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The elicitin-like glycoprotein, ELI025, is secreted by the pathogenic oomycete Pythium insidiosum and evades host antibody responses.

Lerksuthirat T, Lohnoo T, Inkomlue R, Rujirawat T, Yingyong W, Khositnithikul R, Phaonakrop N, Roytrakul S, Sullivan TD, Krajaejun T - PLoS ONE (2015)

Bottom Line: Elicitins are secretory proteins and are found only in the oomycetes, primarily in Phytophthora and Pythium species.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT
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.

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Cloning and expression of ELI025.(A) Plasmid DNA map of pET28b-ELI025 shows the cloning sites (Nde-I and EcoR-I) of ELI025. Expression of ELI025 is under the control of the T7 promoter. The numbers in parentheses indicate a location of each plasmid component; (B) Protein structure of ELI025 shows a signal peptide (SP; amino acid position 1–20), an elicitin domain (amino acid position 25–110), three disulfide bonds (C1, cysteine position 27 and 91; C2, cysteine position 47 and 76; C3, cysteine position 71 and 110), two predicted N-linked glycosylation sties (N; amino acid position, 22 and 87), and three predicted O-linked glycosylation sties (O; amino acid position 49, 51, and 54).
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pone.0118547.g001: Cloning and expression of ELI025.(A) Plasmid DNA map of pET28b-ELI025 shows the cloning sites (Nde-I and EcoR-I) of ELI025. Expression of ELI025 is under the control of the T7 promoter. The numbers in parentheses indicate a location of each plasmid component; (B) Protein structure of ELI025 shows a signal peptide (SP; amino acid position 1–20), an elicitin domain (amino acid position 25–110), three disulfide bonds (C1, cysteine position 27 and 91; C2, cysteine position 47 and 76; C3, cysteine position 71 and 110), two predicted N-linked glycosylation sties (N; amino acid position, 22 and 87), and three predicted O-linked glycosylation sties (O; amino acid position 49, 51, and 54).

Mentions: The full-length ELI025-encoding sequence (NCBI accession number: HS975204) was amplified from the pCR4-blunt-TOPO vector harboring PinsEST#025 cDNA [34], in a 50-μl PCR reaction containing 1.5 μl of PCR product, 1 μl of the Elongase and its buffer mixture (buffer A:B ratio = 1:4) (Invitrogen), 200 μM of dNTPs, and 0.4 μM each of the primer ELI025_NdeI (5’-GGCATCACATATGtacaacgagaccaagccg-3’) and ELI025_EcoRI (5’-CAAGAATTCCTAGGCCTTGCAGCTCGTC-3’). The reaction was carried out in a MyCycler (Biorad) with the following conditions: initial denaturation at 94°C for 30 s, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 1.10 min, and final extension at 68°C for 5 min. The PCR product was double digested with NdeI and EcoRI (New England Biolabs), and directionally cloned into pET28b (Novagen), yielding an in-frame His-tag fusion on the N-terminus of ELI025. The resulting plasmid, pET28b-ELI025 (Fig. 1A), was propagated in the Escherichia coli strain DH5α. The sequence of the ELI025-coding region of the plasmid was confirmed using primers, T7-promoter (5’-TAATACGACTCACTATAGGG-3’) and T7-terminator (5’-GCTAGTTATTGCTCAGCGG-3’).


The elicitin-like glycoprotein, ELI025, is secreted by the pathogenic oomycete Pythium insidiosum and evades host antibody responses.

Lerksuthirat T, Lohnoo T, Inkomlue R, Rujirawat T, Yingyong W, Khositnithikul R, Phaonakrop N, Roytrakul S, Sullivan TD, Krajaejun T - PLoS ONE (2015)

Cloning and expression of ELI025.(A) Plasmid DNA map of pET28b-ELI025 shows the cloning sites (Nde-I and EcoR-I) of ELI025. Expression of ELI025 is under the control of the T7 promoter. The numbers in parentheses indicate a location of each plasmid component; (B) Protein structure of ELI025 shows a signal peptide (SP; amino acid position 1–20), an elicitin domain (amino acid position 25–110), three disulfide bonds (C1, cysteine position 27 and 91; C2, cysteine position 47 and 76; C3, cysteine position 71 and 110), two predicted N-linked glycosylation sties (N; amino acid position, 22 and 87), and three predicted O-linked glycosylation sties (O; amino acid position 49, 51, and 54).
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Related In: Results  -  Collection

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pone.0118547.g001: Cloning and expression of ELI025.(A) Plasmid DNA map of pET28b-ELI025 shows the cloning sites (Nde-I and EcoR-I) of ELI025. Expression of ELI025 is under the control of the T7 promoter. The numbers in parentheses indicate a location of each plasmid component; (B) Protein structure of ELI025 shows a signal peptide (SP; amino acid position 1–20), an elicitin domain (amino acid position 25–110), three disulfide bonds (C1, cysteine position 27 and 91; C2, cysteine position 47 and 76; C3, cysteine position 71 and 110), two predicted N-linked glycosylation sties (N; amino acid position, 22 and 87), and three predicted O-linked glycosylation sties (O; amino acid position 49, 51, and 54).
Mentions: The full-length ELI025-encoding sequence (NCBI accession number: HS975204) was amplified from the pCR4-blunt-TOPO vector harboring PinsEST#025 cDNA [34], in a 50-μl PCR reaction containing 1.5 μl of PCR product, 1 μl of the Elongase and its buffer mixture (buffer A:B ratio = 1:4) (Invitrogen), 200 μM of dNTPs, and 0.4 μM each of the primer ELI025_NdeI (5’-GGCATCACATATGtacaacgagaccaagccg-3’) and ELI025_EcoRI (5’-CAAGAATTCCTAGGCCTTGCAGCTCGTC-3’). The reaction was carried out in a MyCycler (Biorad) with the following conditions: initial denaturation at 94°C for 30 s, 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 68°C for 1.10 min, and final extension at 68°C for 5 min. The PCR product was double digested with NdeI and EcoRI (New England Biolabs), and directionally cloned into pET28b (Novagen), yielding an in-frame His-tag fusion on the N-terminus of ELI025. The resulting plasmid, pET28b-ELI025 (Fig. 1A), was propagated in the Escherichia coli strain DH5α. The sequence of the ELI025-coding region of the plasmid was confirmed using primers, T7-promoter (5’-TAATACGACTCACTATAGGG-3’) and T7-terminator (5’-GCTAGTTATTGCTCAGCGG-3’).

Bottom Line: Elicitins are secretory proteins and are found only in the oomycetes, primarily in Phytophthora and Pythium species.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Program, Multidisciplinary Unit, Faculty of Science, Mahidol University, Bangkok, Thailand.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus