Leishmania-macrophage cellular interactions in vitro

描述：（由申请人提供）：我们的长期目标是 阐明利什曼原虫毒力的多个决定因素。具体阐述 这些寄生虫的侵入性/逃避性决定因素， 实现细胞内进入、存活和复制， 感染宿主细胞或巨噬细胞。 提出用于研究的相关利什曼原虫分子是核苷 二磷酸激酶（NdK）和可能的ATP酶。据推测，利什曼原虫的秘密 这些酶消耗细胞外ATP（eATP）积累的网站， 感染，从而防止eATP诱导的巨噬细胞及其 激活-对寄生虫生存有害的事件。NdK和ATP酶 在培养的寄生虫的用过的培养基中检测到活性。酶 部分纯化，并发现逆转ATP依赖的细胞溶解的J774 体外巨噬细胞。NdK基因在基因组中被组织为两个 不同的串联重复簇。用此转染野生型细胞 基因增加NdK的释放，促进它们感染巨噬细胞 in vivo. 建议继续进行以下研究：（1）进一步努力， 从利什曼原虫野生型细胞、ndk转染的利什曼原虫 和E.大肠杆菌转化子的生化和生物学特性， （2）对该酶的基因和基因间区进行了分析。 两个基因组簇的序列异质性和评估其 通过特别强调分泌的转染的差异功能 NdK：（3）进一步评价分泌型利什曼原虫的生物学功能 NdK作为eATP的清除剂用于：（A）减少eATP诱导的细胞因子的产生。 （B）保持这些的完整性 通过防止宿主细胞过早的eATP诱导的细胞溶解;和（4） 通过分子遗传学方法进一步评估利什曼原虫NdK功能。 了解这些和其他的细胞和分子机制 利什曼原虫的侵入性/逃避性决定因素将帮助我们更好地发展 治疗策略，以更有效地处理利什曼病。

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.