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Published on: March 26, 2014
Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity
Hailong Zhao1, Jun Qiao1, Qingling Meng2
1Key Laboratory of Control and Prevention of Animal Disease of Xinjiang Production & Construction Corps, College of Animal Science and Technology, Shihezi University, North Street No.4, Shihezi, 832003, Xinjiang, China.
Abstract:
The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186.
Insights
This study isolated and expressed serine proteinase 186 from Arthrobotrys oligospora, demonstrating its potent nematode-degrading activity. The recombinant enzyme effectively killed Caenorhabditis elegans and Haemonchus contortus, highlighting its potential as a biocontrol agent.
Area of Science:
- Mycology
- Biochemistry
- Molecular Biology
Background:
- Nematode-trapping fungi, like Arthrobotrys oligospora, are natural predators of nematodes.
- Serine proteinases are crucial virulence factors for these fungi during nematode invasion.
Purpose of the Study:
- To clone, express, and characterize serine proteinase 186 (reP186) from Arthrobotrys oligospora.
- To evaluate the in vitro nematode-degrading activity and bioactivity of the purified recombinant enzyme.
Main Methods:
- Gene amplification using reverse transcription polymerase chain reaction (RT-PCR).
- Cloning into pPIC9K vector and expression in Pichia pastoris.
- Purification via Ni-affinity chromatography, SDS-PAGE, and Western blot analysis.
- In vitro degradation assays with various protein substrates and nematode species (C. elegans, H. contortus).
Main Results:
- Successfully obtained and purified recombinant serine proteinase 186 (reP186) with a molecular weight of 33 kDa.
- reP186 demonstrated degradation of protein substrates like casein, gelatin, BSA, collagen, and nematode cortical layers.
- Optimal activity of reP186 was observed at pH 8.0 and 55°C, with sensitivity to PMSF inhibitor.
- Significant killing rates were achieved for C. elegans (up to 100%) and H. contortus (up to 84%) after 36 hours of treatment.
Conclusions:
- Recombinant serine proteinase 186 from Arthrobotrys oligospora possesses strong nematode-degrading bioactivity.
- The enzyme's efficacy against both free-living and parasitic nematodes suggests potential applications in biological control strategies.
- Further research into reP186 could lead to novel nematode management solutions in agriculture and veterinary medicine.
