATGGACAGCGTCATCTCTGG-3'; ELI025_promoter_R1, 5'-GCGTCAAGATGAGAAACGAGG-3'). Each amplification reaction was performed in a 50-μl reaction containing 100 ng of genomic DNA template, 0.02 U/μl of DNA polymerase (Phusion), 1x Phusion buffer, 200 μM of dNTPs, and 0.5 μM each of the primer. The amplification was carried out in a Mastercycler Nexus thermal cycler (Eppendorf), with the following conditions: an initial denaturation at 98°C for 30 s, 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR products were purified using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and assessed for amount and size by 1% gel electrophoresis.

Bioinformatic analysis
The molecular weight of ELI025 was calculated by ProtParam [47]. Signal peptide, transmembrane domain, N-and O-linked glycosylation and GPI-anchor of ELI025 were predicted using the SignalIP program version 4.0 [48], the TMHMM program version [49], the NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc), and NetOGlyc [50] programs, and the big-PI predictor [51], respectively. The promoter and ELI025-coding sequences from all P. insidiosum strains used in this study were aligned and compared using the ClustalX program version 2 and the GeneDoc program [52,53].

Nucleotide sequence accession numbers
All ELI025-coding sequences from P. insidiosum strain Pi-S, MCC18, and P01 have been submitted to the DNA data bank of Japan (DDBJ), under accession numbers AB971191 to AB971193, respectively.
The 5'-untranslated and -flanking DNA sequences of the ELI025 gene from the three P. insidiosum strains were compared with that of various genes from several oomycetes and parasites (Fig. 2). These sequences share a 19-nucleotide oomycete core-promoter sequence, located between 9 and 79 nucleotides upstream of the start codon (Fig. 2). Two putative core-promoter components, an initiator element (Inr; 5'-TCATTCC-3') and a flanking promoter region (FPR; 5'-CAACCTTCC-3'), were identified in this region of ELI025 (Fig. 2). A predicted transcription start site (+1; Fig. 2) of the ELI025 gene is within the Inr element. A TATA box was not observed in the promoter region of ELI025.
The predicted full-length ELI025 protein sequences (112 amino acids long) from the three P. insidiosum strains were 100% identical. A predicted signal peptide of ELI025 covered the first 20 N-terminal amino acids (Fig. 1B). The calculated molecular weights of ELI025, with and without the signal peptide, were 12 and 10 kDa, respectively. The elicitin domain spanned from amino An Elicitin-Like Protein of Pythium insidiosum acid position 25 to 110. By analogy to known elicitins [34], three disulfide bonds (Cys27/Cys91, Cys47/Cys76, and Cys71/Cys110) were identified within the elicitin domain of ELI025 (C1, C2, C3; Fig. 1B). There are two predicted N-linked glycosylation sites at amino acid positions 22 and 87, and three predicted O-linked glycosylation sites at amino acid positions 49, 51, and 54 ( Fig. 1B). Neither a GPI anchor nor a transmembrane region are predicted for ELI025.

ELI025 is a major secreted non-immunogenic glycoprotein
The recombinant protein, rELI025, was successfully expressed and purified from E. coli (protein yield: 2 mg per 1 liter of bacterial culture). Purity of rELI025 was at least 99%, as demonstrated by silver staining analysis of SDS-PAGE gel. The molecular weight of rELI025 in the SDS-PAGE gel was estimated to be 12.4 kDa (Fig. 4A). rELI025 appeared as an intense 12.4-kDa Western blot band, when reacted with the mouse anti-6x histidine-tag antibody (data not shown) or the rabbit anti-rELI025 antibodies (Fig. 4B). Gel-separated total proteins from crude extracts or supernatants of three P. insidiosum strains, had molecular weights ranging from 6 to 115 kDa (Fig. 4A). In Western Blots, rabbit  (Table 1) were aligned and compared. C1-C6 indicate conserved cysteine residues; (B) Phylogenetic analysis of elicitins by the neighbor-joining method. The phylogenetic tree, constructed from elicitin domain sequences of P. insidiosum ELI025 and the top BLAST hit proteins of 15 other oomycetes (Table 1), shows three major clades (as indicated by Gr1, Gr2 and Gr3; containing multiple sequences per clade) and two minor clades (as indicated by Gr4 and Gr5; containing one sequence per clade). Only the branch support values of 70% or more are shown at corresponding nodes. doi:10.1371/journal.pone.0118547.g003 An Elicitin-Like Protein of Pythium insidiosum anti-rELI025 serum reacted only with the 10-and 15-kDa bands in CFA (culture filtrate antigen), which contains secreted proteins of P. insidiosum (Fig. 4B). The rabbit anti-rELI025 serum did not react any proteins in SABH (soluble antigens from broken hyphae), which contains intracellular proteins (Fig. 4B). The rabbit pre-immune serum did not detect any proteins in SABH or CFA. If the rabbit anti-rELI025 serum is pre-absorbed with rELI025 protein prior to Western Blot detection, the band intensities for the 10-and 15-kDa proteins were reduced by~85% (data not shown).
The native ELI025 (nELI025) in CFA was treated with protein deglycosylases to remove either N-or O-linked glycosyl adducts. The SDS-PAGE and Western blot profiles (probed with the rabbit anti-rELI025 serum) show that the 15-kDa band disappears in the CFA treated with the N-linked deglycosylation enzyme (either alone or in combination with the O-linked deglycosylase; Fig. 5). In contrast, the 10-and 15-kDa bands were both present in CFA treated with O-linked deglycosylase or in the no enzyme control.
The 10-and 15-kDa bands excised from SDS-PAGE gel (Fig. 3A) and Western blot membrane (Fig. 3B)   An Elicitin-Like Protein of Pythium insidiosum derived 686.9 peak, showed that the corresponding peptides had nearly-identical spectra (Fig. 6B), and matched the same peptide sequence (KNQQCLALLDAVKA) predicted for ELI025. Similarly, MASCOT analyses of MS/MS data of the 10-and 15-kDa SDS-PAGE bandderived 853.9 peak, revealed that the corresponding peptides had nearly-identical spectra (data not shown), and matched another peptide sequence (KATNPSDCVLVFNDVRL) predicted for ELI025.
Three serum samples each from pythiosis patients and normal individuals (control) were used as primary antibodies in Western blot to detect rELI025 or nELI025 in SABH and CFA. All control sera did not detect any proteins in SABH and CFA (data not shown). While pythiosis sera detected relatively-high molecular weight proteins of SABHs and CFAs (> 30 kDa), they failed to detect many lower molecular weight proteins, including the 10-and 15-kDa (representing nELI025). The patient sera also failed to react with the 12.4-kDa rELI025 band (Fig. 4C).   An immunohistochemical staining assay, using the rabbit anti-rELI025 serum, was used to target cellular localization of the P. insidiosum nELI025 in an infected arterial tissue. nELI025 markedly localized at the cell surface and surrounding areas (Fig. 7). No signal was detected with the rabbit pre-immune serum.

Discussion
The elicitin domain of ELI025 was predicted to contain three disulfide bonds (Fig. 1B), which is a crucial characteristic of elicitin [34]. The E. coli strain rosetta-gami2 (DE3) was used to express ELI025 based on its reported facility in proper disulfide bond formation. The rabbit anti-rELI025 antibody, detected two proteins in CFA (10-and 15-kDa), but not in SABH (Fig. 4B). The rabbit anti-rELI025 serum, pre-absorbed with rELI025, failed to effectively detect any proteins in CFA, indicating that the rabbit anti-rELI025 antibodies were specific to ELI025. The 10-and 15-kDa bands could be different proteins (i.e., other elicitins) or different isoforms of Fig 7. Cellular location of ELI025. Infected arterial tissue from a pythiosis patient was sequentially stained with rabbit anti-rELI025 serum, as the primary antibody, and then mouse anti-rabbit IgG antibody conjugated with horseradish-peroxidase, as the secondary antibody (see Materials and Methods). Images of the hyphae and location of ELI025 (indicated by arrows) were captured with a bright-field microscope. The scale bar represents 10 μm.
doi:10.1371/journal.pone.0118547.g007 the same protein (ELI025 contains several predicted glycosylation linkages). Deglycosylation of CFA proteins indicated that the 10-kDa band represents nELI025 without glycosylation, while the 15-kDa band represents nELI025 with predominant N-linked glycosylation (Fig. 5). Thus, nELI025 is a secreted glycoprotein, with two isoforms. The slightly-larger size of rELI025 (12.4 kDa) compared to the non-glycosylated nELI025 (10 kDa) is expected based on its expression in E. coli as a fusion with Thrombin and a His tag (Fig. 1A).
Mass spectrometric analyses were used to confirm the identity of ELI025 in the 10-and 15-kDa bands of SDS-PAGE gel (Fig. 4A). The sequences, determined by MS and MS/MS analyses, of the peptide mass 686.9 (KNQQCLALLDAVKA) and 853.9 (KATNPSDCVLVFNDVRL) of either the 10-or 15-kDa SDS-PAGE bands matched perfectly with the ELI025 predicted protein sequence. However, no peptide masses matching ELI025 were detected in the 10-and 15-kDa Western blot bands. This may result from the Western blot bands being contaminated with blocking reagent, primary antibody, secondary antibody, enzyme, and substrate, which may compromise detection sensitivity. As an alternative method to determine the identity of the 10-and 15-kDa bands in PVDF membrane, we used rabbit anti-ELI025 antibodies against rELI025 in Western blot analysis. The rabbit anti-rELI025 antibodies reacted only with the 10and 15-kDa bands (Fig. 4B), suggesting that the protein detected in the Western blots is ELI025.
Until recently, there were few genetic and molecular studies done in P. insidiosum, and to date, there is no transformation system for introducing foreign or modified P. insidiosum genes into the organism. In other oomycetes, transformations systems have been developed, and some of these depend on the hsp70 and ham34 promoters from the oomycete Bremia lactucae for transgene expression [78][79][80][81]. Since the upstream region of ELI025 has conserved sequence found in the core promoter elements of many oomycete genes including hsp70 and ham34 (Fig. 2), it may be possible to use already-developed transformation vectors such as pTH210, pHAMT34H, pHAMT35N/SK, and pHAMT35G [79], which utilize the hsp70 and ham34 promoters, for developing transformations systems in P. insidiosum. In addition, since ELI025 is highly expressed, its upstream region could be used as a driving promoter for DNA transformation in P. insidiosum.
Elicitins form a unique group of proteins that have been found previously in two oomycete genera (Phytophthora spp. and some Pythium spp.), but not in fungi or bacteria [22,82,83]. In this study, we performed a similarity search of elicitin domain-containing proteins in the publicly-available genome, transcriptome, and proteome databases of various oomycetes (Table 1). In addition to Phytophthora and Pythium species, elicitin homologs were also found in oomycete genera Pseudoperonospora, Hyaloperonospora, and Albugo (Table 1). As expected, phylogenetic analysis grouped the top BLAST hit elicitins of these oomycetes according to their genera based on previous classifications: Phytophthora species in Gr1 (with an exception for P. vexans), Pythium species in Gr2 and Gr3 (with an exception for H. arabidopsis), Albugo species in Gr4, and Pseudoperonospora species in Gr5 (Fig. 3B). The conserved homology of elicitins among the closely related species also extended to both their core promoter sequences (Fig. 2) and their signal sequences (Fig. 3A). It should be noted that elicitins found thus far are in the more closely-related subgroups Pythiales, Peronosporales, and Albuginales. In contrast, no elicitin homologs were detected in Aphanomyces and Saprolegnia species, which belong to Saprolegnales, a more distantly-related oomycete lineage. This finding suggests that the origin and expansion of elicitins occurred after splitting off the oomycetes from its ancient progenitor and between the Saprolegnales lineage, and the ancestor lineage of the Pythiales, Peronosporales, and Albuginales.
Based on an extensive genome search (Table 1), elicitins are found in many oomycetes, but absent in all non-oomycete organisms, such as, fungi. Thus, elicitins are a signature character of the oomycetes. Among oomycetes, P. insidiosum is a notorious human pathogen. It shares microscopic features with some pathogenic fungi (such as, Aspergillus species, Fusarium species, and Zygomycetes). This can lead to misdiagnosis of pythiosis as a fungal infection [46,84], and results in delayed and improper treatment of patients. Because of the uniqueness of the elicitins to P. insidiosum among human pathogens, detection of ELI025 or its gene product could aid in the development of more specific diagnostic tests for pythiosis, such as using the anti-rELI025 antibodies to detect P. insidiosum in infected tissue.
The detection of ELI025 in CFA, together with the predicted amino acid sequence harboring a signal peptide, indicate that ELI025 is a secreted protein. Additionally, the immunohistochemical staining assay of the infected tissue from a pythiosis patient showed localization of ELI025 at P. insidiosum's cell surface and surrounding areas (Fig. 7). This evidence suggest that, in P. insidiosum, ELI025 is expressed and secreted, both during in vitro growth and during infection of host tissue. Elicitins, secreted by the plant-pathogenic oomycetes, are beneficial to the pathogens by effecting host response and triggering programed cell death [23,24]. The role of elicitin secreted by P. insidiosum in humans is unknown. Pythium species are thought to be sterol auxotrophic microorganisms [22,32,33]. Like the elicitins from the plant-pathogenic oomycetes, P. insidiosum ELI025 has been predicted to contain a hydrophobic cavity that can bind a sterol molecule, implying that it can function as a sterol-carrying protein [31,34,[85][86][87][88]. Western blot assays showed that the small proteins (< 30 kDa) in CFA, including nELI025, were not recognized by sera from patients with pythiosis (Fig. 4C). Poor immunogenicity could prevent the elimination of ELI025 by host antibody responses, and therefore, it could allow ELI025 to act in sterol acquisition inside host tissue.
In conclusion, ELI025 has been successfully cloned and expressed in E. coli. Genetic, biochemical, and immunological characterization showed that ELI025 is a small glycoprotein, abundantly secreted by P. insidiosum. ELI025 had two isoforms (glycosylated and nonglycosylated form), and was not recognized by host antibodies. The upstream region of ELI025 shared core promoter elements with the promoters of other oomycete genes. Among human fungal and oomycete pathogens, ELI025 is unique to P. insidiosum, and therefore, it is a potential target for development of more specific diagnostic tests. Characterization of ELI025 provided a new insight into the biology and pathogenesis of the understudied microorganism, P. insidiosum, and it could lead to a discovery of a new strategy for infection control.