当前位置: X-MOL 学术J. Proteomics › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
SILAC-based quantitative proteomics reveals pleiotropic, phenotypic modulation in primary murine macrophages infected with the protozoan pathogen Leishmania donovani.
Journal of Proteomics ( IF 3.3 ) Pub Date : 2019-12-14 , DOI: 10.1016/j.jprot.2019.103617
Despina Smirlis 1 , Florent Dingli 2 , Pascale Pescher 3 , Eric Prina 3 , Damarys Loew 2 , Najma Rachidi 3 , Gerald F Späth 3
Affiliation  

Leishmaniases are major vector-borne tropical diseases responsible for great human morbidity and mortality, caused by protozoan, trypanosomatid parasites of the genus Leishmania. In the mammalian host, parasites survive and multiply within mononuclear phagocytes, especially macrophages. However, the underlying mechanisms by which Leishmania spp. affect their host are not fully understood. Herein, proteomic alterations of primary, bone marrow-derived BALB/c macrophages are documented after 72 h of infection with Leishmania donovani insect-stage promastigotes, applying a SILAC-based, quantitative proteomics approach. The protocol was optimised by combining strong anion exchange and gel electrophoresis fractionation that displayed similar depth of analysis (combined total of 6189 mouse proteins). Our analyses revealed 86 differentially modulated proteins (35 showing increased and 51 decreased abundance) in response to Leishmania donovani infection. The proteomics results were validated by analysing the abundance of selected proteins. Intracellular Leishmania donovani infection led to changes in various host cell biological processes, including primary metabolism and catabolic process, with a significant enrichment in lysosomal organisation. Overall, our analysis establishes the first proteome of bona fide primary macrophages infected ex vivo with Leishmania donovani, revealing new mechanisms acting at the host/pathogen interface. SIGNIFICANCE: Little is known on proteome changes that occur in primary macrophages after Leishmania donovani infection. This study describes a SILAC-based quantitative proteomics approach to characterise changes of bone marrow-derived macrophages infected with L. donovani promastigotes for 72 h. With the application of SILAC and the use of SAX and GEL fractionation methods, we have tested new routes for proteome quantification of primary macrophages. The protocols developed here can be applicable to other diseases and pathologies. Moreover, this study sheds important new light on the "proteomic reprogramming" of infected macrophages in response to L. donovani promastigotes that affects primary metabolism, cellular catabolic processes, and lysosomal/vacuole organisation. Thus, our study reveals key molecules and processes that act at the host/pathogen interface that may inform on new immuno- or chemotherapeutic interventions to combat leishmaniasis.

中文翻译:

基于SILAC的定量蛋白质组学揭示了感染原生动物病原体利什曼原虫多诺万尼的原代小鼠巨噬细胞的多效性,表型调节。

利什曼原虫病是主要的媒介传播的热带疾病,由利什曼原虫属的原生动物,锥虫等寄生虫引起,导致人类高发病率和高死亡率。在哺乳动物宿主中,寄生虫在单核吞噬细胞(尤其是巨噬细胞)中存活并繁殖。然而,利什曼原虫的潜在机制。影响他们的主人还不完全了解。在本文中,使用基于SILAC的定量蛋白质组学方法,记录了利什曼原虫donovani昆虫阶段前鞭毛体感染72小时后,原代骨髓衍生的BALB / c巨噬细胞的蛋白质组学改变。通过结合强阴离子交换和凝胶电泳分离对方案进行优化,该分离显示出相似的分析深度(总共6189个小鼠蛋白)。我们的分析揭示了响应多形利什曼原虫感染的86种差异调节蛋白(其中35种增加,51种减少)。蛋白质组学结果通过分析所选蛋白质的丰度得到了验证。细胞内利什曼原虫多诺万尼感染导致各种宿主细胞生物学过程发生变化,包括初级代谢和分解代谢过程,并显着丰富了溶酶体组织。总的来说,我们的分析建立了第一个真正的原代巨噬细胞蛋白质组,该蛋白在体外被利什曼原虫感染,揭示了作用于宿主/病原体界面的新机制。意义:对多发利什曼原虫感染后原代巨噬细胞中发生的蛋白质组变化知之甚少。这项研究描述了一种基于SILAC的定量蛋白质组学方法,以表征感染了多巴尼原鞭毛体72小时的骨髓衍生巨噬细胞的变化。随着SILAC的应用以及SAX和GEL分级分离方法的使用,我们已经测试了用于初级巨噬细胞蛋白质组学定量的新途径。此处开发的协议可适用于其他疾病和病理。此外,这项研究为感染巨噬细胞的“蛋白质组重编程”提供了重要的新启示,该巨噬细胞响应了影响初生代谢,细胞分解代谢过程和溶酶体/真空组织的L. donovani前鞭毛体。因此,我们的研究揭示了作用于宿主/病原体界面的关键分子和过程,这些分子和过程可能为抗击利什曼病的新的免疫或化学疗法干预提供了信息。
更新日期:2019-12-17
down
wechat
bug