Lipid Rafts and Signaling in the Human Protozoan Parasite, Entamoeba Histolytica

Non-Technical Summary:<br/>
Amoebic dysentery is a food- and water-borne illness that is prevalent in the developing world. It is acquired by ingesting the parasite, Entamoeba histolytica. Globally, it is the 2nd leading cause of morbidity and mortality attributable to parasitic infections. The prevalence of E. histolytica infection has been estimated to range from 1% to 40% of the population in Central and South America, Africa and Asia and from 0.2% to 10% in developed nations such as the USA. Interestingly, many infections are asymptomatic or go unreported. Thus, the magnitude of the problem is difficult to discern. None-the-less, as of 2010, it is estimated that 2.6 billion people worldwide do not use modern sanitation practices, and 886 million do not have access to clean drinking water sources. Thus, there is considerable global risk for acquiring E. histolytica infection. The pathogen can bind to human cells via its lectin, a surface complex consisting of 3 protein subunits. The subunits are proposed to reside in lipid rafts which are membrane regions rich in cholesterol. However, the mechanism by which the protein subunits assemble is poorly understood. Our studies are designed to gain insight into the way in which this cell surface receptor assembles and what regulates its ability to bind to human cells. In particular, we will study if GGL becomes activated when it is found in rafts, what proteins bind to this receptor, what parts of the receptor control its location to raft or non-raft regions, and what cellular signaling pathways become activated after the receptor enters rafts. We expect to gain significant understanding of the way E. histolytica causes infection. The studies are important for several reasons. First, since E. histolytica is a water-borne pathogen, the proposed work will provide insight into this threat to the global water supply. Threats to water supplies, even those outside the US, are of concern to South Carolinians as contaminated water may negatively impact travelers or military personnel stationed abroad. Second, E. histolytica is classified as a bioterrorism agent. Thus, the research is of interest from a national and state security perspective.
<P>
Approach:<br/>
In the first Aim, we will determine how raft-localization activates the GGL. We will address this question by following the movement of the GGL into rafts and simultaneously measuring activity of the GGL. This will allow us to correlate localization to rafts and activity temporally. We will examine the role of actin in activating the GGL in rafts using raft disrupting agents. Cells will be treated with cytochalasin or latrunculin and the activity and localization of the GGL will be assessed. We will identify other proteins that localizing to rafts with the GGL by mass spectroscopy. In the second Aim, we will study the mechanisms that regulate the physical localization of the GGL to rafts. Here we will use single molecule tracking to follow movement of the GGL in rafts and to record the interactions among the GGL subunits. To determine the role of post-translational modification we will define the E. histolytica raft palmytoylome by isolating and identifying by mass spectroscopy all palmitoylated proteins in rafts. We will also disrupt GPI-anchorage genetically and biochemically and determine the localization of the GGL subunits. To study signaling (third Aim) we will track the localization of PIP2 and PIP3 after the GGL subunits enter rafts using fluorescent PIP-biosensors that we developed for use in this system. We will also generate mutants with altered levels of PIP2 and PIP3 and follow GGL-raft interactions in these cell lines. Finally, we will determine what proteins interact with PIP2 and PIP3 in rafts by mass spectroscopy.
<P>
Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: During the funding period we continued to carry out research to gain insight into the cell and molecular mechanisms regulating virulence in the human protozoan parasite, Entamoeba histolytica. This food-borne pathogen is the causative agent of amoebic dysentery and ranks second in the world for deaths due to parasitic disease. In particular, we have characterized the role of cholesterol and phosphatidylinositol-containing lipids in virulence, and the role of PI 3-kinase signaling in drug resistance. We have submitted NIH R03 and R21 grant proposals to fund our research. These proposals are pending review at the NIH. Dissemination: The results of our studies related to our gene discovery project were published in PLoS ONE. The results of our studies on the role of host cell components in the submembrane distribution of the Gal/GalNAc lectin were published in Eukaryotic Cell. It was subsequently awarded Editor's Choice Recognition by the journal editorial board. We also published a review article in Trends in Parasitology on lipid rafts in parasites. The results of our studies were also presented at 4 international conferences. The results of our studies on the relationships between phosphatidylinositol, cholesterol and virulence of E. histolytica were recently submitted as a manuscript to Infection and Immunity. Our findings provide significant insight into the cell biology of this parasite.
<br/>PARTICIPANTS: Not relevant to this project.
<br/>TARGET AUDIENCES: Not relevant to this project.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
<P>
IMPACT: Summary of a project published in PLoS ONE: Entamoeba histolytica is a protozoan parasite for which forward genetics approaches have not been extensively applied. Given that the E. histolytica genome has been sequenced, it should be possible to apply genomic approaches to discover gene function. We used a genome-wide over-expression screen to uncover genes regulating an important virulence function of E. histolytica, namely phagocytosis. We developed an episomal E. histolytica cDNA over-expression library, transfected the collection of plasmids into trophozoites, and applied a high-throughput screen to identify phagocytosis mutants in the population of over-expressing cells. The screen was based on the phagocytic uptake of human red blood cells loaded with the metabolic toxin, tubercidin. Expression plasmids were isolated from trophozoites that survived exposure to tubercidin-charged erythrocytes (phagocytosis mutants), and the cDNAs were sequenced. We isolated the gene encoding profilin, a well-characterized cytoskeleton-regulating protein with a known role in phagocytosis. This supports the validity of our approach. We assigned a phagocytic role to several genes not previously known to function in this manner. To our knowledge, this is the first genome-wide forward genetics screen to be applied to this pathogen. The study demonstrates the power of forward genetics in revealing genes regulating virulence in E. histolytica. The study validates an E. histolytica cDNA over-expression library as a valuable tool for functional genomics. Summary of project published in Eukaryotic Cell: Entamoeba histolytica cell surface receptors, such as the Gal/GalNAc lectin, facilitate attachment to host cells and extracellular matrix. The Gal/GalNAc lectin binds to galactose or N-acetylgalactosamine residues on host components and is composed of heavy (Hgl), intermediate (Igl), and light (Lgl) subunits. Although Igl is constitutively localized to lipid rafts (cholesterol-rich membrane domains), Hgl and Lgl transiently associate with this compartment in a cholesterol-dependent fashion. In this study, trophozoites were exposed to biologically relevant ligands to determine if ligand binding influences the submembrane distribution of the subunits. Exposure to human red blood cells (hRBCs) or collagen was correlated with enrichment of Hgl and Lgl in rafts. This enrichment was abrogated in the presence of galactose, suggesting that direct lectin-ligand interactions are necessary to influence subunit location. Using a cell line that is able to attach to, but not phagocytose, hRBCs, it was shown that physical attachment to ligands was not sufficient to induce the enrichment of lectin subunits in rafts. Finally, intracellular calcium levels increased upon attachment to collagen; this increase was essential for the enrichment of lectin subunits in rafts. Together, these data provide evidence that ligand-induced enrichment of lectin subunits in rafts may be the first step in a signaling pathway that involves both PIP(2) and calcium signaling.