VERMONT COBRE: PROJECT 3: MECHANISM OF ENTAMOEBA HISTOLYTICA PHAGOCYTOSIS

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A. Specific Aims The ability of the intestinal ameba Entamoeba histolytica to phagocytose host cells correlates with parasite virulence, but the mechanism underlying this process and its specific contribution to pathogenesis remains unknown. The original application had two specific aims, with the overall goal of understanding E. histolytica phagocytosis. Aim 1 was to extend preliminary data demonstrating that phosphatidylserine is exposed on the host cell surface during amebic cell killing, by delineating its role as a potential ligand that stimulates phagocytosis and the mechanism by which it becomes exposed. During the first two years of this project, we attempted unsuccessfully to adapt methods used to study the mechanisms of lymphocyte caspase activation and phosphatidylserine exposure during apoptosis to our system. During this time we also made the exciting new discovery that C1q- and host collectins are strong stimulants of E. histolytica phagocytosis. Based in part on the technical problems we encountered with the original aim and in part on a desire to pursue this interesting finding, we have changed aim 1. Aim2 has not been changed. The current specific aims are: Aim 1: Test the hypothesis that E. histolytica has a phagocytosis receptor specific for the collagenous tail of C1q and the collectins. Aim 2: Test the hypothesis that the serine-rich E. histolytica protein (SREHP) and/or the related asparagine rich Ariel proteins function as receptors for phagocytosis of killed cells. B. Studies and Results Aim1: We discovered that C1q is a potent stimulant of E. histolytica phagocytosis. Towards understanding the significance of this finding, we developed a method to construct "single-ligand" fluorescent particles by biotinylating protein ligands of interest and using them to coat streptavidin-latex beads. Additional reagents developed include purification of the collagenous C1q-tail domain (which is conserved amongst collectin family members), and fluorescent-labeling of C1q, MBL, and purified collagenous C1q-tails. We have shown that C1q, MBL, and purified C1q tails are strong stimulants for phagocytosis using a flow cytometry-based phagocytosis assay, and that they are strong chemoattractants using a transwell migration assay. We have also shown that C1q binds both healthy and apoptotic lymphocytes, but that C1q binding is concentrated to apoptotic blebs. Furthermore, phagocytosis of C1q-coated particles depends on ligand density, suggesting one explanation for preferential engulfment of apoptotic cells by E. histolytica. We submitted a manuscript reporting these findings that was favorably reviewed, and we expect it to be accepted following minor revisions. Aim2: We have now published some of our results (publication #2 below) in a paper which describes a monoclonal antibody screen we conducted to identify phagocytosis inhibitory antibodies. In this screen, we identified the SREHP as the target of an inhibitory antibody. Ongoing studies are focused on delineating further the function of the SREHP. Recombinant SREHP made in Escherichia coli did not bind specifically to host cells and had no effect on phagocytosis. The protein is known to be glycosylated, so, to insure normal post-translational modification, we are attempting to express the SREHP on the surface of adherent mammalian cells which we can then use for binding studies. We cloned the SREHP into the expression vector pcDNA3.1 and attempted to express the protein on the surface of Chinese Hamster Ovary Cells. The protein was expressed (as determined by Western blots), but did not localize to the cell surface. We have now cloned the SREHP gene into the vector pDisplay, which incorporates a mammalian signal peptide and membrane anchor, and we plan to use this vector to express the SREHP. Also as noted last year, we found that the Fab fragment of the 10D11Ab that inhibits phagocytosis has no effect on phagocytosis despite retention of its binding activity. We hypothesize that the 10D11Ab may inhibit phagocytosis by capping the SREHP, and, thereby, interfering with E. histolytica cell signaling. We optimized a method to measure calcium fluxes in E. histolytica using flow cytometry and Fura 2-AM. This method worked well for our positive control (i.e., ionomycin treatment), but we found no change in cytosolic calcium levels following 10D11Ab treatment. Finally, we continue to try to silence expression of the SREHP using RNA-mediated interference. Our approach has focused on expression of small hairpin RNAs under control of the E. histolytica RNA Pol III promoter that is optimized for expression of small RNAs. Towards this end, we have constructed a new vector for use in E. histolytica that contains a Neomycin selection cassette with flanking actin 5' and 3' UTRs and the E. histolytica U6 promoter with an associated multi-cloning site. We are currently trying to ligate double-stranded RNAs based on 4 different SREHP sequences (and a scrambled control sequence) and designed to form hairpins into this vector. C. Significance: The mechanisms that underlie amebic phagocytosis remain unknown, and the precise contribution of phagocytosis to pathogenesis remains unclear. Successful completion of these studies promises to shed light on both. In aim 1, we will continue to clarify the ligands on apoptotic cells that trigger E. histolytica phagocytosis. This will assist in identification of additional phagocytosis receptors, which may be candidates for inclusion in a vaccine to prevent amebiasis. Our work in aim 2 implicating the SREHP as an E. histolytica phagocytosis receptor is important because we have assigned a function to the SREHP for the first time. This protein is a leading vaccine candidate that has been studied extensively, but was previously without a known function. Successful silencing of SREHP expression in aim 2 will extend the results of our antibody inhibition studies; furthermore, SREHP silencing should produce amebae with a defined defect in phagocytosis that can be used to determine the contributions of phagocytosis to pathogenesis of disease. D. Plans: The aims will remain unchanged during the coming year. For aim 1, we are now using fluorescently-labeled C1q tails and flow cytometry to conduct binding studies. These studies will allow us to determine if E. histolytica has a saturable surface receptor specific for the collagenous collectin tail. Inhibition with anti-C1q antibodies will be used to determine binding specificity. Cross inhibition with free collectins (i.e., mannose binding lectin), furthermore, will determine if E. histolytica recognizes C1q- and MBL-coated particles via the same mechanism. These specificity studies will lay the groundwork for cross-linking studies to identify the amebic collectin receptor. For aim 2, we are continuing to try to express the SREHP on the surface of mammalian cells. Once successful, we will assess binding of fluorescently-labeled apoptotic cells and bacteria to SREHP-expressing cell monolayers compared to control cells. This will directly test if the SREHP functions in adherence to apoptotic cells and bacteria. In addition, we are continuing to try to silence the SREHP gene using RNAi. For this, we are cloning constructs to express small hairpin RNAs into the vector we built. E. Publications (since the 2007 progress report): 1.Boettner DR, Huston CD, Linford AS, Buss SN, Houpt E, Sherman NE, Petri WA. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family. PLoS Pathogens. 2008. 4(1):122-33. 2.Teixeira JE, Huston CD. Participation of the serine-rich Entamoeba histolytica protein in amebic phagocytosis of apoptotic host cells. Infection and Immunity. 2008. 76:959-66. 3.Teixeira JE, Huston CD. Evidence of a continuous endoplasmic reticulum in the protozoan parasite Entamoeba histolytica. Eukaryotic Cell. 2008. Feb 15; [Epub ahead of print] PMID: 18281599. Mentoring Summaries: Gary Ward Dr. Ward meets biannually one-on-one with Dr. Huston to discuss data and the overall direction of Dr. Huston's work, and more frequently on an informal basis when issues related research or personnel management arise. Dr. Ward critically reads and provides feedback to Dr. Huston on his manuscripts and grant applications, and serves on the dissertation committee of one of Dr. Huston's PhD students, Brad Heron. Dr. Ward and Dr. Huston participate in a joint, biweekly lab meeting, which also includes one of the other junior investigators on the COBRE grant, Dr. Matrajt. This data-centered meeting is highly interactive, and an excellent way for Dr. Huston and his students and postdocs to receive regular feedback on the course of their research. Dr. Ward has made an effort to introduce Dr. Huston to - and facilitate his interactions with - others on campus or in the immediate area that might be helpful to him in his research. For example, Dr. Ward recently nominated Dr. Huston to speak at the annual Dartmouth College Molecular Pathogenesis retreat; his talk was very well received, and he now has a number of new contacts in the Dept of Microbiology and Immunology at Dartmouth Medical School. Dr. Ward also provides every opportunity to Dr. Huston to meet with visiting scientists and seminar speakers who come to UVM. Cory Teuscher Chris was concerned that failing a student would reflect poorly on him and impact his promotion and tenure. I told him that since he teaches in the medical school curriculum and clinic that he shouldn't worry about this, particularly since his medical school teaching evaluations are strong (at least that's what he said). I also told him that in the long run it is better to fail or drop a poor student rather than coddling them along since he will be judge by the product he puts out. It demonstrates higher academic integrity to drum out a student rather than to support one whose long term performance will ultimately reflect poorly on him. Markus Thali Dr. Thali meets with Dr. Huston occasionally on an informal basis to discuss research and personnel management issues. He serves on the thesis committee of one of Dr. Huston's graduate student (Brad Heron) and, as the director of the Cell and Molecular Biology Graduate Program, he is somewhat familiar with the progress of the other graduate student in Dr. Huston's lab (Archana Vaithilingam). In addition, since fall 2007, the Huston and the Thali groups meet once a month for a joint group meeting.