A forward genetics screen for genes regulating phagocytosis and signaling in Enta

描述(申请人提供)：溶组织内阿米巴是阿米巴痢疾和肝脓肿的病原体。由于这种病原体被归类为B类生物恐怖主义因子，因此了解调节其毒力的分子和细胞机制被提升为优先事项。可能在致病过程中起作用的两个细胞过程是吞噬作用和基于PI3K的信号转导。因此，更好地了解这些过程可能会揭示反阿米巴策略的新靶点。我们提出了一种正向遗传学方法，利用蛋白质的过度表达来识别调节吞噬作用和PI3-激酶信号的基因。在目标1中，我们将在可诱导表达系统中构建一个溶组织肠杆菌的cDNA文库，并将该文库导入溶组织肠杆菌滋养体中。然后，我们将使用基于人类红细胞内化(加载了代谢毒素Tubersidin)或第二种代谢毒素亚甲基二膦酸盐内化的筛选，从转化子群体中选择吞噬突变体和吞噬突变体。从幸存者身上重新分离表达质粒并对cDNA插入物进行测序将有助于鉴定调控这些内吞过程的基因。在目标2中，我们将从转化子群体中选择抗Wortmannin的突变体。Wortmannin是一种PI 3-激酶的抑制剂，表达增加水平的Wortmannin靶点或其下游效应物的细胞应该耐受更高水平的药物。对幸存者过度表达的cDNA的鉴定将揭示Wortmannin的靶标和PI 3-Kinase的下游效应因子。这将提供一个全球视野中的PI 3-激酶信号网络在组织溶解。这些研究的完成将为调节吞噬作用和PI 3-激酶信号的基因提供重要的洞察，这些基因是溶组织埃希氏菌的重要毒力功能。这些研究的完成还将导致开发一种新的研究工具，即可诱导的cdna表达文库。公共卫生相关性：溶组织内阿米巴是阿米巴痢疾和肝脓肿的病原体。由于这种病原体也被归类为B类生物恐怖主义因子，因此了解调节其毒力的分子和细胞机制被提升为优先事项。拟议中的研究将确定调节这种有机体的吞噬作用和信号转导的基因。由于这些细胞过程对溶组织埃希氏菌的毒力是重要的，这项工作将极大地提高我们对致病性的理解。

DESCRIPTION (provided by applicant): Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. Two cellular processes that may play a role in pathogenicity are phagocytosis and PI 3-kinase-based signal transduction. Therefore, a better understanding of these processes may reveal new targets for anti-amoebic strategies. We propose a forward genetics approach that uses overexpression of proteins to identify genes that regulate phagocytosis and PI 3-kinase signaling. In Aim 1, we will generate an E. histolytica cDNA library in an inducible E. histolytica expression system and transfect this library into E. histolytica trophozoites. We will then select for phagocytosis mutants and pinocytosis mutants from the population of transformants using screens based on the internalization of human red blood cells (loaded with a metabolic toxin, tubercidin) or on the internalization of a second metabolic toxin, methylenediphosphonate. Re-isolation of the expression plasmids from survivors and sequencing of the cDNA inserts will allow for the identification of genes that regulate these endocytic processes. In Aim 2, we will select for wortmannin-resistant mutants from the population of transformants. Wortmannin is an inhibitor of PI 3-kinases and cells that express increased levels of wortmannin targets or their downstream effectors should tolerate higher levels of the drug. Identification of cDNAs overexpressed in survivors will reveal targets of wortmannin and downstream effectors of PI 3-kinases. This will provide a global view of PI 3-kinase signaling networks in E. histolytica. Completion of these studies will provide significant insight into the genes that regulate phagocytosis and PI 3-kinase signaling, important virulence functions in E. histolytica. Completion of these studies will also result in the development of a new research tool, an inducible cDNA expression library. PUBLIC HEALTH RELEVANCE: Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess. Since this pathogen is also categorized as a Class B bioterrorism agent, there is elevated priority to understand the molecular and cellular mechanisms that regulate its virulence. The proposed studies will identify genes that regulate phagocytosis and signal transduction in this organism. Since these cellular processes are important to E. histolytica virulence, this work will significantly enhance our understanding of pathogenicity.