Degradation of Nematodes by Chitinase AO-492 from Arthrospora oligospora

doi:10.13560/j.cnki.biotech.bull.1985.2024-0010

Abstract

Abstract:

【Objective】To explore the biological function of secreted protein AO-492 in the process of chitin induction from nematode-trapping fungus Arthrobotrys oligospora.【Method】The coding region of the main domain of chitinase AO-492 from A. oligospora was cloned and analyzed, and expressed in Pichia pastoris. The recombinant protein ReAO-Z492 was purified by nickel column affinity chromatography. The enzymatic activity of the recombinant protein under different temperature, pH and metal ion conditions was analyzed by NAG assay, and its biological function was analyzed by acting on Caenorhabditis elegans and eggs.【Result】The chitinase AO-492 had a signal peptide, no transmembrane domain, contained two chitin binding domains and a glycoside hydrolase 18 family domain, and the highly conserved substrate binding site of the glycoside hydrolase 18 family chitinase -SVGGWT- and the hydrolase active site -FDGGDLDWE-, as well as a typical TIM barrel molecular structure. Phylogenetic analysis showed that the protein had the closest genetic relationship with chitinase（EPS35099.1）. SDS-PAGE and Western Blot analysis showed that the molecular weight of ReAO-Z492 was about 59 kD, which specifically reacted with mouse polyclonal antibody against A. oligospora. The optimal temperature and pH of ReAO-Z492 were 40℃ and 7.0, respectively. Mg²⁺ promoted the enzyme activity, while Ag⁺, Cu²⁺, Fe³⁺ and Zn²⁺ inhibited it. ReAO-Z492 had strong degrading activity on the body wall and egg shell of C. elegans.【Conclusion】The chitinase AO-492 of A. oligospora has a strong degradation effect on C. elegans and its eggs.

Key words: Arthrobotrys oligospora, chitinase, ReAO-Z492, enzymatic activity, nematode-trapping fungi

ZHANG Jia-hua, ZHANG Hui-mei, MA Xi-xi, SUN Yan-sen, LI Ruo-bing, LI Ning-xing, CAI Xue-peng, QIAO Jun, MENG Qing-ling. Degradation of Nematodes by Chitinase AO-492 from Arthrospora oligospora[J]. Biotechnology Bulletin, 2024, 40(5): 261-268.

Figures/Tables 10

Table 1 Primers’ information used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	产物大小 Product size/bp
492-F 492-R 492-ZF 492-ZR	ATGAGATCTCTGTTTCTACGAG TCAACAGTCTCTCGGCAGTAAG GCTACGTAATGCATCATCATCATCATCATAGATCTCTGTTTCTACGA GCGGCCGCTCAACAGTCTCTCGGCAGTAAG	1 620 1 654

Fig. 1 PCR amplification of AO-492 gene of A. oligospora Xj-2 strain M: DL2000 DNA marker; 1: negative control; 2-3: amplified products of AO-492 gene

Fig. 2 Domain pattern of chitinase AO-492 from A. oligospora

Fig. 3 Genetic evolution analysis of chitinase AO-492

Fig. 4 Identification of recombinant plasmid pPIC9K-AO492 by double-nzyme digestion M: DL8000 DNA marker; 1: undigested pPIC9K-AO492; 2-3: double-enzyme digested products of pPIC9K-AO492

Fig. 5 Screening of recombinant yeast strains A: MD plate; B: YPD medium containing 1 mg/mL G418; C: YPD medium containing 2 mg/mL G418; D: YPD medium containing 3 mg/mL G418

Fig. 6 Identification of recombinant protein ReAO-Z492 by SDS-PAGE (A) and Western blot (B) M: Protein molecular quality standard ; 1: culture supernatant of pPIC9K empty vector induced by methanol for 72 h ; 2-6: culture supernatants induced by methanol for 24,48,72,96 and 120 h, respectively. (B) 1-2: Purified and concentrated ReAO-Z492

Fig. 7 Determination of relative enzymatic activity of recombinant chitinase AO-492 A: NAG standard curve. B: The effect of different pH on the activity of AO-492. C: The effect of different temperatures on the activity of AO-492. D: The effect of different metal ions on the activity of AO-492. * * * * indicates P ≤ 0.0001, ns indicates P > 0.05

Fig. 8 Degradation effect of ReAO-Z492 on C. elegans Control: A1-A3: C. elegans were treated with inactivated ReAO-Z492 for 0, 3, and 6 h. Experimentl: B1-B3, C1-C3 and D1-D3: C. elegans were treated with ReAO-Z492 for 0, 3, and 6 h. The arrow refers to the part of the body wall degradation of nematodes

Fig. 9 Degradation effect of ReAO-Z492 on C. elegans eggs Control: A1-A3: C. elegans eggs were treated with inactivated ReAO-Z492 for 0, 6, and 12 h. Experiment: B1-B3: C. elegans eggs were treated with ReAO-Z492 for 0, 6, and 12 h. The arrow refers to the degradation part of egg shell

References 30

[1]	Freitas LA, Savegnago RP, Menegatto LS, et al. Cluster analysis to explore additive-genetic patterns for the identification of sheep resistant, resilient and susceptible to gastrointestinal nematodes[J]. Vet Parasitol, 2022, 301: 109640.
[2]	Hou B, Yong R, Wuen JY, et al. Positivity rate investigation and anthelmintic resistance analysis of gastrointestinal nematodes in sheep and cattle in Ordos, China[J]. Animals, 2022, 12(7): 891.
[3]	Mendoza-de Gives P, Braga FR, de Araújo JV. Nematophagous fungi, an extraordinary tool for controlling ruminant parasitic nematodes and other biotechnological applications[J]. Biocontrol Sci Technol, 2022, 32(7): 777-793.
[4]	Zhang F, Boonmee S, Monkai J, et al. Drechslerella daliensis and D.xiaguanensis(Orbiliales, Orbiliaceae), two new nematode-trapping fungi from Yunnan, China[J]. Biodivers Data J, 2022, 10: e96642.
[5]	Niu XM, Zhang KQ. Arthrobotrys oligospora: a model organism for understanding the interaction between fungi and nematodes[J]. Mycology, 2011, 2(2): 59-78.
[6]	Huang XW, Zhao NH, Zhang KQ. Extracellular enzymes serving as virulence factors in nematophagous fungi involved in infection of the host[J]. Res Microbiol, 2004, 155(10): 811-816. pmid: 15567274
[7]	Yang JK, Tian BY, Liang LM, et al. Extracellular enzymes and the pathogenesis of nematophagous fungi[J]. Appl Microbiol Biotechnol, 2007, 75(1): 21-31. pmid: 17318531
[8]	Wernet N, Wernet V, Fischer R. The small-secreted cysteine-rich protein CyrA is a virulence factor participating in the attack of Caenorhabditis elegans by Duddingtonia flagrans[J]. PLoS Pathog, 2021, 17(11): e1010028.
[9]	Gomaa EZ. Microbial chitinases: properties, enhancement and potential applications[J]. Protoplasma, 2021, 258(4): 695-710. doi: 10.1007/s00709-021-01612-6 pmid: 33483852
[10]	Qiu ST, Zhou SP, Tan Y, et al. Biodegradation and prospect of polysaccharide from crustaceans[J]. Mar Drugs, 2022, 20(5): 310.
[11]	Le B, Yang SH. Microbial chitinases: properties, current state and biotechnological applications[J]. World J Microbiol Biotechnol, 2019, 35(9): 144.
[12]	Yang JK, Yu Y, Li J, et al. Characterization and functional analyses of the chitinase-encoding genes in the nematode-trapping fungus Arthrobotrys oligospora[J]. Arch Microbiol, 2013, 195(7): 453-462.
[13]	Li C, Li XP, Bai CZ, et al. A chitinase with antifungal activity from naked oat(Avena chinensis)seeds[J]. J Food Biochem, 2019, 43(2): e12713.
[14]	Du JH, Duan S, Miao JY, et al. Purification and characterization of chitinase from Paenibacillus sp[J]. Biotechnol Appl Biochem, 2021, 68(1): 30-40.
[15]	Meerupati T, Andersson KM, Friman E, et al. Genomic mechanisms accounting for the adaptation to parasitism in nematode-trapping fungi[J]. PLoS Genet, 2013, 9(11): e1003909.
[16]	Yang JK, Wang L, Ji XL, et al. Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation[J]. PLoS Pathog, 2011, 7(9): e1002179.
[17]	Chen W, Jiang X, Yang Q. Glycoside hydrolase family 18 chitinases: the known and the unknown[J]. Biotechnol Adv, 2020, 43: 107553.
[18]	Huang QS, Xie XL, Liang G, et al. The GH18 family of chitinases: their domain architectures, functions and evolutions[J]. Glycobiology, 2012, 22(1): 23-34.
[19]	Houston DR, Recklies AD, Krupa JC, et al. Structure and ligand-induced conformational change of the 39-kDa glycoprotein from human articular chondrocytes[J]. J Biol Chem, 2003, 278(32): 30206-30212. doi: 10.1074/jbc.M303371200 pmid: 12775711
[20]	Tjoelker LW, Gosting L, Frey S, et al. Structural and functional definition of the human chitinase chitin-binding domain[J]. J Biol Chem, 2000, 275(1): 514-520. doi: 10.1074/jbc.275.1.514 pmid: 10617646
[21]	Xie XH, Fu X, Yan XY, et al. A broad-specificity chitinase from Penicillium oxalicum k10 exhibits antifungal activity and biodegradation properties of chitin[J]. Mar Drugs, 2021, 19(7): 356.
[22]	Suryawanshi N, Eswari JS. Purification and characterization of chitinase produced by thermophilic fungi Thermomyces lanuginosus[J]. Prep Biochem Biotechnol, 2022, 52(9): 1087-1095.
[23]	Zargar V, Asghari M, Dashti A. A review on chitin and chitosan polymers: structure, chemistry, solubility, derivatives, and applications[J]. ChemBioEng Rev, 2015, 2(3): 204-226.
[24]	Moussian B. Chitin: structure, chemistry and biology[M]// Targeting Chitin-containing Organisms. Singapore: Springer, 2019: 5-18.
[25]	Tharanathan RN, Kittur FS. Chitin—the undisputed biomolecule of great potential[J]. Crit Rev Food Sci Nutr, 2003, 43(1): 61-87.
[26]	Chen Q, Peng DL. Nematode chitin and application[M]// Targeting Chitin-containing Organisms. Singapore: Springer, 2019: 209-219.
[27]	Adrangi S, Faramarzi MA. From bacteria to human: a journey into the world of chitinases[J]. Biotechnol Adv, 2013, 31(8): 1786-1795. doi: 10.1016/j.biotechadv.2013.09.012 pmid: 24095741
[28]	Zhong WQ, Chen Y, Gong SS, et al. Enzymological properties and nematode-degrading activity of recombinant chitinase AO-379 of Arthrobotrys oligospora[J]. Kafkas Universitesi Veteriner Fakultesi Dergisi, 2019, 25(4): 435-444.
[29]	贡莎莎, 孟庆玲, 乔军, 等. 少孢节丛孢菌XJ-A1几丁质酶AO-483基因的克隆及生物学活性[J]. 浙江农业学报, 2019, 31(2): 222-228. doi: 10.3969/j.issn.1004-1524.2019.02.07
	Gong SS, Meng QL, Qiao J, et al. Gene cloning and bioactivity analysis of chitinase gene AO-483 from Arthrobotrys oligospora XJ-A1[J]. Acta Agric Zhejiangensis, 2019, 31(2): 222-228.
[30]	Gong SS, Meng QL, Qiao J, et al. Biological characteristics of recombinant Arthrobotrys oligospora chitinase AO-801[J]. Korean J Parasitol, 2022, 60(5): 345-352.