Die parasitophore Vakuole des Mikrosporidiums Encephalitozoon cuniculi: Biogenese und Metabolitaustausch

Abstract

Microsporidia possess a unique invasion mechanism, which is based on the explosive extrusion of a hollow tube, the so called polar tube, from the microsporidian spore. The common infection model postulates that during extrusion the polar tube pierces the plasma membrane of an adjacent host cell and the infectious sporoplasma is susequently injected through the polar tube into the cytosol of the host cell. After invasion, microsporidia of the genus Encephalitozoon are very quickly surrounded by a parasitophorous vacuole membrane (PVM). Due to the special invasion mechanism of microsporidia and the absence of host cell marker proteins in the PVM directly after invasion (Fasshauer et al., 2005), it was unclear up to now, if the PVM is transferred with the sporoplasm and therefore derived from the microsporidium itself, or if the PVM is derived from the host cell. In this work, a selective labelling of either E. cuniculi membranes or the host cell plasma membrane was used to investigate microsporidia and host cell participation on PVM biogenesis. When E. cuniculi membranes were labelled with the fluorescent lipid BODIPY-500/510-C12-phosphocholine and the labelled spores were used to infect host cells, the fluorescent signal was only detected in the emerging meronts but not in the parasitophorous vacuole membrane. Therefore, E. cuniculi membranes do not significantly participate in the biogenesis of the PVM. In contrast, if the host cell plasma membrane was labelled with the raft and nonraft specific lipids DiIC16 and SPEEDY DiO, both tracers were detectable in the nascent vacuole membrane shortly after invasion. The lipids for the PVM thus originate from the host cell and raft as well as nonraft lipids participate in the biogenesis. To understand the chronology of PVM biogenesis, the invasion of E. cuniculi into living fluorescently labelled host cells was analysed by time lapse microscopy. When a polar tube striked a cell, a fluorescently labelled plasma membrane invagination emerged at the contact site shortly before PVM formation. This observation suggests a modification for the invasion mode of E. cuniculi. According to the new model, the plasma membrane is not pierced but forced to form a long channel-like invagination. Afterwards, the sporoplasm is not injected into the cytosol but into this invagination, followed by parasitophorous vacuole formation from plasma membrane material. Previous colocalization studies showed that the PVM is devoid of the transferrin receptor, a marker for early endosomes and the cell surface (Fasshauer et al., 2005). Since our investigations reveal that the PVM originates from the host cell plasma membrane, the absence or presence of other host cell surface proteins in the PVM was analyzed. A biotinylation of host cell surface proteins and subsequent infection with E. cuniculi showed that host cell surface proteins are absent from the majority (>90%) of the emerging vacuoles. It is likely that during PVM formation or in the first minutes after invasion an exclusion mechamism for host cell surface proteins exists. Exclusion of host cell proteins from the PVM in E. cuniculi is as in Toxoplasma gondii most likely responsible for the fusion incompetence with endosomes and lysosomes, since the PVM lacks proteins necessary for membrane fusion. The genome of E. cuniculi is with ~ 2.9 million basepairs one of the smallest eucaryotic genomes known so far. Due to adaptation to the intracellular lifestyle, E. cuniculi lacks many biosynthetic pathways, for instance amino acid biosynthesis (Katinka et al., 2001). This suggests that E. cuniculi is metabolically extremely dependent on its host cell. Nutrients initially have to pass the parasitophorous vacuole membrane to become available for E. cuniculi. Microinjection of different membrane impermeable dextran dyes and fluorescently labelled peptides into the cytosol of infected host cells revealed that the PVM possesses pores with an exclusion size of 3-10 kDa. The PVM should therefore be permeable to nutrients like carbohydrates, amino acids and ATP. The PVM permeability properties are independent of age or size of the vacuole. During intracellular development, E. cuniculi has thus permanent access to the host cell nutrient pool.