Developmental Biology Of Leishmania Promastigotes

利什曼原虫前鞭毛体的发育生物学

• 批准号: 6503181
• 负责人: David 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/6503181
• 关键词: Leishmania; Psychodidae; clinical research; delayed hypersensitivity; disease vectors; flow cytometry; glycolipids; host organism interaction; human subject; interferon gamma; interleukin 2; laboratory mouse; leishmaniasis; life cycle; mutant; polymerase chain reaction; secretory protein; skin hypersensitivity; skin infection; transfection

We have developed the first reproducible model of Leishmania infection transmitted by actual sand fly bites, and have anlalyzed the host response in the skin at the site of an infective bite. Compared to naive mice, mice pre-exposed to the bites of uninfected flies showed a reduction in lesion pathology, a reduction in parasite load in the skin and a reduction in the ability to transmit Leishmania back to uninfected flies. The protection was associated not with the neutralization of an infection enhancing component, but with a strong saliva-specific DTH response at the site of the bite. This host response appears to reproduce the strong DTH reaction to sand fly bites that is well known amongst humans living in endemic regions. The protection in mice was also associated with a strong up-regulation of INF-g and IL-12 at the site of bite, suggesting that within this inflammatory setting, infected macrophages might be activated for early killing of the parasites. A salivary protein extracted from an SDS gel, having an apparent mol wt of 15kD (named SP15), was able to protect vaccinated mice challenged with parasites plus salivary gland homogenate. The aminoterminal sequence of the SP15 protein was determined by Edman degradation, and used to screen a 3-frame translation of a cDNA database originating from randomly picked clones from a unidirectionally cloned salivary gland cDNA library. The SP15 gene coding for the mature protein was inserted in the VR1020 vector, and the plasmid was used to vaccinate mice. Vaccinated mice developed a strong DTH reaction to sand fly bites, and were protected against challenge using L. major plus SGH. Leishmania promastigotes synthesize an abundance of phosphoglycans which are either present on the cell surface anchored by PI (lipophosphoglycan, LPG) or secreted as protein-containing glycoconjugates. The relative contributions of different phosphoglycan-containing molecules in Leishmania/sand fly interactions have been tested using mutants specifically deficient in either total phosphoglycans or LPG alone. These mutants have revealed dual roles for phosphoglycans as virulence molecules in the sand fly vector: the sereted molecules protect the parasite from proteolytic digestion in the bloodfed midgut, and the surface LPG mediates attachment of the parasite to the gut wall so as to prevent loss of infection during bloodmeal excretion. The role of LPG in mediating attachment to the midgut suggests that gut-associated lectins or lectin-like molecules, which have been described for sand flies, serve as parasite attachment sites, and that these molecules can vary between phlebotomine species. We have established cDNA libraries of sand fly midguts from newly emergent sand flies of different species, including P. papatasi, P. argentipes, P. sergenti, and Lutzomyia Longipaplpis. The libraries were constructed using the SMART cDNA library construction kit. The library of P. papatasi has been completed. About 500 randomly picked genes were sequenced from the 5' end of the gene followed by sequence similarity searches using the BLAST program. This was followed by blasting each sequence within the library with the remaining genes, thus grouping them in clusters. An important finding was the discovery of the gene encoding for galectin, a galactose-binding lectin. This gene was only found in the midgut of P. papatasi when compared to first round libraries of P. sergenti, P. argentipes and Lu. Longipalpis.

我们已经开发了第一个可重复的模型，利什曼原虫感染传播的实际沙蝇叮咬，并分析了主机的反应，在皮肤上的感染叮咬的网站。与未感染的小鼠相比，预先暴露于未感染苍蝇叮咬的小鼠显示病变病理学减轻，皮肤中寄生虫负荷减少，以及将利什曼原虫传播回未感染苍蝇的能力降低。这种保护与中和感染增强成分无关，但与叮咬部位的唾液特异性DTH反应有关。这种宿主反应似乎再现了对白蛉叮咬的强烈DTH反应，这在生活在流行地区的人类中是众所周知的。在小鼠中的保护作用也与叮咬部位的INF-g和IL-12的强烈上调有关，这表明在这种炎症环境中，感染的巨噬细胞可能被激活以早期杀死寄生虫。从SDS凝胶中提取的一种表观分子量为15 kD的唾液蛋白（命名为SP 15）能保护用寄生虫和唾液腺匀浆攻击的免疫小鼠。SP15蛋白的氨基末端序列通过Edman降解测定，并用于筛选源自从单向克隆的唾液腺cDNA文库随机挑选的克隆的cDNA数据库的3-框翻译。将编码成熟蛋白的SP15基因插入到VR 1020载体中，并将该质粒用于接种小鼠。接种疫苗的小鼠对白蛉叮咬产生了强烈的DTH反应，并被保护免受L。专业加SGH。利什曼原虫前鞭毛体合成丰富的磷酸聚糖，其存在于由PI锚定的细胞表面上（脂磷酸聚糖，LPG）或作为含蛋白质的糖缀合物分泌。利什曼原虫/白蛉相互作用中不同的含磷酸聚糖的分子的相对贡献已经使用特异性缺乏总磷酸聚糖或LPG的突变体进行了测试。这些突变体揭示了磷酸聚糖在白蛉载体中作为毒力分子的双重作用：sereted分子保护寄生虫免受血饲中肠中的蛋白水解消化，并且表面LPG介导寄生虫附着于肠壁，以防止在血粉排泄期间感染损失。LPG在介导附着到中肠中的作用表明，肠道相关的凝集素或凝集素样分子，这已被描述为白蛉，作为寄生虫附着位点，这些分子可以在白蛉物种之间变化。我们建立了新出现的不同种类白蛉中肠的cDNA文库，包括P. papatasi，P. argentipes，P. sergenti和Lutzomyia Longipaplpis。使用SMART cDNA文库构建试剂盒构建文库。P. papatasi的图书馆已经完成。从基因的5'端对约500个随机挑选的基因进行测序，然后使用BLAST程序进行序列相似性搜索。随后用剩余的基因爆破文库中的每个序列，从而将它们分组为簇。一个重要的发现是发现了半乳糖凝集素（一种半乳糖结合凝集素）的编码基因。当与P. sergenti、P. argentipes和Lu的第一轮文库相比时，该基因仅在P. papatasi的中肠中发现。长须龙