Observations by Immunofluorescence Microscopy and Electron Microscopy on the Cytopathogenicity of Naegleria Fowleri in Mouse Embryo-Cell Cultures

Abstract

IT has been suggested (Brown, , 1979 that Naegleria fowleri may destroy cultured mouse-embryo (ME) cells by a phagocytosis-like mechanism alone, although the evidence for this is not conclusive. ME cells in contact with vigorously motile trophozoites were seen by phase-contrast microscopy to suffer gradual loss of cytoplasm , but observations at high magnification were hampered by peripheral image halos that obscured fine detail and the process of engulfment itself could not be discerned. Further studies of the cytopathogenicity of N . fowleri were carried out, therefore, by immunofluorescence and electron-microscopic techniques designed to show clearly how amoebae attack and destroy mammalian cells in vitro. MATERIALS AND METHODS Cultures.Trophozoites of Naegleria fo wleri strain HB-1 were maintained at 37°C in Fulton's medium A (Fulton, 1970) and harvested for inoculation of cell cultures as described previously .Secondary ME-cell cultures and associated media were prepared according to . M E-cell coverslip cultures for immunofluorescence studies were established in Leighton tubes. For electron microscopy, the technique of Persijn and Scherft (1965) was used whereby ME-cell monolayers were cultured on 25 x 17-mm thin mica slips (Mica and Micanite Supplies Ltd, London) in 30-mm plastic petri dishes. The freshly seeded dishes were incubated for 2 days at 37°C in a humidified atmosphere of 5% (v/v) CO2 in air, then changed to maintenance medium (MM).Preparation of ME-cell antiserum. Two-day-old secondary ME-cell monolayers in 20-oz (568 ml) screw-capped glass bottles were trypsinised and the cells washed by centrifugation at 800 g for 5 min. in three changes of phosphate-buffered saline (PBS), pH 7.3. The cells were suspended at a concentration of 1 x 106/ml in 0.1% (v/v) formol saline and the suspension was divided into two equal parts. One part was sonicated for 2 min. in a Mullard 50-watt ultrasonic disintegrator (Medical and Scientific Equipment Ltd, Crawley, Sussex); the other was centrifuged at 800 g for 5 min. and the deposit resuspended in the sonicated preparation. The formolised antigen, containing whole cells and sub-cellular components, was stored at 4°C in small volumes for up to 18 days until required for inoculation of animals.New Zealand White rabbits were given intravenous inoculations of ME-cell antigen according to a schedule similar to that described previously for the preparation of amoeba-specific

In vitro Destruction of Nerve Cell Cultures by Acanthamoeba spp.: A Transmission and Scanning Electron Microscopy Study

“…In the present study, food-cups were observed on the surface of all 4 species of Acanthamoeba. The process of ingestion of target cells by Acanthamoeba, however, differed from that described for the free-living amoeba, N. fowleri, which have been shown to ingest target cells in a piece-meal fashion using food-cups by a process termed trogocytosis (Brown, 1979; Marciano-Cabral and John, 1983). In contrast, Acanthamoeba trophozoites ingested whole cells or cellular debris via food-cups, although these structures were neither as numerous nor as prominent as on Naegleria trophozoites (Marciano-Cabral, 1988).…”

Taking a bite: Amoebic trogocytosis in Entamoeba histolytica and beyond

“…These amoebae also ingested nucleated cells in a nibbling fashion, but did not ingest the nuclei (11, 12). The term trogocytosis was later coined in more detailed studies of the process in N. fowleri (Figure 3B) (13). After co-incubation with target cells, it was shown that N. fowleri trophozoites contained distinct “bites” of target cells, and in ultrastructural studies, target cell mitochondria could be identified within the ingested bites (13).…”

“…The term trogocytosis was later coined in more detailed studies of the process in N. fowleri (Figure 3B) (13). After co-incubation with target cells, it was shown that N. fowleri trophozoites contained distinct “bites” of target cells, and in ultrastructural studies, target cell mitochondria could be identified within the ingested bites (13). During N. fowleri trogocytosis and prior to target cell death, targeted cells appeared morphologically normal and their cell membranes remained intact.…”

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

“…In vitro, it has been shown that trophozoites destroy their target cells by a combination of phagocytosis and the cytolytic action of secreted enzymes (Visvesvara & Callaway, 1974). Brown (1978Brown ( , 1979 suggests that N. fowleri injures target cells by piecemeal ingestion of cells, a process he called 'trogocytosis', using 'food-cup' structures on its surface (Fulford & Marciano-Cabral, 1986). Furthermore, a diverse array of proteins has been associated with Naegleria-mediated cell lysis (Marciano-Cabral & Cabral, 2007).…”