Amebiasis ranks second as a protozoan cause of death. Entamoeba histolytica, the etiologic agent, is an intestinal ameba that acquires nutrients by phagocytosis of colonic bacteria, and phagocytosis of host cells by E. histolytica is a prominent feature of invasive amebiasis. Despite its importance, little is known about the mechanism of E. histolytica phagocytosis. Entamoeba histolytica induces host cell apoptosis, resulting in host cell surface changes that engage unknown amebic phagocytosis receptors. In mammals, collectin family members (e.g., mannose binding lectin (MBL)) and the structurally related protein C1q bind to apoptotic cells, and initiate phagocytosis by interaction of their conserved collagenous "tails" with calreticulin. Calreticulin, which has no transmembrane domain, serves as a bridge between collectins and the macrophage receptor CD91. Preliminary data show that: 1) C1q, MBL, and collectin tails stimulate E. histolytica phagocytosis, 2) C1q and MBL compete for binding to the surface of E. histolytica, 3) calreticulin is present on the amebic surface and re-localizes to the phagocytic cup during interaction with apoptotic cells, 4) human C1q and the collectins MBL and SP-A compete for binding to immobilized amebic calreticulin, and 5) specific E. histolytica surface proteins interact with calreticulin. Therefore, host collectins bound to apoptotic cells and bacteria may stimulate E. histolytica phagocytosis by interaction of their collagenous tail domain with an amebic receptor. Calreticulin is one logical candidate, and, if calreticulin participates in amebic phagocytosis, it is hypothesized to do so by bridging between collectins and a calreticulin receptor on the amebic surface.
The specific aims test these hypotheses.
In aim 1, purified collectins, C1q, and collagenous collectin "tails" will be used to test if E. histolytica has a receptor for the collectin tail that mediates engulfment of apoptotic cells. Except for C1q, the collectins also opsonize bacteria in mucosal secretions. Therefore, studies will be performed to test if E. histolytica engulfs apoptotic cells and bacteria via the same mechanism.
In aim 2, RNA-mediated interference and an alternative gene silencing method will be used to determine if calreticulin participates in E. histolytica phagocytosis. Recombinant calreticulin will be used for binding studies to determine if E. histolytica has a calreticulin receptor.
In aim 3, either a collectin or a calreticulin receptor will be identified. The decision of which to pursue will depend on the results from aims 1 and 2. In either case, cross-linking methods and complementary affinity-based methods will be combined with mass spectrometry to identify interacting amebic surface proteins. Successful completion of the proposed studies will provide a molecular understanding of E. histolytica phagocytosis, and will substantially augment our knowledge of how E. histolytica interacts with the host and with colonic bacteria. Furthermore, new insights into the pathogenesis of amebiasis that result from these studies may suggest novel methods for its treatment and prevention.
Entamoeba histolytica, a single-celled intestinal parasite that causes invasive amebiasis (a disease Characterized by bloody diarrhea and liver abscesses), engulfs killed cells during invasion through host tissues. The goal of this project is to determine the molecular mechanisms underlying E. histolytica's ability to recognize and engulf killed cells. This will provide novel insights into how E. histolytica causes invasive infections, possibly suggesting new methods to treat or prevent amebiasis.
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