Summary

使用共聚焦显微镜研究 利什曼原虫 的吞噬作用

Published: July 29, 2021
doi:

Summary

什曼原虫 感染中与吞噬作用相关的机制仍然知之甚少。在这里,我们描述了评估 利什曼原虫 与宿主细胞相互作用期间发生的早期事件的方法。

Abstract

吞噬作用是一个精心策划的过程,涉及不同的步骤:识别,结合和内化。专业吞噬细胞通过吞噬作用吸收 利什曼原虫 寄生虫,包括通过多个宿主细胞受体识别寄生虫表面上的配体。 利什曼原虫 与巨噬细胞膜的结合通过补体受体1型(CR1)和补体受体3型(CR3)和模式识别受体发生。脂磷聚糖(LPG)和63 kDa糖蛋白(gp63)是参与巨噬细胞 – 利什曼原虫 相互作用的主要配体。在宿主细胞受体最初识别寄生虫配体之后,寄生虫在寄生液泡内化,存活并繁殖。 利什曼原虫诱导的液泡的成熟过程涉及从细胞内囊泡中获取分子,包括单体G蛋白Rab 5和Rab 7,溶酶体相关膜蛋白1(LAMP-1),溶酶体相关膜蛋白2(LAMP-2)和微管相关蛋白1A / 1B-轻链3(LC3)。

在这里,我们描述了使用共聚焦显微镜评估 利什曼原虫 与宿主细胞相互作用期间发生的早期事件的方法,包括(i)结合(ii)内化和(iii)吞噬体成熟。通过增加围绕这些感染结果决定因素的知识体系,我们希望提高对 利什曼原虫 感染发病机制的理解,并支持最终寻找新的化疗靶点。

Introduction

利什曼病是一种被忽视的热带病,由 利什曼原虫属的原生动物寄生虫引起,导致脊椎动物宿主的广泛临床表现,包括皮肤利什曼病、皮肤黏膜利什曼病和内脏利什曼病1。世界卫生组织(WHO)估计,超过10亿人处于危险之中,每年报告的新病例超过100万2

利什曼原虫 属是专性细胞内原生动物,在宿主细胞内存活,包括单核细胞、巨噬细胞和树突状细胞3. 利什曼原虫 – 巨噬细胞相互作用是一个复杂的过程,通过直接相互作用或通过涉及补体受体的调理作用涉及多个宿主细胞受体和寄生虫配体45。经典的表面受体,如CR1,CR3,甘露糖 – 岩藻糖,纤连蛋白,Toll样和清道夫受体,介导寄生虫附着到巨噬细胞678。这些受体识别 利什曼原虫表面的分子,包括63 kDa糖蛋白(gp63)和糖脂脂脂磷酸聚糖(LPG)9。这些是前鞭毛虫表面最丰富的分子,在破坏宿主免疫反应中起着至关重要的作用,有利于在哺乳动物细胞中建立寄生虫感染10。在寄生虫表面配体与巨噬细胞受体结合后,F-肌动蛋白积聚在哺乳动物细胞表面,在寄生虫被吞噬时围绕它们。随后,这导致形成寄生虫诱导的隔室,称为寄生液泡(PV),其呈现吞噬溶酶体特征11。一旦进入这些吞噬溶酶体,寄生虫就会经历生存和繁殖所必需的几种改变3

PV的生物发生是一种高度调控的膜运输过程,对这种病原体12的细胞内存活至关重要。该区室的形成是由吞噬体和宿主内吞途径的区室之间的连续融合事件引起的。经典细胞生物学研究表明,PV的成熟涉及单体G蛋白Rab 5和Rab 7蛋白的获取,这主要与早期和晚期内体成熟有关,分别是13。此外,这些区室获取溶酶体相关膜蛋白1和2(LAMP 1,LAMP 2),溶酶体膜的主要蛋白质成分和微管相关蛋白1A / 1B-轻链3(LC3),自噬体标记物14。尽管有明显的相似性,但PV形成1516 的动力学和这些区室的形态因 利什曼原虫 物种而异。例如,由 墨西哥乳杆菌 亚马逊乳杆菌 引起的感染诱导形成含有大量寄生虫的大隔室17。相比之下,其他物种,如 巴西乳杆菌婴儿乳杆菌, 形成较小的液泡,通常每个液泡18中只含有一到两个寄生虫。

尽管对宿主细胞-利什曼原虫 相互作用有这种了解,但由宿主受体和寄生虫配体之间接触引发的初始事件尚未完全阐明。已知这些事件是寄生虫感染结果的决定因素,并且取决于寄生虫种类,招募以识别寄生虫的宿主细胞受体的类型以及巨噬细胞信号通路的激活1920。因此,必须鉴定参与 利什曼原虫诱导的PV的生物发生的分子,并确定这些分子在感染建立和结果中的作用。在这里,我们描述了一种监测 利什曼原虫吞噬过程中发生的早期事件的方法,包括结合,内化,吞噬体形成和成熟。这项工作有助于澄清PLC,Akt,Rab5,Rab7和LC3参与由不同 利什曼原虫 物种诱导的PP的形成。重要的是,该方案可用于研究参与PV成熟的其他蛋白质的参与。未来的研究将扩大利 什曼原虫 – 宿主细胞相互作用所涉及的机制的知识,并有助于设计新的化疗策略。

Protocol

细胞是在国家研究伦理委员会批准程序后从健康供体获得的(ID:94648218.8.0000.0040)。 1. 细胞培养 人单核细胞来源的巨噬细胞注意:要获得用于 体外 分化为巨噬细胞的人单核细胞,请从健康供体收集血液并纯化D.英语和B.R.Andersen21描述的外周血单核细胞(PBMC)。收集外周血(50毫升)后,将其倒入肝素化管中,然后在室温下在磷酸盐?…

Representative Results

本报告旨在评估从呈现 巴西乳杆菌 -LCL或 巴西乳杆菌-DL形式的CL的患者中分离出的巴西 乳杆菌吞噬过程中发生的早期事件。我们首先评估了 人单核细胞来源的巨噬细胞对 巴西猪笼草-LCL或巴西猪笼草-DL的结合和吞噬作用。数据显示, 巴西乳杆菌-LCL和 巴西乳杆菌-DL与巨噬细胞的结合相似(图1)。此外,没有观察到宿主细胞对 <…

Discussion

利什曼原虫 – 巨噬细胞相互作用是一个复杂的过程,涉及几个可能影响疾病发展的步骤5。为了更好地了解未被置 信化的利什曼原虫 和宿主细胞相互作用所涉及的机制,我们描述了一种方案,该方案采用共聚焦荧光显微镜来评估 利什曼原虫 感染早期至晚期的吞噬作用。使用涉及两个或多个荧光团的荧光技术来研究细胞生物学机制,包括免疫标记和荧光标记?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

我们感谢巴西菲奥克鲁斯巴伊亚的贡萨洛·莫尼兹研究所和显微镜系的援助。这项工作得到了国际志愿者协会-FIOCRUZ号79700287000的支持,P.S.T.V.持有CNPq(305235/2019-2)的研究生产力补助金。质粒由加利福尼亚州多伦多大学的毛里西奥·特雷比兹尼克提供。作者要感谢安德里斯·沃尔特的英语语言修订和手稿文案编辑帮助。

Materials

2-mercaptoethanol Thermo Fisher Scientific 21985023
AlexaFluor 488-conjugated goat anti-rabbit IgG Thermo Fisher Scientific Tem varios no site
anti-LC3 antibody Novus Biologicals NB600-1384
Bovine serum albumin (BSA) Thermo Fisher Scientific X
CellStripper Corning 25-056-CI
CellTracker Red (CMTPX) Dye Thermo Fisher Scientific C34552
Centrífuga Thermo Fisher Scientific
Ciprofloxacin Isofarma X
CO2 incubator Thermo Fisher Scientific X
Confocal fluorescence microscope (Leica SP8) Leica Leica SP8
Fetal Bovine Serum (FBS) Gibco 10270106
Fluorescence microscope (Olympus Lx73) Olympus Olympus Lx73
Gentamicin Gibco 15750045
Glutamine Thermo Fisher Scientific 35050-061
HEPES (N- 2-hydroxyethyl piperazine-N’-2-ethane-sulfonic acid) Gibco X
Histopaque Sigma 10771
M-CSF Peprotech 300-25
NH4Cl Sigma A9434
Normal goat serum Sigma NS02L
Nucleofector 2b Device Lonza AAB-1001
Nucleofector solution Lonza VPA-1007
Paraformaldehyde Sigma 158127
Phalloidin Invitrogen A12379
Phorbol myristate acetate (PMA) Sigma P1585
Phosphate buffer solution (PBS) Thermo Fisher Scientific 10010023
ProLong Gold Antifade kit Life Technologies P36931
Roswell Park Memorial Institute (RPMI) 1640 medium Gibco 11875-093
Saponin Thermo Fisher Scientific X
Schneider's Insect medium Sigma S0146
Sodium bicarbonate Sigma S5761
Sodium pyruvate Sigma S8636
Triton X-100 Sigma X

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Paixão, A. R., Dias, B. R. S., Palma, L. C., Tavares, N. M., Brodskyn, C. I., de Menezes, J. P. B., Veras, P. S. T. Investigating the Phagocytosis of Leishmania using Confocal Microscopy. J. Vis. Exp. (173), e62459, doi:10.3791/62459 (2021).

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