Genetic Manipulation of Entamoeba Virulence

描述（来自申请人的摘要）：半胱氨酸蛋白酶是关键 E.并在组织侵袭中起中心作用 破坏宿主防御系统我们已经证明纯化的半胱氨酸 E.溶组织菌降解细胞外基质成分 并切割IgG、伊加、补体的C3和C5组分， 过敏毒素C3a和C5a，限制宿主对阿米巴感染的反应。E. histolytica和E. dispar在形态上与高度同源的 包括半胱氨酸蛋白酶基因，但只有E.溶组织菌可以侵入 主持人我们建议测试的假设，表面和细胞外 半胱氨酸蛋白酶对阿米巴入侵至关重要， 具体目标： 目的1：我们将测试假设，半胱氨酸蛋白酶，这是 关键的入侵，不同的位置，释放，或特异性， 印刷受体.这些研究将确定主要的胞外蛋白酶， 表达活性的重组半胱氨酸蛋白酶，并鉴定 对肽和生理底物的特异性。 目的2：我们将测试抑制关键半胱氨酸的假设， 蛋白酶将阻止入侵。这些研究将比较 特异性肽抑制剂、反义构建体和插入蛋白酶 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 在蛋白酶缺陷型菌株中表达将恢复毒力。我们将使用 可选择的表达载体，以过表达特定的半胱氨酸蛋白酶， E. dispar和L6来评估对侵袭的影响。 这些研究应该进一步我们了解一个重要的毒力因子 大肠并建立关键的半胱氨酸蛋白酶，这是连接 并可能成为未来新型药物治疗的靶点。

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.