Invasion of Toxoplasma Gondii Occurs by Active Penetration of the Host Cell
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Journal of Cell Science 108, 2457-2464 (1995) 2457 Printed in Great Britain © The Company of Biologists Limited 1995 Invasion of Toxoplasma gondii occurs by active penetration of the host cell J. Hiroshi Morisaki1, John E. Heuser1 and L. David Sibley2,* 1Department of Cell Biology and Physiology, and 2Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110, USA *Author for correspondence SUMMARY Toxoplasma gondii is an obligate intracellular parasite that phagosome that formed over a period of 2-4 minutes. infects a wide variety of vertebrate cells including Phagocytosis involved both reorganization of the host macrophages. We have used a combination of video cytoskeleton and tyrosine phosphorylation of host proteins. microscopy and fluorescence localization to examine the In some cases, parasites that were first internalized by entry of Toxoplasma into macrophages and nonphagocytic phagocytosis, were able to escape from the phagosome by host cells. Toxoplasma actively invaded host cells without a process analogous to invasion. These studies reveal that inducing host cell membrane ruffling, actin microfilament active penetration of the host cell by Toxoplasma is funda- reorganization, or tyrosine phosphorylation of host mentally different from phagocytosis or induced endocytic proteins. Invasion occurred rapidly and within 25-40 uptake. The novel ability to penetrate the host cell likely seconds the parasite penetrated into a tight-fitting vacuole contributes to the capability of Toxoplasma-containing formed by invagination of the plasma membrane. In vacuoles to avoid endocytic processing. contrast, during phagocytosis of Toxoplasma, extensive membrane ruffling captured the parasite in a loose-fitting Key words: parasite, invasion, phagocytosis, endocytosis INTRODUCTION Internalization of antibody-coated Toxoplasma also leads to lysosome fusion in CHO cells transfected with FcRII receptors Toxoplasma is a protozoan parasite with an extremely broad by a process that it mediated by the FcR cytoplasmic tail host range (Dubey and Beattie, 1988) and unique capacity to (Joiner et al., 1990). The diametrically opposed fates of Toxo- invade virtually all nucleated cell types from warm-blooded plasma parasites that are phagocytosed versus those which vertebrate hosts (Werk, 1985). Early cinematographic studies invade actively, suggests that their respective fates are largely indicate that invasion proceeds rapidly and that during inter- determined by a critical difference at the time of internaliza- nalization the parasite passes through a visible constriction at tion (Jones and Hirsch, 1972; Sibley et al., 1985b; Joiner et al., the host cell plasma membrane (Hirai et al., 1966; Bommer, 1990). 1969; Nguyen and Stadtsbaeder, 1979). Electron microscopy A number of bacterial pathogens gain entry into vertebrate studies have revealed that invasion is an ordered process that cells by exploiting a normal host cell process for internaliza- initiates with binding of the parasite at its apical end followed tion (Falkow et al., 1992). Salmonella typimurium induces a by invagination of the host cell membrane to form a vacuole process similar to macropinocytosis, thereby enabling the surrounding the parasite (Jones et al., 1972; Aikawa et al., bacterium to enter cells which are normally not phagocytic, 1977; Nichols and O’Connor, 1981). Despite the efficiency such as epithelial cells (Francis et al., 1993; Alpuche-Aranda with which Toxoplasma enters its host cell, it remains unre- et al., 1994). To determine if a similar process of induced solved if invasion occurs by direct penetration of the parasite endocytic uptake might account for the ability of Toxoplasma or by induction of a host-mediated endocytic event. to enter cells, we have used time-lapse video microscopy to Toxoplasma is also able to survive in professional phago- examine the entry of Toxoplasma into phagocytic and non- cytes where it resides within a specialized compartment called phagocytic host cells. These studies reveal important func- the parasitophorous vacuole (PV) that avoids acidification tional differences between phagocytosis and Toxoplasma (Sibley et al., 1985b), and lysosome fusion (Jones and Hirsch, invasion and suggest that intracellular survival is dependent on 1972). Despite the ability of Toxoplasma to efficiently survive the active penetration of the host cell by the parasite. in a wide range of cell types, this delicate balance is tipped in favor of the phagocyte when parasites are opsonized with specific antibody. Phagocytosis of antibody-coated parasites MATERIALS AND METHODS by mouse macrophages leads to an elevated respiratory burst (Wilson et al., 1980), rapid acidification (Sibley et al., 1985a), Reagents and fusion with endosomes/lysosomes (Sibley et al., 1985a). Cell culture reagents were obtained from Gibco BRL (Gaithersburg, 2458 J. H. Morisaki, J. E. Heuser and L. David Sibley MD) except for FCS which was obtained from Hyclone (Logan, UT). RESULTS Chemicals were obtained from Sigma (St Louis, MO) unless otherwise noted. Time-lapse video microscopy was used to examine the inter- action of Toxoplasma with host cells in vitro and to analyze Parasite and cell cultures the mechanism(s) of cell entry. Internalization of Toxoplasma RH strain Toxoplasma tachyzoites were propagated by serial passage by BM cells in vitro occurred by two distinct processes that in human fibroblasts (HF) monolayers. HF and CHO cells were differed in kinetics, morphology, and intracellular fate of the cultured in 5% CO2 at 37°C in DMEM containing 10% FCS, 2 mM glutamine, and 20 µg/ml gentamicin. Bone marrow-derived vacuole. These two alternative forms of internalization are macrophages (BM) were obtained from the femurs of Balb/c mice and classified as ‘active invasion’ and ‘phagocytosis’ as described cultured in DMEM supplemented with 10% FCS and 20% condi- in detail below. tioned L929 cell medium. BM cells were used in the first or second passage after reaching confluence. Active invasion of Toxoplasma into BM and CHO Parasites were isolated from freshly lysed monolayers, separated cells using 3.0 micron polycarbonate filters, and washed in HBSS contain- When freshly lysed Toxoplasma parasites were added to mono- ing 1 mM EGTA and 10 mM HEPES. For opsonization experiments, layers of BM cells, many parasites remained loosely adherent parasites were incubated at room temperature for 30 minutes with to the cell surface without provoking a phagocytic response monoclonal antibody (mAb) DG52 or rabbit polyclonal antisera to p30 (RPcαp30) followed by extensive washing in PBS. and without entering the host cell. When invasion did occur, it was typically preceded by helical rotation and gliding In vitro invasion motility of the parasite across the substrate or cell surface Unlike receptor mediated phagocytosis, unopsonized Toxoplasma cells do not bind to host cells at temperatures below 20°C, conse- quently all invasion and phagocytosis experiments were conducted at 37°C. For video microscopy, monolayers cultured on coverslips were mounted in Ringer’s buffer containing 1% BSA on glass slides supported by lateral strips of vacuum grease (so called Zigmond chambers). Freshly isolated Toxoplasma parasites were used to infect these monolayers by infusing Ringer’s buffer containing parasites across the coverslip. The slide was incubated upside down at room temperature for 5 minutes to allow the parasites to settle on the monolayer and then re-righted and mounted on the microscope stage which was prewarmed to 37°C. Alternatively, monolayers grown on coverslips were infected at a low multiplicity of infection for 1 hour, washed, and recultured at 37°C overnight in DMEM 10% FCS. Following 18-36 hours of culture, coverslips were mounted on glass slides and examined for lysis of infected cells and invasion of new host cells within the same monolayer. Time-lapse video microscopy and image analysis Zigmond chamber slides were maintained at 37°C and examined by phase contrast microscopy using an upright microscope equipped with a 63× NA 1.4 objective lens. Active invasion was strongly inhibited when examined in bright transmitted light. For time-lapse recording, the microscope was equipped with a low-light level silicon-intensi- fied video camera (model C2400, Hamamatsu Photonics, Japan). Video images were averaged for 8 frames with an ARGUS-10 image processor (Hamamatsu) and stored at 1 frame/s on 12″ optical discs using a TQ-3038F recorder (Panasonic, Secaucus, NJ). Images were printed using a UP-870MD video printer (Sony Corp., Japan). Fluorescence localization of actin and Y-PO4 residues Monolayers grown on LabTek chamber slides were infected with freshly isolated Toxoplasma, rinsed, and fixed in 2.5% formalin and 0.1% glutaraldehyde in PBS for 10 minutes followed by extraction in cold acetone for 5 minutes. To examine the distribution of actin-rich microfilaments, monolayers were stained with bodipy phalloidin according to manufacturers instructions (Molecular Probes, Eugene, OR). Immunolocalization of actin was also performed on fixed, extracted monolayers using the mAb C4 (Boehringer Mannheim Co., Fig. 1. Active invasion of Toxoplasma into BM cells recorded by Indianapolis, IN) followed by FITC-conjugated goat anti-mouse IgG. time-lapse video microscopy. The parasite actively penetrates into a The distribution of phosphotyrosine residues was detected using the tight-fitting vacuole formed by invagination of the plasma mAb PY20 (ICN