Henriques C, Atella G C, Bonilha V L, de Souza W
Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Parasitol Res. 2003 Jan;89(2):123-33. doi: 10.1007/s00436-002-0728-y. Epub 2002 Sep 24.
One fundamental step of Leishmania-macrophage interaction is the phase of parasite internalization through an endocytic process, with the formation of the parasitophorous vacuole (PV). The present study analyzed this process using two approaches. First, to investigate the host cell proteins which take part in this compartment, the macrophage surface was biotinilated and allowed to interact with both Leishmania forms, the PV was then isolated, and the biotinilated proteins were analyzed by Western blot. The results obtained showed that the isolated PV from macrophages infected for 60 min with infective promastigotes displayed high molecular weight proteins, 220 kDa and 180 kDa, contrary to the isolated PV obtained from amastigotes. The isolated PV from amastigotes, after 60 min interaction, displayed a faint, biotinilated protein profile, in contrast to the PV containing amastigote which, after 30 min interaction, displayed a strong protein profile in the range of 120 kDa and 40-60 kDa. The biotinilated protein profile may represent proteins distributed in the PV membrane and may also correspond to biotinilated proteins incorporated by the intracellular parasite, as observed by confocal microscopy. In a second approach, to investigate the PV phospholipid composition, macrophages were incubated with (32)P, allowed to interact with the parasites, and the isolated PV was then processed for phospholipid analysis by thin layer chromatography and scintillation counting. An increase in the levels of lysophosphatidylcholine was observed in infected macrophages. The isolated PV from infective promastigotes and amastigotes, after 60 min interaction, displayed high levels of phosphatidylcholine. Then the PV was ruptured and the intravacuolar parasite's (32)P phospholipid composition was analyzed by TLC; and labeling of the parasites was found, suggesting that phospholipids from the macrophage are transferred to the parasite. Taken together, the results obtained show that several proteins and phospholipids found in the plasma membrane of the macrophage are also found in the PV compartment.
利什曼原虫与巨噬细胞相互作用的一个基本步骤是寄生虫通过内吞过程内化的阶段，同时形成吞噬泡（PV）。本研究使用两种方法分析了这一过程。首先，为了研究参与这个区室的宿主细胞蛋白，将巨噬细胞表面用生物素化，使其与利什曼原虫的两种形态相互作用，然后分离出吞噬泡，通过蛋白质免疫印迹法分析生物素化的蛋白。获得的结果表明，用感染性前鞭毛体感染60分钟的巨噬细胞分离出的吞噬泡显示出高分子量蛋白，220 kDa和180 kDa，这与从无鞭毛体获得的分离吞噬泡相反。无鞭毛体感染60分钟后分离出的吞噬泡显示出微弱的生物素化蛋白谱，而含有无鞭毛体的吞噬泡在相互作用30分钟后显示出在120 kDa和40 - 60 kDa范围内的强蛋白谱。生物素化蛋白谱可能代表分布在吞噬泡膜上的蛋白，也可能对应于细胞内寄生虫掺入的生物素化蛋白，这通过共聚焦显微镜观察到。在第二种方法中，为了研究吞噬泡的磷脂组成，将巨噬细胞用（32）P孵育，使其与寄生虫相互作用，然后分离出吞噬泡，通过薄层色谱法和闪烁计数法对其进行磷脂分析。在感染的巨噬细胞中观察到溶血磷脂酰胆碱水平增加。感染性前鞭毛体和无鞭毛体相互作用60分钟后分离出的吞噬泡显示出高水平的磷脂酰胆碱。然后使吞噬泡破裂，通过薄层色谱法分析泡内寄生虫的（32）P磷脂组成；发现了寄生虫的标记，表明巨噬细胞的磷脂被转移到了寄生虫。综上所述，获得的结果表明，在巨噬细胞质膜中发现的几种蛋白质和磷脂也存在于吞噬泡区室中。
