VERMONT COBRE: PROJECT 3: MECHANISM OF ENTAMOEBA HISTOLYTICA PHAGOCYTOSIS

佛蒙特州 COBRE：项目 3：溶组织内阿米巴吞噬机制

• 批准号: 7959818
• 负责人: CHRISTOPHER D HUSTON
• 金额: $ 16.64万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959818
• 关键词: Addendum; Affinity; Amebiasis; Amino Acid Sequence; Amoeba genus; Antibodies; Apoptotic; Applications Grants; Area; Asparagine; Bacteria; Bacterial Adhesins; Binding; Biological; Biological Assay; Biology; Biotin; Biotinylation; Bovine Serum Albumin; C3bi; Cell Line; Cell division; Cell surface; Cells; Centers of Research Excellence; Chinese Hamster Ovary Cell; Clinical; Collagen; Collectins; Communicable Diseases; Complement; Complement 1q; Complement 3b; Computer Retrieval of Information on Scientific Projects Database; Data; Drosophila genus; Endoplasmic Reticulum; Entamoeba dispar; Entamoeba histolytica; Environment; Escherichia coli; Fab Immunoglobulins; Faculty; Family member; Feedback; Flow Cytometry; Funding; Gel; Genes; Genome; Glycocalyx; Grant; Group Meetings; Homologous Gene; Human; Immunobiology; Immunoglobulin G; Immunoglobulins; Immunology; Infection; Institution; Intestines; Joints; Journals; Label; Latex Bead; Lectin; Leishmania; Leishmania glycoprotein gp63; Ligands; Malignant Epithelial Cell; Mannose Binding Lectin; Mannose-Binding Lectins; Manuscripts; Mass Spectrum Analysis; Membrane; Membrane Proteins; Mentors; Metalloproteases; Methods; Microbiology; Molecular; Molecular Biology; Monoclonal Antibodies; Names; National Institute of Allergy and Infectious Disease; Open Reading Frames; Paper; Parasites; Pathogenesis; Pathway interactions; Peptide Sequence Determination; Peptide Signal Sequences; Personnel Management; Phagocytosis; Plasmids; Play; Process; Productivity; Progress Reports; Proteins; Protocols documentation; Publications; Publishing; Pulmonary Surfactant-Associated Protein A; Reading; Recombinants; Research; Research Personnel; Resources; Role; Running; Scientist; Serine; Silver Staining; Sorting - Cell Movement; Source; Streptavidin; Students; Surface; System; Tail; Testing; Time; United States National Institutes of Health; Vaccines; Vermont; Virulence; Visit; Work; base; cell killing; cell motility; college; crosslink; falls; follow-up; graduate student; immune resistance; insight; interest; lung Carcinoma; macrophage; medical schools; meetings; member; migration; novel; particle; phagocytosis receptor; prevent; programs; protein function; protein profiling; receptor; receptor binding; research study; small hairpin RNA; vaccine candidate; vector; vector control; ward

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 mechanisms underlying this process and its specific contribution to pathogenesis remain unknown. The specific aims were not changed during the last year. 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 Aim 1: As noted last year, we discovered that E. histolytica phagocytoses apoptotic cells coated with C1q more efficiently than apoptotic cells coated with control proteins. To follow up on this, we developed a method to construct "single-ligand" fluorescent particles by biotinylating protein ligands of interest and using them to coat streptavidin-latex beads. With this system, we demonstrated that C1q alone is a potent stimulant of E. histolytica phagocytosis. Mannose binding lectin (MBL), a collectin family member that is structurally related to C1q by virtue of a common collagen-like tail domain, also stimulated E. histolytica phagocytosis, as did purified collagenous tails from C1q and surfactant protein A (SP-A). Entamoeba histolytica also migrated towards these proteins in transwell assays. These findings were published last October. We have now used flow cytometry to assay binding of FITC-labeled collectins to the surface of E. histolytica. The results showed that C1q-FITC binds to the amebic surface in a saturable manner, and that binding of C1q-FITC can be partially competed with free MBL. These data provide additional evidence of an amebic receptor specific for the collagenous collectin tail. We are actively engaged in identifying this receptor. Our first approach was to use purified C1q as the bait in cross-linking experiments using the multifunctional cross-linker Sulfo-SBED, which transfers biotin to proteins in close proximity. Our initial experiments were unsuccessful, because the C1q bait forms multimers resulting in overwhelming biotinylation of other bait molecules. A similar approach using purified collagenous tails might work, as might affinity-based methods. An additional approach we have taken is to use flow cytometry to sort amebic trophozoites that are positive or negative for phagocytosis of C1q-coated beads. We have compared the membrane protein profiles of trophozoites positive for phagocytosis of C1q-coated beads to the membrane protein profiles from non-phagocytic trophozoites and those that phagocytosed BSA-coated control beads. Enrichment of specific membrane proteins is observable on silver-stained SDS-PAGE gels. After optimizing our FACS sorting protocol further, we will identify the reproducible bands using mass spectrometry. Although we found that collagenous tails from SP-A significantly stimulate E. histolytica phagocytosis, intact SP-A actually inhibited phagocytosis. Phagocytosis of SP-A-coated particles was approximately five times lower than basal phagocytosis of beads coated with bovine serum albumin (BSA, negative control). This raises the possibility that the lectin domain of SP-A binds to an inhibitory receptor on the ameba surface, while the SP-A tail stimulates phagocytosis via a common collectin receptor. Consistent with this possibility, SP-A can inhibit or enhance macrophage phagocytosis based on context. That is, in the absence of bacteria or cellular debris, SP-A binds an inhibitory receptor via its lectin domain, but in the presence of bacteria or cellular debris it preferentially binds the debris, which exposes its collagenous tail and stimulates phagocytosis. Our ability to pursue this possibility further has been limited by inadequate quantities of SP-A. Thus, we have developed a method to isolate SP-A from a human lung carcinoma cell line, which we are in the process of optimizing. Once we have adequate amounts of SP-A, we plan to confirm its biological activity with bacterial binding studies and then to use it to further examine if SP-A may have the ability to either stimulate or inhibit E. histolytica phagocytosis depending on the local environment. Aim 2: Some of our results from aim 2 were published in 2008 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. In follow-up work, we found that the Fab fragment of the inhibitory antibody had no effect on phagocytosis and that recombinant SREHP produced in Escherichia coli had no specific binding activity. The SREHP is a glycoscylated protein, so we want to express it on the surface of Chinese Hamster Ovary Cells and conduct binding studies. The gene has been cloned into the vector pDisplay (which incorporates a mammalian signal peptide and membrane anchor) for this purpose, but the experiment has not yet been completed. In addition, we want to silence expression of the SREHP in amebic trophozoites. We have constructed four plasmids for expression of small hairpin RNAs based on four different SREHP sequences (and a scrambled control vector), but we have not been able to transfect E. histolytica successfully with these constructs. C. Significance: Entamoeba histolytica's phagocytic ability correlates with virulence and these studies promise to clarify its underlying mechanisms. Aim 1 is focused on clarifying the ligands on apoptotic cells that trigger E. histolytica phagocytosis and on the amebic receptors that bind them. These receptors may be candidates for inclusion in a vaccine to prevent amebiasis. Furthermore, if we confirm that SP-A inhibits or enhances amebic phagocytosis depending on context, then it may suggest a completely novel mechanism by which E. histolytica senses its environment and this could provide critical insights into why E. histolytica is invasive in only about 10% of infections. Successful silencing of the SREHP in aim 2 would extend results of our antibody study, and demonstration of binding of apoptotic cells and/or bacteria to CHO cells expressing the SREHP would definitively demonstrate that this protein functions as an adhesin. Though the SREHP is a leading vaccine candidate, its function is unknown; therefore, this would be very significant. D. Plans: An R01 grant on the phagocytosis studies in aim 1 was funded by the NIAID in 9/2008. Accordingly, the specific aims will be changed entirely in the coming year if I am allowed to remain on the COBRE grant. The new aims will focus on an E. histolytica homologue to leishmanolysin. Leishmanolysin is an immunodominant surface protein of leishmania promastigotes, and is an M8-type metalloproteinase that degrades complement and immunoglobulins. Interestingly, the Drosophila orthologue invadolysin plays critical roles in cell division and cell migration. We identified two leishmanolysin homologues in the E. histolytica genome, which we named E. histolytica leishmanolysin-like proteins 1 and 2 (EhLMLP-1 and -2). The EhLMLP-1 gene is not present in Entamoeba dispar, a non-pathogenic ameba that is closely related to E. histolytica. Few differences in the protein coding regions have been identified between E. histolytica and E. dispar. This and the importance of leishmanolysin in leishmania virulence make it important to determine the function of EhLMLP-1 and the specific role(s) it plays in E. histolytica biology. We plan to: 1) determine if EhLMLP-1 is a cell surface metalloproteinase; 2) determine if EhLMLP-1 contributes to immune resistance by degrading IgG and converting complement C3b to iC3b; and 3) determine if EhLMLP-1 and -2 function in cell division and migration. E. Publications (since the 2008 progress report) Teixeira JE, Heron B, Huston CD. C1q- and collectin-dependent phagocytosis of apoptotic host cells by the intestinal protozoan Entamoeba histolytica. Journal of Infectious Diseases. 2008. 198:1062-1070. Vaithilingam A, Teixeira JE, Huston CD. Addenda article: Endoplasmic reticulum continuity in the protozoan parasite Entamoeba histolytica: evolutionary implications and a cautionary note. Communicative and Integrative Biology. 2008. 1(2):172-174. Mentoring Summaries: Dr. Markus Thali Dr. Thali meets with Dr. Huston on a monthly basis to discuss research and personnel management issues. He also served 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. Dr. Cory Teuscher Chris previously had issues with a graduate student that he successfully resolved. We discussed on several occasions his interests and desire to submit a second R01 application. We talked in depth about the two fundamental approaches to successfully running an academic research enterprise: highly focused with sustained productivity in a single area/molecule/pathway vs. broad based with multiple mutually non-exclusive areas of interests and expertise. The strengths and weaknesses of each approach were discussed, particularly from the perspective of a junior faculty member with clinical responsibilities. Dr. 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 Master's 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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 A.特定目标 肠道阿米巴结构症对吞噬细胞宿主细胞的肠道肠结构性的能力与寄生虫的毒力相关，但是该过程及其对发病机理的特定贡献的机制尚不清楚。 去年，具体目标没有改变。 目标1：检验溶酶体溶血性大肠杆菌具有针对C1q和Collectins胶原蛋白的吞噬受体的假设。 AIM 2：检验以下假设：丝氨酸富含丝氨酸的溶组织溶质蛋白（SHEP）和/或相关的富含天冬酰胺的Ariel蛋白充当吞噬细胞吞噬的受体。 B.研究和结果 AIM 1：如去年所述，我们发现与涂有对照蛋白的凋亡细胞相比，用C1Q涂有C1Q的凋亡细胞更有效地吞噬了凋亡细胞。 为了跟进这一点，我们开发了一种方法来通过感兴趣的生物素化蛋白配体来构建“单配体”荧光颗粒，并使用它们覆盖链霉亲素 - 莱尔特克斯珠。 使用该系统，我们证明了单独的C1Q是溶血性大肠杆菌吞噬作用的有效刺激物。 Mannose结合凝集素（MBL）是一种收集蛋白家族成员，它通过常见的胶原蛋白样尾巴结构域在结构上与C1Q相关，也刺激了E. histolixtica吞噬吞噬作用，来自C1Q和表面活性剂蛋白A（SP-A）的纯化胶原尾巴也是如此。 在Transwell分析中，Entamoeba Histolictica也向这些蛋白质迁移。 这些发现于去年10月发布。 现在，我们已经使用流式细胞仪来测定FITC标记的集合蛋白与溶组织溶解性大肠杆菌表面的结合。 结果表明，C1Q-FITC以饱和的方式与Amebic表面结合，并且C1Q-FITC的结合可以与游离MBL部分竞争。 这些数据提供了针对胶原蛋白尾部特有的Amebic受体的其他证据。 我们积极参与识别该受体。 我们的第一种方法是使用纯化的C1Q作为诱饵在交联实验中使用多功能交联硫膜，该硫酸盐含量将生物素转移到蛋白质上，近距离接近蛋白质。 我们的最初实验是不成功的，因为C1Q诱饵形成了多聚体，导致其他诱饵分子的压倒性生物素化。 使用纯化的胶原式尾巴的类似方法可能会像基于亲和力的方法一样起作用。 我们采用的另一种方法是使用流式细胞术来对C1Q涂层珠的吞噬作用为阳性或阴性。 我们已经将C1Q涂覆珠的滋养体呈阳性的滋养体的膜蛋白谱与非吞噬滋养体的膜蛋白谱以及吞噬BSA涂层的对照珠的膜蛋白谱。 在银色染色的SDS-PAGE凝胶上可以观察到特定膜蛋白的富集。 在进一步优化了FACS排序协议之后，我们将使用质谱识别可重复的频段。 尽管我们发现SP-A的胶原尾巴显着刺激了溶血性吞噬作用，但完整的SP-A实际上抑制了吞噬作用。 SP-A涂层颗粒的吞噬作用比涂有牛血清白蛋白（BSA，阴性对照）的珠子的基底吞噬作用低约五倍。 这增加了SP-A的凝集素结构域与Ameba表面上的抑制受体结合，而SP-A尾部通过常见的集群受体刺激吞噬吞噬作用。 与这种可能性一致，SP-A可以基于上下文抑制或增强巨噬细胞吞噬作用。 也就是说，在没有细菌或细胞碎屑的情况下，SP-A通过其凝集素结构域结合抑制性受体，但是在存在细菌或细胞碎屑的情况下，它优先结合碎屑，从而暴露其胶原尾巴并刺激吞噬吞噬作用。 我们进一步追求这种可能性的能力受到数量不足的SP-A的限制。 因此，我们开发了一种将SP-A与人类肺癌细胞系分离的方法，我们正在优化。 一旦我们拥有足够量的SP-A，我们计划通过细菌结合研究来确认其生物学活性，然后使用它进一步检查SP-A是否有能力刺激或抑制E. thisoloselytica吞噬作用，具体取决于局部环境。 AIM 2：我们来自AIM 2的某些结果在2008年发表在一篇论文中，该论文描述了我们进行的单克隆抗体筛选，以鉴定吞噬作用抑制性抗体。 在此屏幕中，我们将其确定为抑制性抗体的靶标。 在后续工作中，我们发现抑制性抗体的FAB片段对吞噬作用没有影响，并且在大肠杆菌中产生的重组shep没有特定的结合活性。 Shep是一种糖蛋白，因此我们想在中国仓鼠卵巢细胞的表面表达并进行结合研究。 为此，将该基因克隆到载体PDISPLAY（其中包含哺乳动物信号肽和膜锚）中，但实验尚未完成。 另外，我们希望在阿米比克滋养体中沉默表达。 我们已经构建了四个质粒，用于基于四个不同的SHERP序列（和一个扰列的控制载体）来表达小发夹RNA，但是我们无法通过这些构建体成功转染E. Histolytica。 C.意义：肠结构性吞噬能力与毒力相关，这些研究有望阐明其潜在的机制。 AIM 1的重点是阐明触发E. E. histoltica吞噬作用的凋亡细胞的配体以及在结合它们的Amebic受体上。 这些受体可能是候选疫苗以预防amebiasis的候选者。 此外，如果我们确认SP-A会根据上下文抑制或增强Amebic吞噬作用，那么它可能暗示了一种完全新颖的机制，通过该机制，E. stolosolytica会感知其环境，这可能会提供有关E. istoloselytica在仅大约10％的感染中具有侵入性的E. istoloselytica的关键见解。 在AIM 2中成功地沉默将扩展我们的抗体研究的结果，并证明了凋亡细胞和/或细菌与表达SHEP的CHO细胞的结合，可以确定证明该蛋白质是粘附蛋白的作用。 尽管SHEP是领先的疫苗候选者，但其功能尚不清楚。因此，这将非常重要。 D.计划：AIM 1的吞噬作用研究的R01赠款是由NIAID在9/2008资助的。 因此，如果我被允许保留在鞋底赠款中，那么特定的目标将在来年完全改变。 新目标将重点放在Leishmanolysin的E. istolytica同源物上。 利什玛诺辛是利什曼原虫前毒剂的免疫主导表面蛋白，是一种M8型金属蛋白酶，可降解补体和免疫球蛋白。 有趣的是，果蝇直系同源蛋白蛋白在细胞分裂和细胞迁移中起关键作用。 我们确定了E. histoltica基因组中的两个Leishmanolysin同源物，我们将其命名为E. stolytica Leishmanolysin样蛋白1和2（EHLMLP-1和-2）。 EHLMLP-1基因不存在于Entamoeba dispar，这是一种与溶组织溶液性大肠杆菌密切相关的非致病性ameba。 蛋白质编码区域的蛋白质编码区域几乎没有差异。 这及其在利什曼原虫毒力中的重要性使得确定EHLMLP-1的功能以及它在E. stolytictica生物学中所扮演的特定作用很重要。 我们计划：1）确定EHLMLP-1是否是细胞表面金属蛋白酶； 2）确定EHLMLP-1是否通过降解IgG并将补体C3B转换为IC3B来有助于免疫抗性； 3）确定EHLMLP -1和-2在细胞分裂和迁移中是否功能。 E.出版物（自2008年进度报告以来） Teixeira JE，Heron B，Huston CD。 肠道原生动物的溶作疗法对凋亡宿主细胞的C1q-和collectin依赖性吞噬作用。 传染病杂志。 2008。198：1062-1070。 Vaithilingam A，Teixeira JE，Huston CD。 Addenda文章：原生动物寄生虫结构的内质网连续性：进化的意义和警告性。 交流和综合生物学。 2008。1（2）：172-174。 指导摘要： Markus Thali博士 Thali博士每月与Huston博士会面，讨论研究和人员管理问题。他还曾在休斯顿博士的一名研究生（Brad Heron）的论文委员会任职，作为细胞和分子生物学研究生课程的主任，他对Huston博士实验室（Archana Vaithilingam）的另一名研究生的进步有些熟悉。此外，自2007年秋季以来，休斯顿和塔利团体每月一次开会一次共同会议。 Cory Teuscher博士 克里斯此前曾与研究生成功解决。我们多次讨论了他的兴趣，并渴望提交第二个R01申请。我们深入讨论了成功运营学术研究企业的两种基本方法：在单个领域/分子/途径中，高度专注于持续的生产力，而基于多个相互非排除的利益和专业知识领域。讨论了每种方法的优势和劣势，特别是从有临床责任的初级教职员工的角度来看。 加里·沃德博士 沃德博士每年一次与休斯顿博士会面，讨论数据和休斯顿博士工作的整体方向，并且在出现相关研究或人员管理时，更频繁地在非正式的基础上进行了讨论。 沃德博士批判性地阅读并提供了休斯顿博士的手稿和赠款申请的反馈，并在休斯顿博士的硕士学生布拉德·赫伦（Brad Heron）的论文委员会任职。 沃德博士和休斯顿博士参加了每两周一次的联合实验室会议，该会议还包括Cobre Grant的其他初级调查员Matrajt博士。这次以数据为中心的会议具有很高的互动性，这是Huston博士及其学生和博士后在他们的研究过程中定期反馈的绝佳方法。 沃德博士努力介绍休斯顿博士，并促进他与校园或附近地区的其他地区的互动，这可能对他的研究有所帮助。例如，沃德博士最近提名休斯顿博士在一年一度的达特茅斯学院分子发病机理上讲话。他的演讲非常受欢迎，他现在在达特茅斯医学院的微生物学和免疫学系有许多新的联系。沃德博士还向休斯顿博士提供了一切机会，与来UVM的来访科学家和研讨会演讲者会面。