Interactions between Toxoplasma gondii and its mammalian host cells

Toxoplasma is a protozoan parasite that is uniquely adapted for invading and surviving within a wide range of host cells. The parasite actively invades the cell, forming a novel vacuole that originates from the host cell plasma membrane. The vacuole membrane is rapidly modified to remove host cell proteins and this compartment subsequently resists fusion with all other host cell endocytic compartments. Shortly after invasion, the parasite secretes a variety of proteins by a process of regulation exocytosis and elaborates an extensive array of membranous tubules that form a network connecting with the vacuolar membrane. Understanding the formation and modification of this unique vacuole may reveal novel mechanisms for subverting host cell endocytic pathways that lead to intracellular survival.     

Cryptosporidium parvum attachment to and internalization by human biliary epithelia in vitro: a morphologic study

To explore the mechanisms by which Cryptosporidium parvum infects epithelial cells, we performed a detailed morphological study by serial electron microscopy to assess attachment to and internalization of biliary epithelial cells by C. parvum in an in vitro model of human biliary cryptosporidiosis. When C. parvum sporozoites initially attach to the host cell membrane, the rhoptry of the sporozoite extends to the attachment site; both micronemes and dense granules are recruited to the apical complex region of the attached parasite. During internalization, numerous vacuoles covered by the parasite's plasma membrane are formed and cluster together to establish a preparasitophorous vacuole. This preparasitophorous vacuole comes in contact with host cell membrane to form a host cell-parasite membrane interface, beneath which an electron-dense band begins to appear within the host cell cytoplasm. Simultaneously, host cells display membrane protrusion along the edge of the host cell-parasite membrane interface, resulting in the formation of a mature parasitophorous vacuole that completely covers the parasite. During internalization, vacuole-like structures appear in the apical complex region of the attached sporozoite, which bud out into host cells. A tunnel directly connecting the parasite to the host cell cytoplasm forms during internalization and remains when the parasite is totally internalized. Immunoelectron microscopy showed that sporozoite-associated proteins were localized along the dense band and at the parasitophorous vacuole membrane. These morphological observations provide evidence that secretion of parasite apical organelles and protrusion of host cell membrane play an important role in the attachment and internalization of host epithelial cells by C. parvum.     

Toxoplasma evacuoles: a two-step process of secretion and fusion forms the parasitophorous vacuole.

Rapid discharge of secretory organelles called rhoptries is tightly coupled with host cell entry by the protozoan parasite Toxoplasma gondii. Rhoptry contents were deposited in clusters of vesicles within the host cell cytosol and within the parasitophorous vacuole. To examine the fate of these rhoptry-derived secretory vesicles, we utilized cytochalasin D to prevent invasion, leading to accumulation of protein-rich vesicles in the host cell cytosol. These vesicles lack an internal parasite and are hence termed evacuoles. Like the mature parasite-containing vacuole, evacuoles became intimately associated with host cell mitochondria and endoplasmic reticulum, while remaining completely resistant to fusion with host cell endosomes and lysosomes. In contrast, evacuoles were recruited to pre-existing, parasite-containing vacuoles and were capable of fusing and delivering their contents to these compartments. Our findings indicate that a two-step process involving direct rhoptry secretion into the host cell cytoplasm followed by incorporation into the vacuole generates the parasitophorous vacuole occupied by TOXOPLASMA: The characteristic properties of the mature vacuole are likely to be determined by this early delivery of rhoptry components.     

Trypanosoma cruzi: mechanisms for entry into host cells

Infective trypomastigote stages of the obligate intracellular protozoan parasite Trypanosoma cruzi are capable of entering virtually any mammalian cell in vitro. Entry is a complex process, involving initial parasite attachment to surface moieties of the target cell, internalization of the parasite via formation of a vacuole, and finally disruption of the vacuolar membrane to permit access of the parasite to the host cell cytoplasm. Attachment requires parasite metabolic energy. At sites of parasite entry recruitment of host cell lysosomes may occur, and lysosomal membrane components contribute prominently to formation of the parasitophorous vacuole. Parasite escape from the vacuole depends upon vacuolar acidification and is mediated by the coordinated action of a parasite-derived neuramindase/trans-sialidase that is capable of desialylating host-derived vacuolar membrane constituents, and a parasite-derived trans-membrane pore-forming protein. Dissection of the entry process at both the organellar and molecular level is providing fundamental and complementary insights into microbial pathogenesis and cell biology.     

Proteases and chaperones are the most abundant proteins in the parasitophorous vacuole of Plasmodium falciparum-infected erythrocytes

After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle.     

Evidence for host cells as the major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells

The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.     

Cholesterol esterification by host and parasite is essential for optimal proliferation of Toxoplasma gondii.

Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.     

An electron microscopic study of Babesia microti invading erythrocytes

Summary Intracellular sporozoan parasites invade the host cell through the invagination of the plasma membrane of the host and a vacuole is formed which accommodates the entering parasite. The vacuole may disappear and the invaginated membrane of the host then becomes closely apposed to that of the parasite's own membrane. As a result the parasite is covered by two membranes. Members of the class Piroplasmea differ from other Sporozoa in that their trophozoites are covered by a single membrane. By screening numerous sections of intraerythrocytic Babesia microti belonging to the class Piroplasmea, it was found that merozoites of Babesia enter the erythrocytes of hamsters in the same way as those of other Sporozoa. When a merozoite touches the red blood cell with its anterior end it becomes attached to the membrane of the host, which starts to invaginate and a parasitophorous vacuole is formed. The vacuolar space disappears rapidly and the membrane of the vacuole and that of the parasite become closely adjacent. At this stage the parasite is surrounded by two plasma membranes. The outer membrane derived from the invaginated host membrane disintegrates quickly and the parasite is left with a single membrane throughout its life span.Supported by Grant AI 08989 from the U.S. Public Health Service. The excellent technical assistance of Ms. Renata Klatt is gratefully acknowledged     

The Toxoplasma gondii rhoptry protein ROP4 is secreted into the parasitophorous vacuole and becomes phosphorylated in infected cells

Many intracellular pathogens are separated from the cytosol of their host cells by a vacuole membrane. This membrane serves as a critical interface between the pathogen and the host cell, across which nutrients are imported, wastes are excreted, and communication between the two cells takes place. Very little is known about the vacuole membrane proteins mediating these processes in any host-pathogen interaction. During a screen for monoclonal antibodies against novel surface or secreted proteins of Toxoplasma gondii, we identified ROP4, a previously uncharacterized member of the ROP2 family of proteins. We report here on the sequence, posttranslational processing, and subcellular localization of ROP4, a type I transmembrane protein. Mature, processed ROP4 is localized to the rhoptries, secretory organelles at the apical end of the parasite, and is secreted from the parasite during host cell invasion. Released ROP4 associates with the vacuole membrane and becomes phosphorylated in the infected cell. Similar results are seen with ROP2. Further analysis of ROP4 showed it to be phosphorylated on multiple sites, a subset of which result from the action of either host cell protein kinase(s) or parasite kinase(s) activated by host cell factors. The localization and posttranslational modification of ROP4 and other members of the ROP2 family of proteins within the infected cell make them well situated to play important roles in vacuole membrane function.     

Overcoating of Toxoplasma Parasitophorous Vacuoles with Host Cell Vimentin Type Intermediate Filaments

The interaction between the Toxoplasma parasitophorous vacuole and vimentin-type intermediate filaments in Vero cells was investigated via immunofluorescence microscopy. A significant rearrangement of host cell vimentin around the Toxoplasma parasitophorous vacuoles occurs throughout the course of infection. Host cell vimentin associates with the parasitophorous vacuoles within an hour after invasion. This vimentin overcoating of the vacuole is initiated at the host cell nuclear surface. During parasite multiplication, vimentin retains a closely defined association with the cytosolic surface of the parasitophorous vacuole. In addition, the vimentin intermediate filaments originating from the host cell nuclear surface are progressively rearranged around the enlarging parasitophorous compartment. During infections, the order of vimentin cytoskeleton is normal throughout the cell and appears redefined only at the vicinity of the parasitophorous vacuole. Depolymerization of the intermediate filaments was achieved with the phosphatase inhibitors okadaic acid and calyculin A. Disruption of the intermediate filament networks resulted in displacement of the parasitophorous vacuoles from the host cell nuclear surface. The data indicate that host cell vimentin binds to the Toxoplasma parasitophorous vacuoles and that the host intermediate filament network serves to dock the parasite compartment to the host cell nuclear surface.     

Invasion factors are coupled to key signalling events leading to the establishment of infection in apicomplexan parasites

Invasion of host cells by apicomplexan parasites is initiated when specialized secretory organelles called micronemes discharge protein complexes onto the parasite surface in response to a rise in parasite intracellular calcium levels. The microneme proteins establish interactions with host cell receptors, engaging the parasite with the host cell surface, and signal for the immediate exocytosis of another set of secretory organelles named the rhoptries. The rhoptry proteins reprogram the invaded host cell and participate in the formation of the parasitophorous vacuole in which the intracellular parasite resides and replicates. Disengagement of the invading parasite from the host cell receptors involves the action of at least one parasite plasma membrane rhomboid protease, which is concomitantly implicated in a checkpoint that signals the parasite to switch from an invasive to a replicative mode.     

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.     

Toxoplasma gondii actively remodels the microtubule network in host cells

Toxoplasma gondii infection triggers host microtubule rearrangement and organelle recruitment around the parasite vacuole. Factors affecting initial stages of microtubule remodeling are unknown. To illuminate the mechanism, we tested the hypothesis that the parasite actively remodels host microtubules. Utilizing heat-killed parasites and time-lapse analysis, we determined microtubule rearrangement requires living parasites and is time dependent. We discovered a novel aster of microtubules (MTs) associates with the vacuole within 1h of infection. This aster lacks the concentrated foci of gamma (gamma)-tubulin normally associated with MT nucleation sites. Unexpectedly, vacuole enlargement does not correlate with an increase in MT staining around the vacuole. We conclude microtubule remodeling does not result from steric constraints. Using nocodazole washout studies, we demonstrate the vacuole nucleates host microtubule growth in-vivo via gamma-tubulin-associated sites. Moreover, superinfected host cells display multiple gamma-tubulin foci. Microtubule dynamics are critical for cell cycle control in uninfected cells. Using non-confluent monolayers, we show host cells commonly fail to finish cytokinesis resulting in larger, multinucleated cells. Our data suggest intimate interactions between T. gondii and host microtubules result in suppression of cell division and/or cause a mitotic defect, thus providing a larger space for parasite duplication.     

Selective inhibition of a two-step egress of malaria parasites from the host erythrocyte

Escape from the host erythrocyte by the invasive stage of the malaria parasite Plasmodium falciparum is a fundamental step in the pathogenesis of malaria of which little is known. Upon merozoite invasion of the host cell, the parasite becomes enclosed within a parasitophorous vacuole, the compartment in which the parasite undergoes growth followed by asexual division to produce 16-32 daughter merozoites. These daughter cells are released upon parasitophorous vacuole and erythrocyte membrane rupture. To examine the process of merozoite release, we used P. falciparum lines expressing green fluorescent protein-chimeric proteins targeted to the compartments from which merozoites must exit: the parasitophorous vacuole and the host erythrocyte cytosol. This allowed visualization of merozoite release in live parasites. Herein we provide the first evidence in live, untreated cells that merozoite release involves a primary rupture of the parasitophorous vacuole membrane followed by a secondary rupture of the erythrocyte plasma membrane. We have confirmed, with the use of immunoelectron microscopy, that parasitophorous vacuole membrane rupture occurs before erythrocyte plasma membrane rupture in untransfected wild-type parasites. We have also demonstrated selective inhibition of each step in this two-step process of exit using different protease inhibitors, implicating the involvement of distinct proteases in each of these steps. This will facilitate the identification of the parasite and host molecules involved in merozoite release.     

Toxoplasma gondii exploits host low-density lipoprotein receptor-mediated endocytosis for cholesterol acquisition

The obligate intracellular protozoan Toxoplasma gondii resides within a specialized parasitophorous vacuole (PV), isolated from host vesicular traffic. In this study, the origin of parasite cholesterol was investigated. T. gondii cannot synthesize sterols via the mevalonate pathway. Host cholesterol biosynthesis remains unchanged after infection and a blockade in host de novo sterol biosynthesis does not affect parasite growth. However, simultaneous limitation of exogenous and endogenous sources of cholesterol from the host cell strongly reduces parasite replication and parasite growth is stimulated by exogenously supplied cholesterol. Intracellular parasites acquire host cholesterol that is endocytosed by the low-density lipoprotein (LDL) pathway, a process that is specifically increased in infected cells. Interference with LDL endocytosis, with lysosomal degradation of LDL, or with cholesterol translocation from lysosomes blocks cholesterol delivery to the PV and significantly reduces parasite replication. Similarly, incubation of T. gondii in mutant cells defective in mobilization of cholesterol from lysosomes leads to a decrease of parasite cholesterol content and proliferation. This cholesterol trafficking to the PV is independent of the pathways involving the host Golgi or endoplasmic reticulum. Despite being segregated from the endocytic machinery of the host cell, the T. gondii vacuole actively accumulates LDL-derived cholesterol that has transited through host lysosomes.     

Nutrient acquisition by intracellular apicomplexan parasites: staying in for dinner

The intracellular forms of the apicomplexan parasites Plasmodium, Toxoplasma and Eimeria reside within a parasitophorous vacuole. The nutrients required by these intracellular parasites to support their high rate of growth and replication originate from the host cell which, in turn, takes up such compounds from the extracellular milieu. Solutes moving from the external medium to the interior of the parasite, are confronted by a series of three membranes --the host cell membrane, the parasitophorous vacuole membrane and the parasite plasma membrane. Each constitutes a potential permeability barrier which must be either crossed or bypassed. It is the mechanisms by which this occurs that are the subject of this review.     

ARF6, PI3-kinase and host cell actin cytoskeleton in Toxoplasma gondii cell invasion

Toxoplasma gondii infects a variety of different cell types in a range of different hosts. Host cell invasion by T. gondii occurs by active penetration of the host cell, a process previously described as independent of host actin polymerization. Also, the parasitophorous vacuole has been shown to resist fusion with endocytic and exocytic pathways of the host cell. ADP-ribosylation factor-6 (ARF6) belongs to the ARF family of small GTP-binding proteins. ARF6 regulates membrane trafficking and actin cytoskeleton rearrangements at the plasma membrane. Here, we have observed that ARF6 is recruited to the parasitophorous vacuole of tachyzoites of T. gondii RH strain and it also plays an important role in the parasite cell invasion with activation of PI3-kinase and recruitment of PIP₂ and PIP₃ to the parasitophorous vacuole of invading parasites. Moreover, it was verified that maintenance of host cell actin cytoskeleton integrity is important to parasite invasion.     

Toxoplasma gondii manipulates host cell signaling pathways via its secreted effector molecules

The obligate intracellular parasite Toxoplasma gondii secretes a vast variety of effector molecules from organelles known as rhoptries (ROPs) and dense granules (GRAs). ROP proteins are released into the cytosol of the host cell where they are directed to the cell nucleus or to the parasitophorous vacuole (PV) membrane. ROPs secrete proteins that enable host cell penetration and vacuole formation by the parasites, as well as hijacking host-immune responses. After invading host cells, T. gondii multiplies within a PV that is maintained by the parasite proteins secreted from GRAs. Most GRA proteins remain within the PV, but some are known to access the host cytosol across the PV membrane, and a few are able to traffic into the host-cell nucleus. These effectors bind to host cell proteins and affect host cell signaling pathways to favor the parasite. Studies on host–pathogen interactions have identified many infection-altered host signal transductions. Notably, the relationship between individual parasite effector molecules and the specific targeting of host-signaling pathways is being elucidated through the advent of forward and reverse genetic strategies. Understanding the complex nature of the host–pathogen interactions underlying how the host-signaling pathway is manipulated by parasite effectors may lead to new molecular biological knowledge and novel therapeutic methods for toxoplasmosis. In this review, we discuss how T. gondii modulates cell signaling pathways in the host to favor its survival.     

Histological and Ultrastructural Studies of the Interaction of Toxoplasma gondii Tachyzoites with Mouse Omentum in Experimental Infection1

Mouse omentum was studied after intraperitoneal challenge with tachyzoites of Toxoplasma gondii. Parasites inhabit omental histiocytes, fibroblasts, mesothelial cells, and free peritoneal macrophages. Recently infected cells showed enhanced metabolic and functional activity. Villous projections of the parasitophorous vacuole wall appeared, usually opposite the anterior pole of the parasite. In mesothelial cells, projections formed terminal swellings not observed in other infected cells. Activation of host cells was followed by reduction of the density of the cytoplasmic matrix, autophagosome formation, and intracellular edema, indicating the damage. The wall of the parasitophorous vacuole loses the supporting host cell endoplasmic reticulum that was attached to the vacuole just after entrance of the parasite into the cell. Then lysis of the parasitophorous vacuole and complete cell destruction occurs. The growth of parasites in undamaged cells does not coincide with the inflammatory response. Inflammation of the peritoneum develops only after the start of mass destruction of infected cells. Thus tachyzoites of Toxoplasma exert significant pathogenic effects by their ability to activate the host cell, causing lysis of the parasitophorous vacuole and subsequent destruction of the entire cell.