Henriques C, de Souza W
Laboratório de Ultraestructura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
Parasitol Res. 2000 Mar;86(3):215-25. doi: 10.1007/s004360050034.
Leishmania amazonensis presents two developmental stages that gain access to the host macrophage through phagocytosis. The protozoan resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which can fuse with the endocytic system. For evaluation of the parasite/host-cell interaction process and of PV biogenesis, the two parasite forms or host-cell membrane whose surface had previously been labeled with specific probes for lipids, proteins, and sialoglycoconjugates were allowed to interact for periods varying from 5 to 15 min for adhesion and from 30 to 60 min for PV formation. The fate of fluorescent probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, DTAF and FITC-thiosemicarbazide, which label membrane lipids, proteins, and sialoglycoconjugates, respectively, interaction with both protozoan forms revealed that adhesion to the macrophage was sufficient for labeling of the parasite surface. In addition, recently formed PVs displayed strongly labeled intravacuolar parasites, except for amastigote-macrophage interaction in a DTAF-labeled macrophage that displayed slight labeling of intravacuolar parasites, with the membrane lining the PV evidently being stained. Therefore, the vacuole modulation presents some particularities such that different host-cell membrane components may be selected, depending on the protozoan form involved. Thereafter, amastigotes labeled with the probes mentioned above displayed a diffuse labeling pattern after interaction with unlabeled macrophages, suggesting the spreading of Leishmania surface molecules during the initial parasite-invasion stages. In particular, intravacuolar DTAF-labeled amastigotes showed a delineating halo around the PV, with the intravacuolar parasite being partially labeled. Promastigotes could not be labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) or with fluorescein-5-thiosemicarbazide, but promastigotes labeled with PKH26 lost the fluorescent probe during the invasion process such that slightly labeled promastigotes were seen inside the PV. These observations indicate the existence of a dynamic process of exchange of membrane-associated glycoproteins and lipids between the parasite and the host cell.
亚马逊利什曼原虫呈现出两个发育阶段，它们通过吞噬作用进入宿主巨噬细胞。这种原生动物存在于一个膜结合的隔室，即寄生泡（PV）中，该寄生泡可与内吞系统融合。为了评估寄生虫/宿主细胞相互作用过程以及PV的生物发生，让两种寄生虫形式或先前已用脂质、蛋白质和唾液酸糖缀合物的特异性探针标记过表面的宿主细胞膜相互作用5至15分钟以进行黏附，30至60分钟以形成PV。通过共聚焦激光扫描显微镜追踪荧光探针的命运。在先前分别用PKH26、DTAF和FITC-氨基硫脲标记膜脂质、蛋白质和唾液酸糖缀合物的宿主细胞中，与两种原生动物形式的相互作用表明，与巨噬细胞的黏附足以标记寄生虫表面。此外，除了在DTAF标记的巨噬细胞中无鞭毛体与巨噬细胞的相互作用显示泡内寄生虫有轻微标记且PV内衬膜明显被染色外，新形成的PV显示泡内寄生虫有强烈标记。因此，液泡调节存在一些特殊性，即根据所涉及的原生动物形式可能会选择不同的宿主细胞膜成分。此后，用上述探针标记的无鞭毛体在与未标记的巨噬细胞相互作用后呈现出弥漫性标记模式，这表明利什曼原虫表面分子在寄生虫初始入侵阶段会扩散。特别是，泡内DTAF标记的无鞭毛体在PV周围显示出一个轮廓分明的光晕，泡内寄生虫被部分标记。前鞭毛体不能用5-（4,6-二氯三嗪基）氨基荧光素（DTAF）或荧光素-5-氨基硫脲标记，但用PKH26标记的前鞭毛体在入侵过程中失去了荧光探针，以至于在PV内可见轻微标记的前鞭毛体。这些观察结果表明在寄生虫和宿主细胞之间存在膜相关糖蛋白和脂质交换的动态过程。
