Abstract
Multiple protozoans produce homologs of the cytokine MIF which play a role in immune evasion, invasion and pathogenesis. However, how parasite-encoded MIF activity is controlled remains poorly understood. Cytokine activity can be inhibited by intracellular binding partners that are released in the extracellular space during cell death. We investigated the presence of an endogenous parasite protein that was capable of interacting and interfering with MIF activity. A screen for protein-protein interaction was performed using immunoaffinity purification of amebic cell lysate with specific anti-Entamoeba histolytica MIF (EhMIF) antibody followed by mass spectrometry analysis, which revealed an E. histolytica-produced JAB1 protein (EhJAB1) as a potential binding partner. JAB1 was found to be highly conserved in protozoans. Direct interaction between the EhMIF and EhJAB1 was confirmed by several independent approaches with GST pull-down, co-immunoprecipitation, and Biolayer interferometry (BLI) assays. Furthermore, the C-terminal region outside the functional JAMM deneddylase motif was required for EhMIF binding, which was consistent with the top in silico predictions. In addition, EhJAB1 binding blocked EhMIF-induced IL-8 production by human epithelial cells. We report the initial characterization of a parasite-encoded JAB1 and uncover a new binding partner for a protozoan-produced MIF protein, acting as a possible negative regulator of EhMIF.
Highlights
Protozoan parasites represent a major threat to health and contribute significantly to morbidity and mortality worldwide
Parasite cell lysates were incubated with a specific antibody against Entamoeba histolytica migration inhibitory factor (MIF) (EhMIF) or IgG control antibody
While a number of putative interacting proteins were co-precipitated with anti-EhMIF antibody, E. histolytica-produced JAB1 protein (EhJAB1) was selected for further analysis given: (i) EhJAB1 was detected in the anti-EhMIF co-IP sample and not detected in the control IP; (ii) EhJAB1 was among the highest percent coverage
Summary
Protozoan parasites represent a major threat to health and contribute significantly to morbidity and mortality worldwide. Entamoeba histolytica is a protozoan parasite that causes colitis[1]. Many of the inflammatory effects of MIF are mediated through direct binding to the CD74 cell surface receptor, causing the secretion of proinflammatory cytokines such as IL-84–6. MIF homologs have been characterized in several pathogenic protozoans including Entamoeba, Plasmodium, Toxoplasma, and Leishmania. These protozoan MIF homologs have demonstrated similar proinflammatory activities to that of human MIF, and play a role in immune evasion, invasion and pathogenesis[12,13,14,15,16,17,18,19]. Which is highly conserved throughout protozoan parasites, as a novel binding partner and potential regulator of the MIF homolog of Entamoeba histolytica
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