Functional expression and characterization of Echinococcus granulosus thioredoxin peroxidase suggests a role in protection against oxidative damage.
A full-length cDNA sequence coding for Echinococcus granulosus thioredoxin peroxidase (EgTPx) was isolated from a sheep strain protoscolex cDNA library by immunoscreening using a pool of sera from mice infected with oncospheres. EgTPx expressed as a fusion protein with glutathione S-transferase (GST) exhibited significant thiol-dependent peroxidase activity that protected plasmid DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Furthermore, the suggested antioxidant role for EgTPx was reinforced in an in vivo assay, whereby its expression in BL21 bacterial cells markedly increased the tolerance and survival of the cells to high concentrations of H2O2 compared with controls. Immunolocalization studies revealed that EgTPx was specifically expressed in all tissues of the protoscolex and brood capsules. Higher intensity of labelling was detected in many, but not all, calcareous corpuscle cells in protoscoleces. The purified recombinant EgTPx protein was used to screen sera from heavily infected mice and patients with confirmed hydatid infection. Only a portion of the sera reacted positively with the EgTPx-GST fusion protein in Western blots, suggesting that EgTPx may form antibody-antigen complexes or that responses to the EgTPx antigen may be immunologically regulated. Recombinant EgTPx may prove useful for the screening of specific inhibitors that could serve as new drugs for treatment of hydatid disease. Moreover, given that TPx from different parasitic phyla were phylogenetically distant from host TPx molecules, the development of antiparasite TPx inhibitors that do not react with host TPx might be feasible.
Li
Jun
J
Molecular Parasitology Laboratory, Australian Centre for International and Tropical Health and Nutrition, The Queensland Institute of Medical Research and The University of Queensland, Brisbane, Queensland 4029, Australia.
Zhang
Wen-Bao
WB
Loukas
Alex
A
Lin
Ren-Yong
RY
Ito
Akira
A
Zhang
Li-Hua
LH
Jones
Malcolm
M
McManus
Donald P
DP
D000818:Animals; D000975:Antioxidants / Q000378:metabolism*; D015153:Blotting, Western; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004446:Echinococcus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D054464:Peroxiredoxins; D010802:Phylogeny; D018510:RNA, Helminth / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D000975:Antioxidants; D018076:DNA, Complementary; D007106:Immune Sera; D018510:RNA, Helminth; D011993:Recombinant Fusion Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D005982:Glutathione Transferase