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To colonize phagocytes, Leishmania subverts microbicidal processes through components of its surface coat that include lipophosphoglycan and the GP63 metalloprotease. How these virulence glycoconjugates are shed, exit the parasitophorous vacuole (PV), and traffic within host cells is poorly understood. Here, we show that lipophosphoglycan and GP63 are released from the parasite surface following phagocytosis and redistribute to the endoplasmic reticulum (ER) of macrophages. Pharmacological disruption of the trafficking between the ER and the Golgi hindered the exit of these molecules from the PV and dampened the cleavage of host proteins by GP63. Silencing by RNA interference of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptors Sec22b and syntaxin-5, which regulate ER-Golgi trafficking, identified these host proteins as components of the machinery that mediates the spreading of Leishmania effectors within host cells. Our findings unveil a mechanism whereby a vacuolar pathogen takes advantage of the host cell's secretory pathway to promote egress of virulence factors beyond the PV.

Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo . Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body. Toxoplasma gondii is an intracellular parasite that reconfigures its host cell to promote pathogenesis. One consequence of Toxoplasma parasitism is increased migratory activity of host cells, which facilitates dissemination. Here, we show that Toxoplasma triggers the unfolded protein response (UPR) in host cells through calcium release from the endoplasmic reticulum (ER). We further identify a novel role for the host ER stress sensor protein IRE1 in Toxoplasma pathogenesis. Upon infection, Toxoplasma activates IRE1, engaging its noncanonical role in actin remodeling through the binding of filamin A. By inducing cytoskeletal remodeling via IRE1 oligomerization in host cells, Toxoplasma enhances host cell migration in vitro and dissemination of the parasite to host organs in vivo . Our study has identified novel mechanisms used by Toxoplasma to induce dissemination of infected cells, providing new insights into strategies for treatment of toxoplasmosis. IMPORTANCE Cells that are infected with the parasite Toxoplasma gondii exhibit heightened migratory activity, which facilitates dissemination of the infection throughout the body. In this report, we identify a new mechanism used by Toxoplasma to hijack its host cell and increase its mobility. We further show that the ability of Toxoplasma to increase host cell migration involves not the enzymatic activity of IRE1 but rather IRE1 engagement with actin cytoskeletal remodeling. Depletion of IRE1 from infected host cells reduces their migration in vitro and significantly hinders dissemination of Toxoplasma in vivo . Our findings reveal a new mechanism underlying host-pathogen interactions, demonstrating how host cells are co-opted to spread a persistent infection around the body.

Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.

A microbe and its host are in constant communication. An emerging platform for direct communication is the membrane contact sites that form between several pathogens and host organelles. Here, we review our progress on the molecular mechanisms underlying contact sites between host mitochondria and the human parasite Toxoplasma gondii . We discuss open questions regarding their function during infection as well as those formed between the host endoplasmic reticulum and Toxoplasma .

Despite marked reductions in morbidity and mortality in the last ten years, malaria still takes a tremendous toll on human populations throughout tropical and sub-tropical regions of the world. The absence of an effective vaccine and resistance to most antimalarial drugs available demonstrate the urgent need for new intervention strategies. Phosphoinositides are a class of lipids with critical roles in numerous processes and their specific subcellular distribution, generated through the action of kinases and phosphatases, define organelle identity in a wide range of eukaryotic cells. Recent studies have highlighted important functions of phosphoinositide kinases in several parts of the Plasmodium lifecycle such as hemoglobin endocytosis and cytokinesis during the erythrocytic stage however, nothing is known with regards to the parasite’s putative phosphoinositide phosphatases. We present the identification and initial characterization of a putative homologue of the SAC1 phosphoinositide phosphatase family. Our results show that the protein is expressed throughout the asexual blood stages and that it localises to the endoplasmic reticulum and potentially to the Golgi apparatus. Furthermore, conditional knockdown and knockout studies suggest that a minimal amount of the protein are likely required for survival during the erythrocytic cycle.

Key Points The endoplasmic reticulum (ER) is a crucial intracellular organelle that is involved in the biosynthesis of cellular molecules, including proteins, carbohydrates and lipids. These become constituents of the ER or are transported to the Golgi, plasma membrane, endosomes and lysosomes. The ER is involved in the transport of both MHC class I and class II complexes to the cell surface. It is the site of peptide loading onto MHC class I molecules, and it is important for regulation of the secretion of soluble immune mediators. Perturbation in ER–Golgi biogenesis affects antigen presentation and immune effector functions, so it is not surprising that the ER is a target for some pathogens. Several pathogens have developed strategies to modulate phagosome maturation and therefore avoid phagosome–lysosome fusion and the subsequent degradation of the pathogen: for example, by generating an ER-derived vacuole. In some cases, this has the added benefit of disrupting ER function. Toxoplasma gondii , Brucella spp. and Legionella pneumophila depend on ER-derived niches for growth, but the precise effects of these pathogen–ER interactions on the immune response are still unclear. Several viruses target the ER and interfere with antigen presentation by MHC class I molecules: for example, hepatitis C virus and human cytomegalovirus. Despite this, the ER contributes to host defence against microorganisms, through its role in autophagy, as well as through the ER stress response and the interferon-induced ER-resident proteins (such as viperin and the p47 GTPases). Different pathogens have evolved distinct strategies to promote their survival in host cells. This Review describes the contribution of the endoplasmic reticulum to host defence and the mechanisms by which pathogens interacting with the endoplasmic reticulum subvert the host immune response. A key determinant for the survival of intracellular pathogens is their ability to subvert the cellular processes of the host to establish a compartment that allows replication. Although most microorganisms internalized by host cells are efficiently cleared following fusion with lysosomes, many pathogens have evolved mechanisms to escape this degradation. In this Review, we provide insight into the molecular processes that are targeted by pathogens that interact with the endoplasmic reticulum and thereby subvert the immune response, ensure their survival intracellularly and cause disease. We also discuss how the endoplasmic reticulum 'strikes back' and controls microbial growth.

Toxoplasma gondii is an important zoonotic parasite with a worldwide distribution. It infects about one-third of the world's population, causing serious illness in immunosuppressed individuals, fetuses, and infants. Toxoplasma gondii biology within the host cell includes several important phases: (1) active invasion and establishment of a nonfusogenic parasitophorous vacuole in the host cell, (2) extensive modification of the parasitophorous vacuolar membrane for nutrient acquisition, (3) intracellular proliferation by endodyogeny, (4) egress and invasion of new host cells, and (5) stage conversion from tachyzoite to bradyzoite and establishment of chronic infection. During these processes, T. gondii regulates the host cell by modulating morphological, physiological, immunological, genetic, and cellular biological aspects of the host cell. Overall, the infection/development predispositions of T. gondii–host cell interactions overtakes the infection resistance aspects. Upon invasion and development, host cells are modulated to keep a delicate balance between facilitating and eliminating the infection.

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum.

Little is known about how the malaria parasite transports and targets proteins into the host erythrocyte. Parasite proteins exported into the host cell not only have to cross the parasite plasma membrane but also must traverse the parasitophorous vacuolar membrane (PVM) that surrounds the parasite. The PVM of Plasmodium chabaudi-infected erythrocytes was analyzed by immunofluorescence using an antibody against a known PVM protein, a fluorescent lipid probe, and electron microscopy. These analyses reveal qualitatively different membranous projections from the PVM. Some PVM projections are uniformly labeled with the antibody and with lipid probes and probably correspond to the Maurer's clefts. In contrast to this uniform labeling of the PVM and projections, a 93-kDa P. chabaudi erythrocyte membrane-associated protein is occasionally detected in vesicle-like structures adjacent to the parasite. These vesicle-like structures are found only coincident with protein synthesis and are located at discrete sites on the PVM. These observations suggest that the 93-kDa protein does not move along the membranous projections of the PVM toward the erythrocyte membrane. It is proposed that the 93-kDa protein is secreted directly into the erythrocyte cytoplasm at discrete PVM domains and then binds to the cytoplasmic face of the erythrocyte membrane.Supplementary material: Additional documentary material has been deposited in electronic form and can be obtained from http://link.springer.de/link/service/journals/00436/index.htm

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories.