DEVELOPMENTAL BIOLOGY OF LEISHMANIA PROMASTIGOTES

利什曼原虫前鞭毛体的发育生物学

• 批准号: 5200423
• 负责人: D L SACKS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200423
• 关键词: Leishmania; N acetylglucosaminidase; Psychodidae; arabinose; chemical structure; chitin; disease vectors; epitope mapping; gene mutation; glycolipids; host organism interaction; in situ hybridization; leishmaniasis; life cycle; molecular cloning

Stage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course of L. major infection in a natural vector, Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associated with arabinose substitution of LPG side-chain oligosaccharides was detected on the surface of a low proportion of midgut promastigotes beginning on day five, and on up to 60% of promastigotes on days 10 and 15. In contrast, 100% of the parasites egested from the mouthparts during forced feeding of 15-day infected flies stained strongly for this epitope. A metacyclic-associated transcript (MAT-1) was used in in situ hybridization studies to demonstrate the positioning of metacyclics in the anterior gut. L. major mutants defective in the expression of side-chain sugars on LPG, which are involved in midgut adhesion, were obtained by negative selection using lectins and antibodies. The absence of specific sidechain sugars was shown by in vitro biosynthetic studies using the phosphoglycan backbone as acceptor to be due to the lack of a galactosyl transferase. Attempts to clone the transferase by functional complimentation of the defective gene have been frustrated by the high frequency of chromosomal integration of the cosmid in the rescued clones. The cosmid DNA has recently been recovered by restriction digests of total DNA from the rescued phenotypes, re-ligation into the cosmid vector for bacterial transformation and cloning. The importance of the sand fly peritrophic matrix to the transformation, growth and development of Leishmania major in P. papatasi was investigated. Sand flies were infected by membrane feeding on amastigotes containing whole blood, with or without added chitinase. Transformation to and growth of promastigotes were observed in dissected midguts from all control flies by 36 hrs, whereas no viable promastigotes were observed in midguts from the chitinase-treated flies. When chitinase-treated flies were infected with logarithmic phase promastigotes instead of amastigotes, viable midgut infections developed. These data suggest an essential role for the peritrophic matrix in protecting the parasite during its early stage of development from the potentially lethal activities of the bloodfed midgut.

阶段特异性分子标记和形态发生标记被用来跟踪 后环前鞭毛体出现、数量和位置的动力学 在L.在自然媒介中的主要感染， 帕帕塔斯白蛉 表面脂磷酸聚糖（LPG）在 转化的前鞭毛体被延迟至第3天，并继续被转化。 此后所有前鞭毛体都大量表达。 的表位 与LPG侧链寡糖的阿拉伯糖取代相关 在中肠前鞭毛体的低比例表面上检测到 在第5天开始，在第10天在多达60%的前鞭毛体上， 15. 相反，100%的寄生虫从口器排出 在强迫喂食15天的感染苍蝇期间， 表位在原位使用了一种与代谢环相关的转录本（MAT-1）， 杂交研究，以证明定位的metacyclics在 前肠 L. LPG上侧链糖表达缺陷的主要突变体， 参与中肠粘连的蛋白质，通过阴性 使用凝集素和抗体进行选择。 没有具体 侧链糖通过体外生物合成研究显示， 作为受体的磷酸聚糖骨架是由于缺乏半乳糖基 转移酶 尝试通过功能性克隆转移酶 缺陷基因的互补性受到了高 拯救克隆中粘粒的染色体整合频率。 粘粒DNA最近被限制性内切酶回收， 来自拯救的表型的总DNA，重新连接到粘粒载体中 用于细菌转化和克隆。 白蛉围食基质对转化的重要性， 主要利什曼原虫在帕帕塔斯体内的生长发育， 研究了 白蛉通过膜取食感染， 含有全血的无鞭毛体，添加或不添加几丁质酶。 解剖观察了前鞭毛体的转化和生长 36小时时，所有对照苍蝇的中肠，而没有存活的前鞭毛体 在几丁质酶处理的果蝇中肠中观察到。 当 几丁质酶处理的果蝇感染对数期 前鞭毛体而不是无鞭毛体，发展出可行的中肠感染。 这些数据表明围食基质在 保护寄生虫在其早期发展阶段， 吸血中肠的潜在致命活动。