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The Parasitophorous Vacuole Is Formed from Host Cell Plasma Membrane and Pinches Off Via a Fission Pore (Cell Capacitance/Intracellular Parasite/Endocytosis) E

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The Parasitophorous Vacuole Is Formed from Host Cell Plasma Membrane and Pinches Off Via a Fission Pore (Cell Capacitance/Intracellular Parasite/Endocytosis) E Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8413-8418, August 1996 Cell Biology Toxoplasma invasion: The parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore (cell capacitance/intracellular parasite/endocytosis) E. SUSS-TOBY*, J. ZIMMERBERG*t, AND G. E. WARDt *Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, and *Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Louis H. Miller, National Institutes of Health, Bethesda, MD, May 1, 1996 (received for review March 10, 1996) ABSTRACT Most intracellular pathogens avoid lysing similar lipid compositions of the host cell membrane and the their host cells during invasion by wrapping themselves in a developing PVM (8). As discussed elsewhere (8, 9), neither vacuolar membrane. This parasitophorous vacuole membrane model can be rejected on the basis of the data thus far reported. (PVM) is often retained, serving as a critical transport The bilayer insertion'model predicts a significant increase in interface between the parasite and the host cell cytoplasm. To surface area during PVM formation, followed by a drop in test whether the PVM formed by the parasite Toxoplasma surface area as the PVM pinches off. The induced invagination gondii is derived from host cell membrane or from lipids model predicts no significant increase in surface area during secreted by the parasite, we used time-resolved capacitance PVM formation, followed by a drop in surface area as the PVM measurements and video microscopy to assay host cell surface pinches off. We report here experiments designed to directly area during invasion. We observed no significant change in test these predictions, using time-resolved capacitance mea- host cell surface area during PVM formation, demonstrating surements to measure host cell surface area (19) while simul- that the PVM consists primarily of invaginated host cell taneously monitoring invasion optically (20). membrane. Pinching off of the PVM from the host cell membrane occurred after an unexpected delay (34-305 sec) and was seen as a 0.219 ± 0.006 pF drop in capacitance, which MATERIALS AND METHODS corresponds well to the predicted surface area of the entire Host Cells and Parasites. COS-1 cells (ATCC CRL 1650) PVM (30-33 Lim2). The formation and closure ofa fission pore were cultured in complete Dulbecco's modified Eagle's me- connecting the extracellular medium and the vacuolar space dium (DMEM) at 37°C, released from the culture dishes by was detected as the PVM pinched off. This final stage of trypsinization (2 min in 0.25% trypsin, 1 mM EDTA) and parasite entry was accomplished without any breach in cell allowed to settle onto a 35 mm AT dish (Bioptechs, Butler, PA) membrane integrity. in complete DMEM for 30-90 min at 37°C prior to use. T gondii (RH strain) tachyzoites were cultured in human foreskin Toxoplasma gondii is an important pathogen of humans, caus- fibroblasts (ATCC CRL 1634) as described (3). Spontaneously ing congenital birth defects when acute infection occurs during released parasites from a massively infected monolayer (mul- pregnancy, and severe encephalitis in immunocompromised tiplicity of infection -10) were passed twice through a 27G persons. Toxoplasmic encephalitis has recently emerged as one needle, filtered through a 10-,um PCI7E filter (Poretics, Liv- of the most serious opportunistic infections associated with ermore, CA), spun for 5 min at 1100 x g, and resuspended in AIDS (1, 2). Like other Apicomplexan parasites (including bath solution (see below). Plasmodium, the causative agent of malaria), T. gondii is an Optical Visualization of Invasion. Experiments were per- obligate intracellular parasite. During invasion, which is an formed in bath solution at 35°C, using a Focht stage heating active process distinctly different from phagocytosis (3), the system (Bioptechs) on a Zeiss IM35 inverted microscope parasite becomes surrounded by the PVM. The PVM pinches equipped with differential interference contrast (DIC) and off from the host cell membrane at the end of invasion, to epifluorescence optics. The parasite suspension was loaded create a vacuole within which the parasite grows and replicates into a beveled parasite delivery pipette (15-,um opening), and to continue the infection. As the interface between the parasite delivered to the COS-1 cells using an Eppendorf 5242 micro- and the host cell, the PVM functions in metabolite uptake, injector. Microscope images were collected using a Dage-MTI nutrient transport, and protein trafficking (4-7). (Michigan City, IN) VE1000 SIT video camera, recorded on a The origin of the PVM in cells infected with Toxoplasma and Sony BVU-950 videocassette recorder, digitized using a PCVI- other Apicomplexan parasites is unknown, and has been the SIONplus capture board, and processed postcapture using subject of considerable controversy (8-11). Two main models Image-Pro Plus (Media Cybernetics, Silver Spring, MD). have been proposed to explain how the PVM is formed. In the For the fluorescence invasion assay, anti-P30 monoclonal bilayer insertion model, the PVM is thought to be formed from antibody (Biogenex Laboratories, San Ramon, CA) was con- lipids that are secreted from apical organelles of the parasite jugated with fluorescein as follows. Sodium bicarbonate (200 and inserted into the host cell membrane during invasion. IlI, 1M) and 6-(fluorescein-5-(and-6)-carboxamido) hexanoic Evidence supporting this model includes the absence of host acid, succinimidyl ester (100 Al, 10 mg/ml) were added se- cell membrane proteins from the forming PVM (12-15), and quentially to 1 ml (250 mg) of anti-P30, and incubated for 90 the presence of membrane-like lamellar structures within and min at 23°C. The reaction was stopped with hydroxylamine emanating from the parasite's apical secretory organelles (100 Al, 1.5 M, pH 8.0), incubated 30 min at 23°C, and passed (16-18). An alternative model, induced invagination, proposes through a PD-10 column (Pharmacia) equilibrated with phos- that the parasite induces the host cell membrane to invaginate phate buffered saline (PBS). The excluded fraction containing to form the PVM. Evidence supporting this model includes the the conjugate (1.4 ml) was collected, sterile filtered, and stored The publication costs of this article were defrayed in part by page charge Abbreviations: PVM, parasitophorous vacuole membrane; EOC, end payment. This article must therefore be hereby marked "advertisement" in of constriction; DIC, differential interference contrast. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8413 8414 Cell Biology: Suss-Toby et al. Proc. Natl. Acad. Sci. USA 93 (1996) at 4°C. To assay invasion using the conjugate, the microscope to quantitate capacitance changes during PVM formation (as stage heater was turned off, and the bath solution was replaced depicted in Fig. 2d, the PVM is formed during the period of with chilled (4°C) PBS containing 0.5% (wt/vol) bovine serum parasite constriction). In the first method, we assumed that any albumin (PBS/BSA). After 4 min, this solution was replaced lipid insertion by the parasite would occur at a linear rate, with 1.5 ml PBS/BSA containing 30 ,ul fluorescein-conjugated based on the constant rate at which the parasite penetrates into anti-P30, and incubated for 12 min at 23°C. The dish was the host cell (G.E.W., unpublished data). This assumption washed four times with chilled PBS/BSA (3 ml each for 3 min) allowed us to fit to our capa1citance data a continuous regres- and viewed with epifluorescence optics. sion curve consisting of two linear segments: one prior to the Electrophysiology. The bath solution contained: 118 mM spike, and one during the period of PVM formation. The NaCl/5.85 mM KCl/1.2 mM MgCl2/2.52 mM CaCl2/11 mM change in capacitance was calculated as the difference between glucose/5 mM Hepes (pH 7.4) with NaOH (21). The pipette the slopes of these two linear segments, multiplied by the solution for whole-cell recordings contained either 122 mM constriction time. By this method, host cell capacitance KCI, or 102 mM Kaspartate and 18 mM KCl, in addition to 2 changed an average of 0.000 ± 0.017 pF (SEM, n = 11). In the mM MgCl2/11 mM EGTA/1 mM CaCl2/5 mM Hepes/1 mM second method of analysis, we made no assumptions about the ATP/0.5 mM GTP (pH 7.26) with KOH. The pipette solution rate of potential lipid insertion. The average capacitance for the perforated patch contained 240 ,ug/ml amphotericin B during a 2-sec period immediately preceding the spike was in 130 mM methanesulfonate/10 mM KCl/8 mM MgCl2/10 subtracted from the average capacitance during the final 2 sec mM Hepes (pH- 7.3) with KOH. Patch pipettes (1.5 MOhm of parasite constriction. By this method, host cell capacitance resistance) were prepared from TW150-4 glass (World Preci- changed an average of 0.000 ± 0.023 pF (SEM; n = 11). sion Instruments, Sarasota, FL). Since the capacitance change associated with pinching off of The imaginary and real parts of the cell admittance were the PVM was found to be -0.219 pF (see below), these data calculated from the output current of an EPC7 patch clamp are clearly inconsistent (Student's t test, a = 0.005) with the amplifier (List Electronics, Darmstadt, Germany) in the volt- hypothesis that the PVM is composed primarily of newly age clamp mode, using a digital lock-in amplifier (software inserted bilayer, which would predict an increase in host cell available upon request). A 1000 Hz 80 mV peak-to-peak sine capacitance during invasion of this magnitude.
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