Instituto de Biotecnología, Grupo de Bioquímica y Parasitología Molecular, Departamento de Parasitología, Universidad de Granada, Granada, Spain.
Instituto de Biopatología y Medicina Regenerativa, Universidad de Granada, Granada, Spain.
PLoS Negl Trop Dis. 2019 Feb 21;13(2):e0007163. doi: 10.1371/journal.pntd.0007163. eCollection 2019 Feb.
Trypanosoma cruzi is the obligate intracellular parasite that causes Chagas disease. The pathogenesis of this disease is a multifactorial complex process that involves a large number of molecules and particles, including the extracellular vesicles. The presence of EVs of T. cruzi was first described in 1979 and, since then, research regarding these particles has been increasing. Some of the functions described for these EVs include the increase in heart parasitism and the immunomodulation and evasion of the host immune response. Also, EVs may be involved in parasite adhesion to host cells and host cell invasion.
METHODOLOGY/PRINCIPAL FINDINGS: EVs (exosomes) of the Pan4 strain of T. cruzi were isolated by differential centrifugation, and measured and quantified by TEM, NTA and DLS. The effect of EVs in increasing the parasitization of Vero cells was evaluated and the ED50 was calculated. Changes in cell permeability induced by EVs were evaluated in Vero and HL-1 cardiomyocyte cells using cell viability techniques such as trypan blue and MTT assays, and by confocal microscopy. The intracellular mobilization of Ca2+ and the disruption of the actin cytoskeleton induced by EVs over Vero cells were followed-up in time using confocal microscopy. To evaluate the effect of EVs over the cell cycle, cell cycle analyses using flow cytometry and Western blotting of the phosphorylated and non-phosphorylated protein of Retinoblastoma were performed.
CONCLUSION/SIGNIFICANCE: The incubation of cells with EVs of trypomastigotes of the Pan4 strain of T. cruzi induce a number of changes in the host cells that include a change in cell permeability and higher intracellular levels of Ca2+ that can alter the dynamics of the actin cytoskeleton and arrest the cell cycle at G0/G1 prior to the DNA synthesis necessary to complete mitosis. These changes aid the invasion of host cells and augment the percentage of cell parasitization.
克氏锥虫是一种必需的细胞内寄生虫，可引起恰加斯病。该疾病的发病机制是一个多因素复杂的过程，涉及大量分子和颗粒，包括细胞外囊泡。克氏锥虫细胞外囊泡的存在于 1979 年首次被描述，此后，对这些颗粒的研究一直在增加。这些 EVs 具有多种功能，包括增加心脏寄生虫感染，以及免疫调节和逃避宿主免疫反应。此外，EVs 可能参与寄生虫与宿主细胞的黏附和宿主细胞的入侵。
方法/主要发现：用差速离心法分离克氏锥虫 Pan4 株 EVs（外泌体），用 TEM、NTA 和 DLS 进行测量和定量。评估 EVs 增加 Vero 细胞寄生虫感染的效果，并计算 ED50。通过台盼蓝和 MTT 检测等细胞活力技术以及共聚焦显微镜评估 EVs 诱导 Vero 和 HL-1 心肌细胞通透性变化。通过共聚焦显微镜跟踪 EVs 诱导的 Vero 细胞内 Ca2+动员和肌动蛋白细胞骨架破坏的时间过程。为了评估 EVs 对细胞周期的影响，通过流式细胞术和磷酸化和非磷酸化视网膜母细胞瘤蛋白的 Western blot 进行细胞周期分析。
结论/意义：用克氏锥虫 Pan4 株的游离体 EVs 孵育细胞，可引起宿主细胞发生多种变化，包括细胞通透性改变和细胞内 Ca2+水平升高，这可能改变肌动蛋白细胞骨架的动力学，并在 DNA 合成之前使细胞周期停滞在 G0/G1 期，以完成有丝分裂所必需的 DNA 合成。这些变化有助于宿主细胞的入侵，并增加细胞寄生虫感染的百分比。
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