Toxoplasma invasion: the parasitophorous vacuole is formed from host cell plasma membrane and pinches off via a fission pore.

Abstract

Most intracellular pathogens avoid lysing their host cells during invasion by wrapping themselves in a vacuolar membrane. This parasitophorous vacuole membrane (PVM) is often retained, serving as a critical transport interface between the parasite and the host cell cytoplasm. To test whether the PVM formed by the parasite Toxoplasma gondii is derived from host cell membrane or from lipids secreted by the parasite, we used time-resolved capacitance measurements and video microscopy to assay host cell surface area during invasion. We observed no significant change in host cell surface area during PVM formation, demonstrating that the PVM consists primarily of invaginated host cell 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 corresponds well to the predicted surface area of the entire PVM (30-33 Lim2). The formation and closure ofa fission pore connecting the extracellular medium and the vacuolar space was detected as the PVM pinched off. This final stage of parasite entry was accomplished without any breach in cell membrane integrity.Toxoplasma gondii is an important pathogen of humans, causing congenital birth defects when acute infection occurs during pregnancy, and severe encephalitis in immunocompromised persons. Toxoplasmic encephalitis has recently emerged as one of the most serious opportunistic infections associated with AIDS (1, 2). Like other Apicomplexan parasites (including Plasmodium, the causative agent of malaria), T. gondii is an obligate intracellular parasite. During invasion, which is an active process distinctly different from phagocytosis (3), the parasite becomes surrounded by the PVM. The PVM pinches off from the host cell membrane at the end of invasion, to create a vacuole within which the parasite grows and replicates to continue the infection. As the interface between the parasite and the host cell, the PVM functions in metabolite uptake, nutrient transport, and protein trafficking (4-7).The origin of the PVM in cells infected with Toxoplasma and other Apicomplexan parasites is unknown, and has been the subject of considerable controversy (8-11). Two main models have been proposed to explain how the PVM is formed. In the bilayer insertion model, the PVM is thought to be formed from lipids that are secreted from apical organelles of the parasite and inserted into the host cell membrane during invasion. Evidence supporting this model includes the absence of host cell membrane proteins from the forming PVM (12-15), and the presence of membrane-like lamellar structures within and emanating from the parasite's apical secretory organelles (16)(17)(18)). An alternative model, induced invagination, proposes that the parasite induces the host cell membrane to invaginate to form the PVM. Evidence supporting this model includes the similar lipid compositions of the host cell membrane and the developing PVM (8). As discussed elsewhere (8, 9), neither model can be ...

Rhoptries: an arsenal of secreted virulence factors

“…Evacuoles are also seen during normal invasion and may be formed from the initial burst of secretion from the rhoptries that occurs in the initial stages of invasion [14] (Figure 1B). During this initial burst, the host plasma membrane is transiently disrupted potentially allowing access to the cytosol before the envelope reseals to envelope the parasite [15,16]. Based largely on these studies, it was proposed that: "secretion of rhoptries at the time of invasion served as a means to deliver effector proteins into the host cell" [14].…”

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

“…Joiner (36) and Sinai and Joiner (37) suggested that insertion of parasite lipids into the PVM might affect PVM interaction with other cell membranes, preventing fusion with endocytic compartments. Suss-Toby (38) reported that a small amount of parasite-derived material (0-18.5% of the total surface area of the PVM) may be inserted into the host cell plasma membrane. Our observations cannot definitively rule out bulk insertion of lipids We used DTAF, which reacts both with primary and secondary amine groups (20), to label membrane proteins of the macrophages before interaction with parasites in order to follow the fate of the labeled proteins during the parasite-host cell interaction process.…”

Conditional Expression ofToxoplasma gondiiApical Membrane Antigen-1 (TgAMA1) Demonstrates That TgAMA1 Plays a Critical Role in Host Cell Invasion

“…After the apical end of a parasite comes in contact with the host cell membrane, apical secretory organelles (micronemes and rhoptries) sequentially discharge their contents (Dubremetz et al, 1993;Carruthers and Sibley, 1997) and a zone of tight interaction forms between the two cells (Nichols and O'Connor, 1981;Dubremetz et al, 1985;Grimwood and Smith, 1995). An invagination in the host cell plasma membrane develops at the point of entry and progressively deepens, ultimately surrounding the fully internalized parasite (Suss-Toby et al, 1996).…”

“…After the apical end of a parasite comes in contact with the host cell membrane, apical secretory organelles (micronemes and rhoptries) sequentially discharge their contents (Dubremetz et al, 1993;Carruthers and Sibley, 1997) and a zone of tight interaction forms between the two cells (Nichols and O'Connor, 1981;Dubremetz et al, 1985;Grimwood and Smith, 1995). An invagination in the host cell plasma membrane develops at the point of entry and progressively deepens, ultimately surrounding the fully internalized parasite (Suss-Toby et al, 1996).Apical membrane antigen-1 (AMA1) is a microneme protein that is highly conserved among apicomplexan parasites (Waters et al, 1990;Donahue et al, 2000;Hehl et al, 2000;Gaffar et al, 2004). Since its initial discovery Ͼ15 years ago, AMA1 has received considerable attention as a malaria vaccine candidate (Deans et al, 1988;Collins et al, 1994;Anders et al, 1998;Kennedy et al, 2002;Stowers et al, 2002).…”