DEVELOPMENTAL BIOLOGY OF LEISHMANIA PROMASTIGOTES

以血为食的沙蝇媒介中肠具有潜在的致命性。 蛋白水解酶的释放为利什曼原虫创造了环境 与血粉的消化有关。石油气涂层的购置 前鞭毛体保护这些细胞免受蛋白分解攻击，因为 缺乏表面LPG的前鞭毛体突变体对 在血液喂养的中肠内杀死，而这些突变体的转基因 能够恢复LPG表达的基因拯救了它们的早期中肠 生死存亡。在感染者周围形成围食膜(PM) 血粉似乎能保护转化的寄生虫免受诱导 外源几丁质酶破坏质膜后的蛋白水解酶 在这些苍蝇身上几乎完全和早期地失去了感染。一本小说 提出了PM在媒介能力中的作用，其中遏制 受感染的血粉在围食期内空间内产生了 阻碍肠道蛋白水解酶的快速扩散，并限制 这些酶对易受感染的寄生虫的可及性 生命周期转变，在他们表达液化石油气之前。 LPG缺失突变体及其挽救的转染体也被用于 确认液化石油气在中肠附着中的作用。三种多诺瓦尼乳杆菌 被检测的突变体在体外未能附着在紫球藻的中肠上，以及 未能在苍蝇体内保持消化后的感染 血餐。1个转染体，其LPG表达已恢复 接近正常水平，显示中肠粘连能力恢复 在体外和体内都有。相比之下，GP63的功能分析- 缺乏突变体未能揭示这种丰富的表面的任何作用。 果蝇寄生虫生命周期中的蛋白水解酶。没有任何影响 在细胞的存活、转化、生长和生长过程中观察到零突变 媒介中肠内寄生虫的分化。关于 脊椎动物宿主，零突变体能够附着并在其中存活 小鼠腹膜巨噬细胞体外培养，并产生递增 BALB/c小鼠的皮肤损害。然而，零突变体是， 对补体介导的裂解的抵抗力显著降低，而且他们的 启动病变发展的能力被推迟。 明年的研究将集中在中肠的特性上。 生化与分子相结合的液化石油气受体 克隆技术。

The bloodfed midgut of sand fly vectors is a potentially lethal environment for Leishmania due to the release of proteolytic enzymes involved in bloodmeal digestion. Acquisition of the LPG coat on promastigotes protects these cells from proteolytic attack since promastigote mutants lacking surface LPG were highly susceptible to killing within the bloodfed midgut, whereas transfection of these mutants with genes capable of restoring LPG expression rescued their early midgut survival. Formation of a peritrophic membrane (PM) around the infected bloodmeal appeared to protect transforming parasites from induced proteases since disruption of the PM with exogenous chitinase resulted in almost complete and early loss of infection in these flies. A novel role for the PM in vector competence is suggested, in which containment of the infected bloodmeal within the intra-peritrophic space creates a barrier to the rapid diffusion of gut proteases, and limits the accessibility of these enzymes to the vulnerable forms of parasites in life-cycle transition, prior to their expression of LPG. LPG minus mutants and their rescued transfectants were also used to confirm the role of LPG in midgut attachment. Each of three L. donovani mutants examined failed to attach to P. argentipes midguts in vitro, and failed to maintain infection in the fly after passage of the digested bloodmeal. One transfectant, for which LPG expression had been restored to near normal levels, displayed restored capacity for midgut adhesion both in vitro and in vivo. In contrast, functional analysis of GP63- deficient mutants failed to reveal any role for this abundant surface protease in the life-cycle of the parasite in the fly. No effect of the null mutation was observed on the survival, transformation, growth, and differentiation of parasites in the vector midgut. With respect to the vertebrate host, null mutants were able to attach to and survive within mouse peritoneal macrophages in vitro, and to produce progressive cutaneous lesions in BALB/c mice. Null mutants were, however, significantly less resistant to complement-mediated lysis, and their ability to initiate lesion development was delayed. Studies in the next year will focus on characterization of midgut receptors for LPG using a combination of biochemical and molecular cloning techniques.