title: Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus.
abstract: The larval stage of Echinococcus granulosus causes cystic echinococcosis (CE), a neglected tropical disease that leads to morbidity and mortality in humans and livestock worldwide. Here, we identified and characterized dihydrofolate reductase (Eg-DHFR) from E. granulosus, and evaluated its potential as a diagnostic antigen for sheep CE. Comparison between mammalian (host) DHFR and Eg-DHFR indicates that 45.7% of the 35 active site residues are different. Immunolocalisation analysis showed that native Eg-DHFR was widely distributed in all life-cycle stages of E. granulosus. Recombinant Eg-DHFR (rEg-DHFR) showed typical DHFR enzymatic parameters towards substrate, and was very sensitive to inhibition by methotrexate (IC50= 27.75 ± 1.03 nM) and aminopterin (IC50= 63.67 ± 6.76 nM). However, inhibition of DHFR exhibited little protoscolicidal effect in vitro. As there is no reliable method to monitor sheep CE, the immunogenicity of rEg-DHFR was detected, and we developed an indirect ELISA (iELISA) for CE serodiagnosis. The iELISA exhibited diagnostic specificity of 89.58%, diagnostic sensitivity of 95.83%, and the diagnostic accuracy was 91.67% compared with necropsy. Cross-reactivity assay showed analytical specificity of 85.7%. These suggest that rEg-DHFR is an effective antigen for the diagnosis of sheep CE.
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Xingju
authors.initials: X
authors.lastname: Song
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Dandan
authors.initials: D
authors.lastname: Hu
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Min
authors.initials: M
authors.lastname: Yan
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.
authors.forename: Xiaobin
authors.initials: X
authors.lastname: Gu
authors.affiliation: Department of Parasitology, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China. guangyou1963@aliyun.com.
authors.forename: Guangyou
authors.initials: G
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004443:Echinococcosis / Q000175:diagnosis* / Q000382:microbiology*; D048209:Echinococcus granulosus / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D021381:Protein Transport; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005493:Folic Acid Antagonists; D013762:Tetrahydrofolate Dehydrogenase