Developmental Biology Of Leishmania Promastigotes

利什曼原虫前鞭毛体的发育生物学

• 批准号: 6668897
• 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/6668897
• 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; parasite infection mechanism; polymerase chain reaction; secretory protein; skin hypersensitivity; skin infection; transfection

Plasmid DNA encoding the Phlebotomus paptasi salivary protein SP15 was able to protect vaccinated mice challenged with parasites plus salivary gland homogenate (SGH). Vaccinated mice developed a strong DTH reaction to sand fly bites, and were protected against challenge using L. major plus SGH. The homologue of SP15 has been cloned from a salivary gland cDNA library of Phlebotomus perniciosus, the main vector of canine leishmaniasis in Mediterranean regions. Dogs immunized with SP15DNA develop a DTH response at the site of P. perniciosus bite. Naive dogs tranferred to endemic zone in southern France have been immunized with SP15 or control DNA and will be followed up over 12 months for seroconversion and/or development of visceral leishmaniasis. The analysis of Leishmania mutants deficient in LPG expression 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. 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. Small amounts of properly folded recombinant midgut galectin has been purified from a cell free expression system and used in binding assays to LPG or to cultured promastigotes of different species. Specific binding to L. major LPG was observed that did not occur with L. major mutants lacking the side-chain poly galactose epitope.

编码白蛉唾液蛋白SP15的质粒DNA能够保护用寄生虫加唾液腺匀浆（SGH）攻击的接种小鼠。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。从地中海地区犬利什曼病的主要传播媒介--恶性白蛉（Phlebotomus perniciosus）的唾液腺cDNA文库中克隆了SP15的同源物。用SP15 DNA免疫的狗在P. perniciosus叮咬部位产生DTH反应。转移到法国南部流行区的未处理犬已用SP15或对照DNA免疫，并将随访12个月以上，以观察血清转化和/或内脏利什曼病的发展。对LPG表达缺陷的利什曼原虫突变体的分析揭示了磷酸聚糖作为白蛉载体中的毒力分子的双重作用：分泌的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介导寄生虫与肠壁的附着，从而防止在血粉排泄期间感染的损失。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。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。长须龙少量正确折叠的重组中肠半乳糖凝集素已从无细胞表达系统中纯化，并用于与LPG或不同物种的培养前鞭毛体的结合测定。特异性结合L.观察到主要的LPG，而L.缺少侧链聚半乳糖表位的主要突变体。