Borges V M, Vannier-Santos M A, de Souza W
Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brasil.
Parasitol Res. 1998 Oct;84(10):811-22. doi: 10.1007/s004360050493.
The intracellular fate of human transferrin (HTf) in macrophages infected by Leishmania was investigated. Binding of HTf-gold complexes at 4 degrees C was competitively inhibited by native holoHTf but not by apoHTf. Infected and uninfected macrophages displayed rather distinct HTf trafficking. Pulse-chase experiments using uninfected macrophages loaded with 15-nm gold-conjugated bovine serum albumin (BSA) and then incubated with 5-nm gold-conjugated HTf revealed a remarkable segregation of these tracers in distinct compartments. Nevertheless, Leishmania-infected macrophages presented extensive particle colocalization at both 60 min and 18 h. Light and electron microscopy immunolabeling indicated that HTf was delivered to the parasitophorous vacuole, formed patches on the amastigote surface, and was endocytosed via the flagellar pocket. Double-staining assays showed the colocalization of biotinylated HTf and its receptor in association with the parasitophorous vacuole. To approach the Tf-binding sites of amastigotes we performed HTf-fluorescein isothiocyanate (FITC) assays. Staining was diffuse at 4 degrees C and punctate at 35 degrees C, and only the former was sensitive to ethidium bromide, indicating an eventual temperature-dependent endocytic process. Within parasites, HTf was found in cysteine-proteinase-rich structures, suggesting that the protein can be endocytosed by intracellular amastigotes and sorted to the parasite endosomal-lysosomal compartments rather than being recycled. The treatment of infected macrophages with holoHTf, but not apoHTf, promoted the parasite's intracellular survival. These results suggest that Leishmania amastigotes can exploit and subvert the host-cell endocytic system and indicate the role of Tf-carried iron in the outcome of leishmanial infection.
研究了利什曼原虫感染的巨噬细胞中人转铁蛋白（HTf）的细胞内命运。4℃时HTf-金复合物的结合受到天然全铁转铁蛋白的竞争性抑制，但脱铁转铁蛋白无此作用。感染和未感染的巨噬细胞表现出相当不同的HTf运输。使用负载15纳米金偶联牛血清白蛋白（BSA）的未感染巨噬细胞进行脉冲追踪实验，然后与5纳米金偶联的HTf一起孵育，结果显示这些示踪剂在不同区室中显著分离。然而，感染利什曼原虫的巨噬细胞在60分钟和18小时时均呈现广泛的颗粒共定位。光镜和电镜免疫标记表明，HTf被递送至寄生泡，在无鞭毛体表面形成斑块，并通过鞭毛袋被内吞。双重染色分析显示生物素化的HTf及其受体与寄生泡共定位。为了接近无鞭毛体的转铁蛋白结合位点，我们进行了HTf-异硫氰酸荧光素（FITC）分析。4℃时染色呈弥漫性，35℃时呈点状，只有前者对溴化乙锭敏感，表明最终存在温度依赖性的内吞过程。在寄生虫体内，HTf存在于富含半胱氨酸蛋白酶的结构中，这表明该蛋白可被细胞内无鞭毛体内吞，并分选至寄生虫的内体-溶酶体区室，而不是被循环利用。用全铁转铁蛋白而非脱铁转铁蛋白处理感染的巨噬细胞，可促进寄生虫在细胞内的存活。这些结果表明，利什曼原虫无鞭毛体可以利用和破坏宿主细胞的内吞系统，并表明转铁蛋白携带的铁在利什曼原虫感染结果中的作用。
