Genetic Manipulation of Entamoeba Virulence

内阿米巴毒力的基因操作

• 批准号: 6534324
• 负责人: SHARON L REED
• 金额: $ 22.61万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534324
• 关键词: Arenaviridae; Baculoviridae; Entamoeba histolytica; amebiasis; antibody specificity; antisense nucleic acid; biotechnology; cysteine endopeptidases; enzyme activity; enzyme inhibitors; extracellular matrix; genetic manipulation; host organism interaction; intracellular parasitism; pathologic process; protozoal infection; transfection /expression vector; transposon /insertion element; virulence

DESCRIPTION (from the applicant's abstract): Cysteine proteinases are key virulence factors of E. histolytica and play a central role in tissue invasion and disruption of host defenses. We have shown that purified cysteine proteinases of E. histolytica degrade components of the extracellular matrix and cleave IgG, IgA, the C3 and C5 components of complement, and the anaphylatoxins, C3a and C5a, limiting the host response to amebic infection. E. histolytica and E. dispar are morphologically identical with highly homologous genomes including cysteine proteinase genes, but only E. histolytica can invade the host. We propose to test the hypothesis that surface and extracellular cysteine proteinases are critical for amebic invasion with the following Specific Aims: Aim 1: We will test the hypothesis that the cysteine proteinases, which are critical to invasion, differ in their location, release, or specificity for substrates. These studies will identify the major extracellular proteinases, express active, recombinant cysteine proteinases, and identify differences in specificity against peptide and physiological substrates. Aim 2: We will test the hypothesis that inhibition of the key cysteine proteinases will block invasion. These studies will compare the effect of specific peptide inhibitors, antisense constructs, and insertional proteinase mutants on invasion. Aim 3: We will test the hypothesis that complementation of cysteine proteinase expression in proteinase-deficient strains will restore virulence. We will use selectable expression vectors to over express specific cysteine proteinases in E. dispar and L6 to evaluate the effect on invasion. These studies should further our understanding of an important virulence factor of E. histolytica and establish the key cysteine proteinases, which are linked to invasion and could be targets of novel drug therapy in the future.

描述(摘自申请者的摘要)：半胱氨酸蛋白酶是关键 溶组织性肠杆菌毒力因子及其在组织侵袭中的核心作用 以及破坏宿主防御系统。我们已经证明，提纯的半胱氨酸 溶组织埃希氏菌的蛋白酶降解细胞外基质成分 并切割补体的免疫球蛋白、免疫球蛋白A、补体的C3和C5成分，以及 过敏毒素，C3a和C5a，限制宿主对阿米巴感染的反应。E. 在形态上完全相同，具有高度同源性 包括半胱氨酸蛋白酶基因在内的基因组，但只有溶组织肠杆菌才能入侵 主持人。我们建议检验表面和细胞外的假设 半胱氨酸蛋白酶是阿米巴侵袭的关键，具有以下特征 具体目标： 目标1：我们将测试半胱氨酸蛋白酶的假设，半胱氨酸蛋白酶是 对入侵至关重要，在它们的位置、释放或对 底物。这些研究将确定主要的胞外蛋白水解酶， 表达活性的重组半胱氨酸蛋白酶，并鉴定其在 针对多肽和生理底物的特异性。 目的2：我们将检验抑制关键半胱氨酸的假设 蛋白水解酶可以阻止入侵。这些研究将比较 特异性多肽抑制物、反义构建物和插入蛋白水解酶 变种人入侵。 目的3：我们将检验半胱氨酸蛋白酶的互补作用假说 在缺乏蛋白酶的菌株中表达将恢复毒力。我们将使用 高效表达特异性半胱氨酸蛋白酶的可选择表达载体 E.dispar和L6，以评价其对入侵的影响。 这些研究应该会加深我们对一个重要的毒力因素的理解 并建立了关键的半胱氨酸蛋白酶，它们与 具有侵袭性，可能成为未来新型药物治疗的靶点。