Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem)

We present a short insight into the problem of parasitophorous vacuole (PV) formation as a most peculiar kind of cell vacuolization occurring in the course of intracellular development of coccidian pathogens of the genera Eimeria, Isospora, Toxoplasma, Sarcocystis, Cryptosporidium, Epieimeria, and Karyolysus. The review focuses on the morpho-functional diversity of PVs in these parasites. By the present time, the PVs containing different parasite genera and species have been examined to different extent. The membrane of the PV (PVM) obviously derives from the host cell plasmalemma. But soon after parasite penetration, the morphofunctional organization and biochemical composition of the PVM drastically changes: its proteins are selectively excluded and those of the parasite are incorporated. As the result, the PV becomes not fusigenic for lysosomes or any other vacuoles or vesicles, because host cell surface markers necessary for membrane fusion are eliminated from the PVM during parasite invasion.The pattern of the PVs is parasite specific and demonstrates a broad diversity within the same genera and species and even at different stages of the endogenous development. The PV is far from being an indifferent membrane vesicle containing the parasite. Instead, it represents a dynamic system that reflects the innermost events of host-parasite relationships, thus promoting the accomplishing of the parasite life cycle, which, in its turn, is a necessary prerequisite of the parasite eventual survival as a species.     

Transmembrane insertion of the Toxoplasma gondii GRA5 protein occurs after soluble secretion into the host cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.     

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.     

Apicomplexa in mammalian cells: trafficking to the parasitophorous vacuole

Most Apicomplexa reside and multiply in the cytoplasm of their host cell, within a parasitophorous vacuole (PV) originating from both parasite and host cell components. Trafficking of parasite-encoded proteins destined to membrane compartments beyond the confine of the parasite plasma membrane is a process that offers a rich territory to explore novel mechanisms of protein–membrane interactions. Here, we focus on the PVs formed by the asexual stages of two pathogens of medical importance, Plasmodium and Toxoplasma . We compare the PVs of both parasites, with a particular emphasis on their evolutionary divergent compartmentalization within the host cell. We also discuss the existence of peculiar export mechanisms and/or sorting determinants that are potentially involved in the post-secretory targeting of parasite proteins to the PV subcompartments.     

The Toxoplasma gondii rhoptry protein ROP 2 is inserted into the parasitophorous vacuole membrane, surrounding the intracellular parasite, and is exposed to the host cell cytoplasm

The origin of the vacuole membrane surrounding the intracellular protozoan parasite Toxoplasma gondii is not known. Although unique secretory organelles, the rhoptries, discharge during invasion of the host cell and may contribute to the formation of this parasitophorous vacuole membrane (PVM), no direct evidence for this hypothesis exists. Using a novel approach we have determined that parasite-encoded proteins are present in the PVM, exposed to the host cell cytoplasm. In infected cells incubated with streptolysin-O or low concentrations of digitonin, the host cell plasma membrane was selectively permeabilized without significantly affecting the integrity of the PVM. Antisera prepared against whole parasites or a parasite fraction enriched in rhoptries and dense granules reacted with the PVM in these permeabilized cells, indicating that parasite-encoded antigens were exposed on the cytoplasmic side of the PVM. Parasite antigens responsible for this staining of the PVM were identified by fractionating total parasite proteins by SDS-PAGE and velocity sedimentation, and then affinity purifying "fraction-specific" antibodies from the crude antisera. Proteins responsible for the PVM- staining, identified with fraction-specific antibodies, cofractionated with known rhoptry proteins. The gene encoding one of the rhoptry proteins, ROP 2, was cloned and sequenced, predicting and integral membrane protein. Antibodies specific for ROP 2 reacted with the intact PVM. These results provide the first direct evidence that rhoptry contents participate in the formation of the PVM of T. gondii and suggest a possible role of ROP 2 in parasite-host cell interactions.     

Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma

The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.     

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.     

The Toxoplasma gondii parasitophorous vacuole membrane: transactions across the border

The obligate intracellular protozoan Toxoplasma gondii establishes its replication permissive niche within the infected host cell. This niche, the parasitophorous vacuole (PV), is delimited from the host cell cytoplasm by the PV membrane (PVM). In this chapter we highlight the roles of the PVM in the remodeling of host cell architecture, nutrient acquisition, the manipulation of signaling, and touch upon the potential roles in the parasite developmental cycle. We further present the PVM as a unique and dynamic "organelle" found only within the infected cell where it is established outside the parent organism. Despite its importance little is known about the biology of the PVM. There has, however, been a recent renewal of interest in the PVM, the study of which has become more tractable with the application of both classical approaches as well as genomic and proteomic analyses. In this review we discuss the diverse activities associated with the PVM and present pressing questions that remain to be elucidated regarding this enigmatic organelle.     

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.     

Inverted topology of the Toxoplasma gondii ROP5 rhoptry protein provides new insights into the association of the ROP2 protein family with the parasitophorous vacuole membrane

Toxoplasma gondii, as many intracellular parasites, is separated from the cytosol of its host cell by a parasitophorous vacuole membrane (PVM). This vacuole forms during host cell invasion and parasite apical organelles named rhoptries discharge proteins that associate with its membrane during this process. We report here the characterization of the rhoptry protein ROP5, which is a new member of the ROP2 family. Contrasting with what is known for other ROP2 family proteins, ROP5 is not processed during trafficking to rhoptries. We show here that ROP5 is secreted during invasion and associates with the PVM. Using differential permeabilization of infected cells, we have shown that ROP5 exposes its C-terminus towards the host cell cytoplasm, which corresponds to a reverse topology compared with ROP2 and ROP4. Taken together with recent modelling data suggesting that the C-terminal hydrophobic domain hitherto described as transmembrane may correspond to a hydrophobic helix buried in the catalytic domain of kinase-related proteins, these findings call for a reappraisal of the current view of ROP2 family proteins association with the PVM.     

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.     

Autophagic elimination of intracellular parasites: convergent induction by IFN-gamma and CD40 ligation?

Autophagy has recently been implicated in the immune elimination of the intracellular protozoan parasite, Toxoplasma gondii. Toxoplasma and other apicomplexan parasites actively invade host cells and form nonfusogenic parasitophorous vacuoles. Nevertheless, following entry into IFN-gamma-activated effector macrophages, vesiculation of the parasite vacuole or PV membrane ensues, in a process dependent upon the activity of p47 GTPases induced by IFN-gamma signaling. Subsequent disruption of the plasma membrane of the stripped parasites precedes autophagolysosomal elimination of T. gondii. In contrast, ligation of the CD40 receptor and autocrine signaling by TNF activate a seemingly distinct, p47 GTPase-independent mechanism leading to autophagic elimination of intracellular T. gondii, without prior disruption of the pathogen vacuole. Thus, two key pathways of the cell-mediated immune response, namely IFN-gamma and CD40/CD40L, trigger a common autophagolysosomal endpoint of parasite elimination, via distinct intermediary mechanisms.     

Invasion and Early Development of Sarcocystis muris (Apicomplexa,Sarcocystidae) in Tissue Cultures1

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30–45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Plasmodium berghei sporozoites in nonreplicative vacuole are eliminated by a PI3P‐mediated autophagy‐independent pathway

The protozoan parasite Plasmodium, causative agent of malaria, invades hepatocytes by invaginating the host cell plasma membrane and forming a parasitophorous vacuole membrane (PVM). Surrounded by this PVM, the parasite undergoes extensive replication. Parasites inside a PVM provoke the Plasmodium‐associated autophagy‐related (PAAR) response. This is characterised by a long‐lasting association of the autophagy marker protein LC3 with the PVM, which is not preceded by phosphatidylinositol 3‐phosphate (PI3P)‐labelling. Prior to productive invasion, sporozoites transmigrate several cells and here we describe that a proportion of traversing sporozoites become trapped in a transient traversal vacuole, provoking a host cell response that clearly differs from the PAAR response. These trapped sporozoites provoke PI3P‐labelling of the surrounding vacuolar membrane immediately after cell entry, followed by transient LC3‐labelling and elimination of the parasite by lysosomal acidification. Our data suggest that this PI3P response is not only restricted to sporozoites trapped during transmigration but also affects invaded parasites residing in a compromised vacuole. Thus, host cells can employ a pathway distinct from the previously described PAAR response to efficiently recognise and eliminate Plasmodium parasites.     

Host but not parasite cholesterol controls Toxoplasma cell entry by modulating organelle discharge

Host cell cholesterol is implicated in the entry and replication of an increasing number of intracellular microbial pathogens. Although uptake of viral particles via cholesterol-enriched caveolae is increasingly well described, the requirement of cholesterol for internalization of eukaryotic pathogens is poorly understood and is likely to be partly organism specific. We examined the role of cholesterol in active host cell invasion by the protozoan parasite Toxoplasma gondii. The parasitophorous vacuole membrane (PVM) surrounding T. gondii contains cholesterol at the time of invasion. Although cholesterol-enriched parasite apical organelles termed rhoptries discharge at the time of cell entry and contribute to PVM formation, surprisingly, rhoptry cholesterol is not necessary for this process. In contrast, host plasma membrane cholesterol is incorporated into the forming PVM during invasion, through a caveolae-independent mechanism. Unexpectedly, depleting host cell plasma membrane cholesterol blocks parasite internalization by reducing the release of rhoptry proteins that are necessary for invasion. Cholesterol back-addition into host plasma membrane reverses this inhibitory effect of depletion on parasite secretion. These data define a new mechanism by which host cholesterol specifically controls entry of an intracellular pathogen.     

Membrane potential changes after infection of monocytes by Toxoplasma gondii.

Membrane potential changes in host cell plasma membrane were analyzed and the parasitophorous vacuole membrane (PVM) potential was characterized after infection by Toxoplasma gondii. Human monocytes infested by T. gondii were stained with two membrane potential sensitive dyes, DiOC(6)(3) carbocyanine and DiSBAC(2)(3) bis-oxonol, before fluorescence emission analysis by confocal laser scanning microscopy. After 24 and 48 h of infection, 34 and 39%, respectively, of monocytes showed several parasites (from two to six) per cell. At these infection times, significant decreases in cytoplasmic emissions were observed for both DiOC(6)(3) and DiSBAC(2)(3). Thus, hyperpolarisation of the host plasma membrane would occur consecutively to infection. Inside the parasitophorous vacuole, the fluorescence intensity of DiOC(6)(3) and DiSBAC(2)(3) increased significantly from 6 to 24 h after infection and the PVM became less polarised. Involvement of different ATPases in the membrane potential of infected monocytes was evaluated with ouabain, DCCD, omeprazole and sodium orthovanadate, ATPase inhibitors. All inhibitors induced a depolarisation of the plasma membrane. In the parasitophorous vacuole compartment, DCCD, omeprazole and sodium orthovanadate but not ouabain caused a significant depolarisation of the PVM, suggesting that H(+), H(+)/K(+) and P-type ATPases were at the origin of the PVM potential. This is the first report showing the presence of ion transporters in the T. gondii PVM and the existence of at least two members of the P-type family of ion pumps: an electrogenic H(+)ATPase and an electroneutral H(+)/K(+) ATPase.     

The Spatiotemporal Dynamics and Membranous Features of the Plasmodium Liver Stage Tubovesicular Network

For membrane‐bound intracellular pathogens, the surrounding vacuole is the portal of communication with the host cell. The parasitophorous vacuole (PV) harboring intrahepatocytic Plasmodium parasites satisfies the parasites' needs of nutrition and protection from host defenses to allow the rapid parasite growth that occurs during the liver stage of infection. In this study, we visualized the PV membrane (PVM) and the associated tubovesicular network (TVN) through fluorescent tagging of two PVM‐resident Plasmodium berghei proteins, UIS4 and IBIS1. This strategy revealed previously unrecognized dynamics with which these membranes extend throughout the host cell. We observed dynamic vesicles, elongated clusters of membranes and long tubules that rapidly extend and contract from the PVM in a microtubule‐dependent manner. Live microscopy, correlative light‐electron microscopy and fluorescent recovery after photobleaching enabled a detailed characterization of these membranous features, including velocities, the distribution of UIS4 and IBIS1, and the connectivity of PVM and TVN. Labeling of host cell compartments revealed association of late endosomes and lysosomes with the elongated membrane clusters. Moreover, the signature host autophagosome protein LC3 was recruited to the PVM and TVN and colocalized with UIS4. Together, our data demonstrate that the membranes surrounding intrahepatic Plasmodium are involved in active remodeling of host cells.      

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.