DEVELOPMENTAL BIOLOGY OF LEISHMANIA PROMASTIGOTES

利什曼原虫前鞭毛体的发育生物学

• 批准号: 2566728
• 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/2566728
• 关键词: Leishmania; N acetylglucosaminidase; Psychodidae; arthropod genetics; chitin; digestion; disease vectors; epitope mapping; gene mutation; glycolipids; host organism interaction; laboratory mouse; leishmaniasis; life cycle; proteolysis

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.

白蛉病媒的吸血中肠是一种潜在的致命的 利什曼原虫的环境由于蛋白水解酶的释放 参与血粉的消化 购买液化石油气大衣 前鞭毛体保护这些细胞免受蛋白水解攻击， 缺乏表面LPG的前鞭毛体突变体对 在吸血的中肠内杀死，而转染这些突变体 能够恢复液化石油气表达的基因拯救了它们的早期中肠 生存 在感染的周围形成围食膜（PM） 血粉似乎可以保护转化寄生虫不受诱导 因为用外源几丁质酶破坏PM， 在这些苍蝇中几乎完全和早期的感染消失。 一种新型 作用的PM在载体能力的建议，其中遏制 感染的血粉在围食内间隙内产生了一个 屏障肠道蛋白酶的快速扩散，并限制 这些酶的易受攻击形式的寄生虫， 生命周期过渡，在其表达液化石油气。 还使用LPG减突变体和它们的拯救的转染子， 证实LPG在中肠附着中的作用。 三个L。杜氏 所检测的突变体在体外不能附着于银足拟步行虫中肠， 未能维持感染的苍蝇后，通过消化的 血粉 一个转染子，其LPG表达已恢复 恢复到接近正常水平，显示中肠粘附能力恢复 无论是在体外还是在体内。 与此相反，GP 63的功能分析- 缺陷突变体未能揭示任何作用，这丰富的表面 蛋白酶在果蝇寄生虫的生命周期中的作用。 无影响 观察到无效突变对存活、转化、生长和 寄生虫在载体中肠的分化。相对于 在脊椎动物宿主中，无效突变体能够附着并在其中生存 小鼠腹腔巨噬细胞在体外，并产生进行性 BALB/c小鼠的皮肤病变。 然而， 对补体介导的裂解的抗性显著降低， 开始病变发展的能力被延迟。 明年的研究将集中在中肠的表征上 结合生物化学和分子生物学技术， 克隆技术