Abstract
Hookworms, parasitic nematodes that infect nearly one billion people worldwide, are a major cause of anemia and malnutrition. We hypothesize that hookworms actively manipulate the host immune response through the production of specific molecules designed to facilitate infection by larval stages and adult worm survival within the intestine. A full-length cDNA encoding a secreted orthologue of the human cytokine, Macrophage Migration Inhibitory Factor (MIF) has been cloned from the hookworm Ancylostoma ceylanicum. Elucidation of the three-dimensional crystal structure of recombinant AceMIF (rAceMIF) revealed an overall structural homology with significant differences in the tautomerase sites of the human and hookworm proteins. The relative bioactivities of human and hookworm MIF proteins were compared using in vitro assays of tautomerase activity, macrophage migration, and binding to MIF receptor CD74. The activity of rAceMIF was not inhibited by the ligand ISO-1, which was previously determined to be an inhibitor of the catalytic site of human MIF. These data define unique immunological, structural, and functional characteristics of AceMIF, thereby establishing the potential for selectively inhibiting the hookworm cytokine as a means of reducing parasite survival and disease pathogenesis.
Highlights
Successive molts to the infectious L3 stage
X-ray Crystal Structure of recombinant AceMIF (rAceMIF)—For crystallographic studies, rAceMIF was expressed in BL21(DE3) E. coli cells transformed with the pET-11b expression vector containing the AceMIF cDNA and purified using anion and cation exchange chromatography as described above
RAceMIF Is Not Inhibited by ISO-1—In light of differences within the active sites of the hookworm and human migration inhibitory factor (MIF) orthologues revealed by the crystal structures, we investigated whether rAceMIF might be sensitive in vitro to the inhibitor of recombinant human MIF (rhMIF), ISO-1 [44, 51]
Summary
We report here the molecular cloning of a cDNA corresponding to an orthologue of MIF that is secreted by hostdwelling stages of the hookworm parasite Ancylostoma ceylanicum. In vitro characterization confirms that the recombinant A. ceylanicum MIF (rAceMIF) is an active tautomerase and lymphocyte chemoattractant, similar to the human orthologue. Unlike human MIF, AceMIF is not inhibited by the small molecule ligand ISO-1, and the three-dimensional crystal structure reveals functionally relevant differences between the hookworm and human proteins. Together, these data establish a structural basis for the development of pathogen-specific MIF inhibitors as potential treatments for infectious diseases, including hookworm
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