Redistribution of plasma-membrane surface molecules during formation of the Leishmania amazonensis-containing parasitophorous vacuole

Leishmania amazonensis presents two developmental stages that gain access t o the host macrophage through phagocytosis. The protozoan resides in a memb rane-bound compartment, the parasitophorous vacuole (PV), which can fuse wi th the endocytic system. For evaluation of the parasite/host-cell interacti on process and of PV biogenesis, the two parasite forms or host-cell membra ne whose surface had previously been labeled with specific probes for lipid s, 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 formatio n. The fate of fluorescent probes was followed by confocal laser scanning m icroscopy. In host cells previously labeled with PKH26, DTAF and FITC-thios emicarbazide, which label membrane lipids, proteins, and sialoglycoconjugat es, respectively, interaction with both protozoan forms revealed that adhes ion to the macrophage was sufficient for labeling of the parasite surface. In addition, recently formed PVs displayed strongly labeled intravacuolar p arasites, except for amastigote-macrophage interaction in a DTAF-labeled ma crophage that displayed slight labeling of intravacuolar parasites, with th e membrane lining the PV evidently being stained. Therefore, the vacuole mo dulation presents some particularities such that different host-cell membra ne components may be selected, depending on the protozoan form involved. Th ereafter, amastigotes labeled with the probes mentioned above displayed a d iffuse labeling pattern after interaction with unlabeled macrophages, sugge sting the spreading of Leishmania surface molecules during the initial para site-invasion stages. In particular, intravacuolar DTAF-labeled amastigotes showed a delineating halo around the PV, with the intravacuolar parasite b eing partially labeled. Promastigotes could not be labeled with 5-(4,6-dich lorotriazinyl)aminofluorescein (DTAF) or with fluorescein-5-thiosemicarbazi de, but promastigotes labeled with PKH26 lost the fluorescent probe during the invasion process such that slightly labeled promastigotes were seen ins ide the PV. These observations indicate the existence of a dynamic process of exchange of membrane-associated glycoproteins and lipids between the par asite and the host cell.