Leishmania-macrophage cellular interactions in vitro

简介：(申请人提供)：我们的长远目标是 阐明利什曼原虫毒力的多个决定因素。具体说明 在这个提案中，是这些寄生虫入侵/逃避的决定因素 实现细胞内进入、生存和复制，从而成功 感染宿主细胞或巨噬细胞。 建议研究的利什马尼亚群岛病毒相关分子是核苷 二磷酸酶(NDK)，可能还有ATPase。据推测，利什马尼亚的秘密 这些酶耗尽细胞外的ATP(EATP)积累在 感染，从而阻止eATP诱导的巨噬细胞及其 激活--不利于寄生虫生存的事件。NDK和ATPase 在培养的寄生虫的废液中检测到活性。恩兹梅斯 部分纯化并发现能逆转J774细胞对ATP依赖的细胞溶解 巨噬细胞体外培养。NDK基因在基因组中被组织成两个 不同的串联重复簇。用此基因转导野生型细胞 基因促进NDK的释放，促进其感染巨噬细胞 在活体内。 建议继续进行如下研究：(1)进一步尝试 从野生型利什曼原虫细胞中纯化NDK亚型，转导NDK的利什曼原虫 和大肠杆菌转化子，用于生物化学和生物学特性 分泌酶基因及基因间隔区的分析 序列异质性的两个基因组簇及其评价 通过特别强调分泌的转染法来区分功能 NDK：(3)进一步评价利什曼原虫分泌物的生物学功能 NDK作为eATP的清道夫：(A)减少eATP诱导的 巨噬细胞杀灭微生物的自由基：和(B)保持这些自由基的完整性 宿主细胞通过防止eATP诱导的过早细胞溶解；以及(4) 用分子遗传学方法进一步评价利什曼原虫NDK功能。 了解这些和其他疾病的细胞和分子机制 利什曼病的侵袭性/回避性决定因素将帮助我们更好地发展 更有效地应对利什曼病的治疗策略。

DESCRIPTION: (provided by the applicant): Our long-term objectives are to elucidate multiple determinants of Leishmania virulence. Specifically addressed in this proposal are the invasive/evasive determinants of these parasites to achieve intracellular entry, survival and replication for a successful infection of their host cells or the macrophages. Leishmaniaamazonensis molecules of relevance proposed for study are nucleoside diphosphate kinases (NdK) and possibly ATPase. Presumably, Leishmania secret these enzymes to deplete extracellular ATP (eATP) accumulated at the site of infection, thereby preventing eATP-induced cytolysis of macrophages and their activation - events detrimental to the parasite survival. NdK and ATPase activities were detected in the spent medium of cultured parasites. The enyzmes were partially purified and found to reverse ATPdependent cytolysis of the J774 macrophages in vitro. The NdK genes are organized in the genome as two different tandem-repeated clusters. Transfection of wild type cells with this gene increases the release of NdK, facilitating their infection of macrophages in vivo. It is proposed to continue the studies as follows: (1) Further attempts to purify NdK isoforms from Leishmania wild type cells, ndk transfected Leishmania and E. coli transformants for biochemical and biological characterization of the secreted enzyme; (2) Analysis of the genes and intergenic regions in the two genomic clusters for sequence heterogeneity and assessment of their differential functions by transfection with particular emphasis on secretory NdK: (3) Further evaluating the biological functions of Leishmania secretory NdK as a scavenger of eATP for: (A) reducing the eATP-induced production of the microbicidal radicals by macrophages: and (B) preserving the integrity of these host cells by preventing their premature eATP-induced cytolysis; and (4) Further evaluation of Leishmania NdK functions by molecular genetic approaches. Understanding the cellular and molecular mechanisms of these and other invasive/evasive determinants of Leishmania will help us develop better therapeutic strategy to deal with leishmaniasis more effectively.