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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR AND IMMUNOLOGICAL APPROACHES TO CONTROLLING GI NEMATODE INFECTIONS OF RUMINANTS

Location: Animal Parasitic Diseases

Title: Characterization of Neospora caninum macrophage migration inhibitory factor

Authors
item Qu, G -
item Fetterer, Raymond
item Jenkins, Mark
item Leng, L -
item Shen, Z -
item Murphy, Charles
item Han, W -
item Bucala, R -
item Tuo, Wenbin

Submitted to: Experimental Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 1, 2013
Publication Date: July 11, 2013
Citation: Qu, G., Fetterer, R.H., Jenkins, M.C., Leng, L., Shen, Z., Murphy, C.A., Han, W., Bucala, R., Tuo, W. 2013. Characterization of Neospora caninum macrophage migration inhibitory factor. Experimental Parasitology. 135(2):246-256.

Interpretive Summary: The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). NcMIF was cloned and expressed in Escherichia coli in three forms, NcMIF (mature protein), NcMIFm (mutation at the second proline to a glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). NcMIF had no enzymatic or immunologic regulatory activities typical of mammalian MIFs. The defined NcMIF protein sequence change resulted in changes in oligomerization of this molecule. NcMIF was localized to the apical end of the parasite and present as an intracellular and secretory protein. NcMIF production was increased in a non-pathogenic clone of Neospora caninum, suggesting that this molecule is involved in pathogenesis of the parasite in its host. This research will advance the understanding of host-parasite interactions and development of vaccines. The cattle industry will benefit from this research.

Technical Abstract: The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in three forms, NcMIF (mature protein), NcMIFm (mutation at the second proline to a glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these NcMIFs had tautomerase, oxidoreductase, or immunologic regulatory activities. The mutation at the second proline of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was localized to the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in tachyzoite lysate and was present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more (p<0.05) abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased (p<0.05) in the presence of the calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in pathogenesis of the parasite.

Last Modified: 8/1/2014
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