Forrest David M, Batista Michel, Marchini Fabricio K, Tempone Antonio J, Traub-Csekö Yara Maria
Laboratório de Biologia Molecular de Parasitas e Vetores, Instituto Oswaldo Cruz-Fiocruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil.
Laboratório de Ciências e Tecnologias Aplicadas em Saúde, Instituto Carlos Chagas-Fiocruz, Rua Prof. Algacyr Munhoz Mader 3775, Curitiba 81350-010, PR, Brazil; Plataforma de Espectrometria de Massas-RPT02H, Instituto Carlos Chagas-Fiocruz, Rua Prof. Algacyr Munhoz Mader 3775, Curitiba 81350-010, PR, Brazil.
J Proteomics. 2020 Sep 15;227:103902. doi: 10.1016/j.jprot.2020.103902. Epub 2020 Jul 14.
Leishmania infantum chagasi is the primary etiological agent of visceral leishmaniasis in Latin America, a lethal disease that afflicts hundreds of thousands of people worldwide every year. Previous studies have shown that the parasite releases microvesicles known as exosomes, which prolong and exacerbate infection in the vertebrate vector. However, little is known of their role in the insect vector, the sand fly Lutzomyia longipalpis. Exosomes were isolated from cultured L. i. chagasi in logarithmic (procyclic) (LOG) and stationary phase (metacyclic-like) (STAT) growth stages, which are the parasite stages found in the vector, and submitted to proteomic analysis. Our studies showed that exosomes from LOG and STAT L. i. chagasi display discrete protein profiles. The presence of approximately 50 known virulence factors was detected, including molecules for immunomodulation and evasion (GP63, EF1α, Oligopeptidase), increased pathogenicity (Casein kinase, KMP-11, Cysteine Peptidase and BiP) and parasite protection (Peroxidoxin). Additionally, the majority of ontological terms were associated with both exosome phases, and no substantial ontological enrichment was observed associated with any of the two exosomal stages. We demonstrated that LOG exosomes show a marked increase in protein number and abundance, including many virulence factors, compared to STAT L. i. chagasi exosomes. SIGNIFICANCE: The knowledge of the role of Leishmania exosomes on leishmaniasis opened up a new world of potential and complexity regarding our understanding of the disease. In Brazil the majority of visceral leishmaniasis cases are caused by the parasite Leishmania infantum chagasi and transmitted by the vector Lutzomyia longipalpis. While Leishmania exosomes were found to play an active role in the mammalian host, little is understood about their effects on the sand fly, or how they might impact on the insect infection by the parasite. For this reason, we isolated exosomes from two developmental stages of L. i. chagasi that occur within the insect with a view to identifying and describing the alterations they undergo. We have identified many hundreds of proteins within both exosome phases and have developed a structure by which to examine potential candidates. Our findings regarding the composition of the exosome proteome raise many questions regarding their function and provide compelling evidence that exosomes play an active role in the parasite's development within the sand fly.
婴儿利什曼原虫恰加斯亚种是拉丁美洲内脏利什曼病的主要病原体，这种致命疾病每年折磨着全球数十万人。先前的研究表明，该寄生虫会释放被称为外泌体的微泡，这些微泡会延长并加剧在脊椎动物宿主中的感染。然而，对于它们在昆虫宿主——长须罗蛉中的作用却知之甚少。从处于对数期（前循环期）（LOG）和稳定期（类后循环期）（STAT）生长阶段的培养婴儿利什曼原虫恰加斯亚种中分离出外泌体，这两个阶段是在昆虫宿主中发现的寄生虫阶段，并进行蛋白质组学分析。我们的研究表明，来自LOG期和STAT期婴儿利什曼原虫恰加斯亚种的外泌体呈现出不同的蛋白质谱。检测到大约50种已知的毒力因子，包括免疫调节和逃避相关分子（GP63、EF1α、寡肽酶）、致病性增强相关分子（酪蛋白激酶、KMP-11、半胱氨酸蛋白酶和BiP）以及寄生虫保护相关分子（过氧化物酶）。此外，大多数本体术语与两个外泌体阶段都相关，并且未观察到与任何一个外泌体阶段相关的显著本体富集。我们证明，与STAT期婴儿利什曼原虫恰加斯亚种外泌体相比，LOG期外泌体在蛋白质数量和丰度上显著增加，包括许多毒力因子。意义：利什曼原虫外泌体在利什曼病中的作用的相关知识为我们对该疾病的理解开启了一个充满潜力和复杂性的全新领域。在巴西，大多数内脏利什曼病病例是由婴儿利什曼原虫恰加斯亚种引起，并由长须罗蛉传播。虽然已发现利什曼原虫外泌体在哺乳动物宿主中发挥积极作用，但对于它们对沙蝇的影响，或者它们如何影响寄生虫对昆虫的感染却了解甚少。因此，我们从昆虫体内发现的婴儿利什曼原虫恰加斯亚种的两个发育阶段中分离出外泌体，旨在识别和描述它们所经历的变化。我们在两个外泌体阶段都鉴定出了数百种蛋白质，并构建了一个结构来研究潜在的候选蛋白。我们关于外泌体蛋白质组组成的发现引发了许多关于其功能的问题，并提供了令人信服的证据，证明外泌体在寄生虫在沙蝇体内的发育中发挥着积极作用。
