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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 A.具体目标 肠道阿米巴溶组织阿米巴吞噬宿主细胞的能力与寄生虫毒力相关，但这一过程的机制及其对发病机制的具体贡献仍不清楚。 去年的具体目标没有改变。 目标 1：检验溶组织内阿米巴具有对 C1q 胶原尾和集合素特异的吞噬受体的假设。 目标 2：检验富含丝氨酸的溶组织内阿米巴蛋白 (SREHP) 和/或相关的富含天冬酰胺的 Ariel 蛋白作为吞噬死亡细胞的受体的假设。 B.研究和结果 目标 1：正如去年所指出的，我们发现溶组织内阿米巴吞噬 C1q 包被的凋亡细胞比包被对照蛋白的凋亡细胞更有效。 为了跟进这一点，我们开发了一种通过生物素化感兴趣的蛋白质配体并使用它们包被链霉亲和素乳胶珠来构建“单配体”荧光颗粒的方法。 通过这个系统，我们证明了 C1q 本身就是溶组织内阿米巴吞噬作用的有效刺激剂。 甘露糖结合凝集素 (MBL) 是一种集合素家族成员，由于具有共同的胶原样尾部结构域而在结构上与 C1q 相关，它也刺激溶组织内阿米巴的吞噬作用，就像从 C1q 和表面活性剂蛋白 A (SP-A) 中纯化的胶原尾部一样）。 在 Transwell 实验中，溶组织内阿米巴也向这些蛋白质迁移。 这些调查结果于去年十月发表。 我们现在使用流式细胞术来测定 FITC 标记的集合素与溶组织内阿米巴表面的结合。 结果表明，C1q-FITC以可饱和的方式与阿米巴表面结合，并且C1q-FITC的结合可以部分与游离的MBL竞争。 这些数据提供了胶原集合素尾部特异的阿米巴受体的额外证据。 我们正在积极致力于识别这种受体。 我们的第一个方法是使用纯化的 C1q 作为交联实验中的诱饵，使用多功能交联剂 Sulfo-SBED，将生物素转移到附近的蛋白质上。 我们最初的实验并不成功，因为 C1q 诱饵形成多聚体，导致其他诱饵分子发生压倒性的生物素化。 使用纯化的胶原尾的类似方法可能有效，基于亲和力的方法也可能有效。 我们采取的另一种方法是使用流式细胞术对 C1q 包被珠的吞噬作用呈阳性或阴性的阿米巴滋养体进行分类。 我们将 C1q 包被珠子吞噬作用呈阳性的滋养体的膜蛋白谱与非吞噬滋养体和吞噬 BSA 包被对照珠子的滋养体的膜蛋白谱进行了比较。 在银染的 SDS-PAGE 凝胶上可以观察到特定膜蛋白的富集。 进一步优化我们的 FACS 分选方案后，我们将使用质谱法识别可重复的条带。 尽管我们发现 SP-A 的胶原尾显着刺激溶组织内阿米巴的吞噬作用，但完整的 SP-A 实际上抑制了吞噬作用。 SP-A 包被的颗粒的吞噬作用大约比牛血清白蛋白包被的珠子（BSA，阴性对照）的基础吞噬作用低五倍。 这提出了这样一种可能性：SP-A 的凝集素结构域与阿米巴表面的抑制性受体结合，而 SP-A 尾部通过常见的集合素受体刺激吞噬作用。 与这种可能性一致，SP-A 可以根据情况抑制或增强巨噬细胞的吞噬作用。 也就是说，在不存在细菌或细胞碎片的情况下，SP-A 通过其凝集素结构域结合抑制性受体，但在存在细菌或细胞碎片的情况下，它优先结合碎片，从而暴露其胶原尾并刺激吞噬作用。 我们进一步追求这种可能性的能力受到 SP-A 数量不足的限制。 因此，我们开发了一种从人肺癌细胞系中分离 SP-A 的方法，目前正在对其进行优化。 一旦我们有了足够量的 SP-A，我们计划通过细菌结合研究来确认其生物活性，然后用它来进一步检查 SP-A 是否具有刺激或抑制溶组织内阿米巴吞噬作用的能力，具体取决于局部环境。 目标 2：目标 2 的一些结果发表于 2008 年的一篇论文中，该论文描述了我们为鉴定吞噬作用抑制抗体而进行的单克隆抗体筛选。 在此筛选中，我们将 SREHP 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SREHP没有特异性结合活性。 SREHP是一种糖基化蛋白，因此我们想在中国仓鼠卵巢细胞表面表达它并进行结合研究。 为此目的，该基因已被克隆到载体pDisplay（包含哺乳动物信号肽和膜锚）中，但实验尚未完成。 此外，我们想要沉默阿米巴滋养体中 SREHP 的表达。 我们基于四种不同的 SREHP 序列（和一个乱序的对照载体）构建了四种用于表达小发夹 RNA 的质粒，但我们未能用这些构建体成功转染溶组织内阿米巴。 C. 意义：溶组织内阿米巴的吞噬能力与毒力相关，这些研究有望阐明其潜在机制。 目标 1 的重点是阐明触发溶组织阿米巴吞噬作用的凋亡细胞上的配体以及与其结合的阿米巴受体。 这些受体可能是包含在预防阿米巴病疫苗中的候选受体。 此外，如果我们确认 SP-A 根据具体情况抑制或增强阿米巴吞噬作用，那么它可能表明溶组织阿米巴感知其环境的全新机制，这可以为为什么溶组织阿米巴仅侵入约10%的感染率。 目标 2 中 SREHP 的成功沉默将扩展我们抗体研究的结果，并且凋亡细胞和/或细菌与表达 SREHP 的 CHO 细胞结合的证明将明确证明该蛋白作为粘附素发挥作用。 尽管 SREHP 是一种领先的候选疫苗，但其功能尚不清楚；因此，这将是非常重要的。 D. 计划：NIAID 于 9/2008 为目标 1 中的吞噬作用研究提供了 R01 资助。 因此，如果我能够继续获得 COBRE 补助金，那么来年的具体目标将完全改变。 新的目标将集中于溶组织内阿米巴与利什曼菌溶血素的同源物。 利什曼溶血素是利什曼原虫前鞭毛体的免疫显性表面蛋白，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，果蝇直系同源物入侵溶血素在细胞分裂和细胞迁移中发挥着关键作用。 我们在溶组织内阿米巴基因组中鉴定了两个利什曼溶血素同源物，我们将其命名为溶组织内阿米巴利什曼溶血素样蛋白1和2（EhLMLP-1和-2）。 EhLMLP-1 基因不存在于迪斯帕内阿米巴（Entamoeba dispar）中，迪斯帕内阿米巴是一种与溶组织内阿米巴密切相关的非致病性阿米巴。 溶组织内阿米巴和迪斯帕内阿米巴之间的蛋白质编码区几乎没有差异。 这以及利什曼原虫溶血素在利什曼原虫毒力中的重要性使得确定 EhLMLP-1 的功能及其在溶组织内阿米巴生物学中发挥的具体作用变得重要。 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转化为iC3b来促进免疫抵抗； 3) 确定EhLMLP-1和-2是否在细胞分裂和迁移中发挥作用。 E. 出版物（自 2008 年进度报告以来） 特谢拉 JE、赫伦 B、休斯顿 CD。 肠道原生动物溶组织内阿米巴对凋亡宿主细胞的 C1q 和集合素依赖性吞噬作用。 传染病杂志。 2008。198：1062-1070。 Vaithilingam A、Teixeira JE、休斯顿 CD。 附录文章：原生动物寄生虫溶组织内阿米巴的内质网连续性：进化意义和警告。 交流和整合生物学。 2008.1(2):172-174。 辅导总结： 马库斯·塔利博士 塔利博士每月与休斯顿博士会面，讨论研究和人事管理问题。他还曾担任休斯顿博士的一名研究生（Brad Heron）的论文委员会成员，并且作为细胞和分子生物学研究生项目的主任，他对休斯顿博士实验室的另一名研究生的进展有些熟悉（阿查纳·瓦蒂林加姆）。此外，自2007年秋季以来，休斯顿和塔利小组每月举行一次联合小组会议。 科里·特舍尔博士 克里斯之前曾与一名研究生发生过问题，但他成功解决了。我们多次讨论了他提交第二份 R01 申请的兴趣和愿望。我们深入讨论了成功运营学术研究企业的两种基本方法：高度专注于单一领域/分子/途径的持续生产力，与基础广泛的多个相互非排斥的兴趣和专业领域。讨论了每种方法的优点和缺点，特别是从具有临床责任的初级教员的角度。 加里·沃德博士 沃德博士每两年与休斯顿博士进行一次一对一的会面，讨论数据和休斯顿博士工作的总体方向，并且当出现与研究或人事管理相关的问题时，更频繁地进行非正式的会面。 沃德博士批判性地阅读休斯顿博士的手稿和资助申请并向其提供反馈，并在休斯顿博士的硕士生之一布拉德·赫伦的论文委员会任职。 Ward 博士和 Huston 博士参加每两周一次的联合实验室会议，其中还包括 COBRE 资助的其他初级研究员 Matrajt 博士。这次以数据为中心的会议具有高度互动性，对于休斯顿博士及其学生和博士后来说，这是定期接收有关其研究过程的反馈的绝佳方式。 沃德博士努力将休斯顿博士介绍给校园内或附近地区可能对他的研究有帮助的其他人，并促进他与其他人的互动。例如，沃德博士最近提名休斯顿博士在年度达特茅斯学院分子发病机制务虚会上发表演讲；他的演讲很受欢迎，现在他在达特茅斯医学院微生物学和免疫学系有了一些新的联系人。沃德博士还为休斯顿博士提供了与来 UVM 访问的科学家和研讨会发言人会面的一切机会。