LEISHMANIA/MACROPHAGE CELLULAR INTERACTIONS IN VITRO

我们的长期目标是阐明利什马尼亚是如何 感染巨噬细胞，以及这些寄生虫随后如何分化， 在这些吞噬细胞中生存和繁殖。了解蜂窝和 体外这种宿主-寄生虫相互作用的分子机制是 对于开发更有效的化学和/或免疫预防和 利什曼病的治疗。 亚马逊利什曼原虫分子以前被认为在 因此建议研究的巨噬细胞感染是：(1) 主要表面糖蛋白/锌蛋白酶(Gp63)；和(2)63kb 衣霉素(TM)耐药变异体的染色体外DNA环。更多 重点放在分子方法上。结果表明，L. 并对其进行了克隆和测序。 选择的gp63残基是通过定点突变修饰的 这个物种和另一个物种的基因。Gp63缺陷基因的转染法 带有突变基因的变异体允许在催化和 寄生重要残基。另外两个基因被发现在 野生型基因转染后63kb DNA环的功能分析 带有这种DNA的缺失克隆的细胞。其中一人被发现授予 衣霉素耐药，明显编码N-乙酰氨基葡萄糖-1- 磷酸转移酶(NAGTase)。利用该基因作为选择性标记， 另一个直接位于上游的基因被鉴定出来，并发现过度表达 一个36kd的蛋白质，与Zeta-晶体蛋白/NADPH对苯二酚同源 氧化还原酶(P36)。 提议继续上述工作，同时与 已建立的体外转染法和巨噬细胞感染系统 如下：(1)优化功能基因的转染率 分析gp63和其他相关分子；(2)进一步分析 催化、受体结合等功能的生物学重要性 利什曼病感染中gp63基序的研究：(3)进一步研究利什曼病 翻译后修饰语在调节中的功能意义 利用N-糖基化和GPI锚定突变体表达gp63； 试图通过负性和负性来识别额外的相关分子 阳性表型选择；(5)描述“无毒”的前鞭毛体- 作为细胞内缺陷突变体的衍生无性无鞭毛体 分化和/或复制。 这些拟议研究的结果将不仅有助于我们理解 利什曼原虫分子在其感染巨噬细胞时也具有可比性 其他生物系统中的分子。

Our long term objectives are to elucidate the questions of how Leishmania infect macrophages, and how these parasites subsequently differentiate, survive and multiply in these phagocytes. Understanding the cellular and molecular mechanisms of such host-parasite interactions in vitro is crucial for developing more effective chemo- and/or immuno-prophylaxis and therapy for leishmaniasis. Leishmania amazonensis molecules previously thought to be important in macrophage infection and thus proposed for investigation are: (1) the major surface glycoprotein/zinc proteinase (gp63); and (2) the 63 kb extrachromosomal DNA circles in tunicamycin (TM)-resistant variants. More emphasis has been placed on the molecular approaches. The gp63 gene of L. amazonensis was cloned and sequenced. Selected residues of gp63 were modified by site-specific mutagenesis of the genes from this and another species. Transfection of gp63-deficient variants with mutant genes allows the identification of catalytically and parasitically important residues. Two additional genes were identified in the 63 kb DNA circle by functional analysis after transfection of wildtype cells with deletion clones of this DNA. One was found to confer tunicamycin-resistance, apparently encoding N-acetylglucosamine-1- phosphate transferase (NAGTase). Using this gene as a selective marker, another gene immediately upstream was identified and found to overexpress a 36 kd protein, homologous to zeta-crystallin/NADPH quinone oxidoreductase (P36). It is proposed to continue the above-mentioned work in conjunction with the established in vitro transfection and macrophage infection systems as follows: (1) To optimize the efficiency of transfection for functional analysis of gp63 and other relevant molecules; (2) To further analyze the biological importance of catalytic, receptor-binding and other functional motifs of gp63 in leishmanial infection; (3) To further characterize the functional significance of posttranslational modifications in regulating gp63 expression by using N-glycosylation and GPI-anchor mutants; (4) Attempts to identify additional molecules of relevance by negative and positive phenotype selections; (5) To delineate "avirulent" promastigote- derived axenic amastigotes as mutants defective in intracellular differentiation and/or replication. The results of these proposed studies will help our understanding not only Leishmania molecules in their infection of macrophages but also comparable molecules in other biological systems.