Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites.

The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.

The nucleocytosolic O-fucosyltransferase SPINDLY affects protein expression and virulence in Toxoplasma gondii.

Once considered unusual, nucleocytoplasmic glycosylation is now recognized as a conserved feature of eukaryotes. While in animals, O-GlcNAc transferase (OGT) modifies thousands of intracellular proteins, the human pathogen Toxoplasma gondii transfers a different sugar, fucose, to proteins involved in transcription, mRNA processing, and signaling. Knockout experiments showed that TgSPY, an ortholog of plant SPINDLY and paralog of host OGT, is required for nuclear O-fucosylation. Here we verify that TgSPY is the nucleocytoplasmic O-fucosyltransferase (OFT) by 1) complementation with TgSPY-MYC3, 2) its functional dependence on amino acids critical for OGT activity, and 3) its ability to O-fucosylate itself and a model substrate and to specifically hydrolyze GDP-Fuc. While many of the endogenous proteins modified by O-Fuc are important for tachyzoite fitness, O-fucosylation by TgSPY is not essential. Growth of Δspy tachyzoites in fibroblasts is modestly affected, despite marked reductions in the levels of ectopically expressed proteins normally modified with O-fucose. Intact TgSPY-MYC3 localizes to the nucleus and cytoplasm, whereas catalytic mutants often displayed reduced abundance. Δspy tachyzoites of a luciferase-expressing type II strain exhibited infection kinetics in mice similar to wild-type but increased persistence in the chronic brain phase, potentially due to an imbalance of regulatory protein levels. The modest changes in parasite fitness in vitro and in mice, despite profound effects on reporter protein accumulation, and the characteristic punctate localization of O-fucosylated proteins suggest that TgSPY controls the levels of proteins to be held in reserve for response to novel stresses.

Intravital imaging of host-parasite interactions in organs of the thoracic and abdominopelvic cavities.

Infections with protozoan and helminthic parasites affect multiple organs in the mammalian host. Imaging pathogens in their natural environment takes a more holistic view on biomedical aspects of parasitic infections. Here, we focus on selected organs of the thoracic and abdominopelvic cavities most commonly affected by parasites. Parasitic infections of these organs are often associated with severe medical complications or have health implications beyond the infected individual. Intravital imaging has provided a more dynamic picture of the host-parasite interplay and contributed not only to our understanding of the various disease pathologies, but has also provided fundamental insight into the biology of the parasites.

O-Fucosylation of thrombospondin-like repeats is required for processing of microneme protein 2 and for efficient host cell invasion by Toxoplasma gondii tachyzoites.

Toxoplasma gondii is an intracellular parasite that causes disseminated infections that can produce neurological damage in fetuses and immunocompromised individuals. Microneme protein 2 (MIC2), a member of the thrombospondin-related anonymous protein (TRAP) family, is a secreted protein important for T. gondii motility, host cell attachment, invasion, and egress. MIC2 contains six thrombospondin type I repeats (TSRs) that are modified by C-mannose and O-fucose in Plasmodium spp. and mammals. Here, using MS analysis, we found that the four TSRs in T. gondii MIC2 with protein O-fucosyltransferase 2 (POFUT2) acceptor sites are modified by a dHexHex disaccharide, whereas Trp residues within three TSRs are also modified with C-mannose. Disruption of genes encoding either POFUT2 or the putative GDP-fucose transporter (NST2) resulted in loss of MIC2 O-fucosylation, as detected by an antibody against the GlcFuc disaccharide, and in markedly reduced cellular levels of MIC2. Furthermore, in 10-15% of the Δpofut2 or Δnst2 vacuoles, MIC2 accumulated earlier in the secretory pathway rather than localizing to micronemes. Dissemination of tachyzoites in human foreskin fibroblasts was reduced for these knockouts, which both exhibited defects in attachment to and invasion of host cells comparable with the Δmic2 phenotype. These results, indicating that O-fucosylation of TSRs is required for efficient processing of MIC2 and for normal parasite invasion, are consistent with the recent demonstration that Plasmodium falciparum Δpofut2 strain has decreased virulence and also support a conserved role for this glycosylation pathway in quality control of TSR-containing proteins in eukaryotes.

Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow.

Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito.

Host cell cytosolic immune response during Plasmodium liver stage development.

Recent years have witnessed a great gain in knowledge regarding parasite-host cell interactions during Plasmodium liver stage development. It is now an accepted fact that a large percentage of sporozoites invading hepatocytes fail to form infectious merozoites. There appears to be a delicate balance between parasite survival and elimination and we now start to understand why this is so. Plasmodium liver stage parasites replicate within the parasitophorous vacuole (PV), formed during invasion by invagination of the host cell plasma membrane. The main interface between the parasite and hepatocyte is the parasitophorous vacuole membrane (PVM) that surrounds the PV. Recently, it was shown that autophagy marker proteins decorate the PVM of Plasmodium liver stage parasites and eliminate a proportion of them by an autophagy-like mechanism. Successfully developing Plasmodium berghei parasites are initially also labeled but in the course of development, they are able to control this host defense mechanism by shedding PVM material into the tubovesicular network (TVN), an extension of the PVM that releases vesicles into the host cell cytoplasm. Better understanding of the molecular events at the PVM/TVN during parasite elimination could be the basis of new antimalarial measures.

Differential Roles for Inner Membrane Complex Proteins across Toxoplasma gondii and Sarcocystis neurona Development.

The inner membrane complex (IMC) of apicomplexan parasites contains a network of intermediate filament-like proteins. The 14 alveolin domain-containing IMC proteins in Toxoplasma gondii fall into different groups defined by their distinct spatiotemporal dynamics during the internal budding process of tachyzoites. Here, we analyzed representatives of different IMC protein groups across all stages of the Toxoplasma life cycle and during Sarcocystis neurona asexual development. We found that across asexually dividing Toxoplasma stages, IMC7 is present exclusively in the mother's cytoskeleton, whereas IMC1 and IMC3 are both present in mother and daughter cytoskeletons (IMC3 is strongly enriched in daughter buds). In developing macro- and microgametocytes, IMC1 and -3 are absent, whereas IMC7 is lost in early microgametocytes but retained in macrogametocytes until late in their development. We found no roles for IMC proteins during meiosis and sporoblast formation. However, we observed that IMC1 and IMC3, but not IMC7, are present in sporozoites. Although the spatiotemporal pattern of IMC15 and IMC3 suggests orthologous functions in Sarcocystis, IMC7 may have functionally diverged in Sarcocystis merozoites. To functionally characterize IMC proteins, we knocked out IMC7, -12, -14, and -15 in Toxoplasma. IMC14 and -15 appear to be involved in switching between endodyogeny and endopolygeny. In addition, IMC7, -12, and -14, which are all recruited to the cytoskeleton outside cytokinesis, are critical for the structural integrity of extracellular tachyzoites. Altogether, stage- and development-specific roles for IMC proteins can be discerned, suggesting different niches for each IMC protein across the entire life cycle. IMPORTANCE The inner membrane complex (IMC) is a defining feature of apicomplexan parasites key to both their motility and unique cell division. To provide further insights into the IMC, we analyzed the dynamics and functions of representative alveolin domain-containing IMC proteins across developmental stages. Our work shows universal but distinct roles for IMC1, -3, and -7 during Toxoplasma asexual division but more specialized functions for these proteins during gametogenesis. In addition, we find that IMC15 is involved in daughter formation in both Toxoplasma and Sarcocystis. IMC14 and IMC15 function in limiting the number of Toxoplasma offspring per division. Furthermore, IMC7, -12, and -14, which are recruited in the G1 cell cycle stage, are required for stress resistance of extracellular tachyzoites. Thus, although the roles of the different IMC proteins appear to overlap, stage- and development-specific behaviors indicate that their functions are uniquely tailored to each life stage requirement.

Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei.

The hepatic stage of the malaria parasite Plasmodium is accompanied by an autophagy-mediated host response directly targeting the parasitophorous vacuolar membrane (PVM) harbouring the parasite. Removal of the PVM-associated autophagic proteins such as ubiquitin, p62, and LC3 correlates with parasite survival. Yet, it is unclear how Plasmodium avoids the deleterious effects of selective autophagy. Here we show that parasites trap host autophagic factors in the tubovesicular network (TVN), an expansion of the PVM into the host cytoplasm. In proliferating parasites, PVM-associated LC3 becomes immediately redirected into the TVN, where it accumulates distally from the parasite's replicative centre. Finally, the host factors are shed as vesicles into the host cytoplasm. This strategy may enable the parasite to balance the benefits of the enhanced host catabolic activity with the risk of being eliminated by the cell's cytosolic immune defence.

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.

Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B.

Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites.

Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis.

The final step during cell division is the separation of daughter cells, a process that requires the coordinated delivery and assembly of new membrane to the cleavage furrow. While most eukaryotic cells replicate by binary fission, replication of apicomplexan parasites involves the assembly of daughters (merozoites/tachyzoites) within the mother cell, using the so-called Inner Membrane Complex (IMC) as a scaffold. After de novo synthesis of the IMC and biogenesis or segregation of new organelles, daughters bud out of the mother cell to invade new host cells. Here, we demonstrate that the final step in parasite cell division involves delivery of new plasma membrane to the daughter cells, in a process requiring functional Rab11A. Importantly, Rab11A can be found in association with Myosin-Tail-Interacting-Protein (MTIP), also known as Myosin Light Chain 1 (MLC1), a member of a 4-protein motor complex called the glideosome that is known to be crucial for parasite invasion of host cells. Ablation of Rab11A function results in daughter parasites having an incompletely formed IMC that leads to a block at a late stage of cell division. A similar defect is observed upon inducible expression of a myosin A tail-only mutant. We propose a model where Rab11A-mediated vesicular traffic driven by an MTIP-Myosin motor is necessary for IMC maturation and to deliver new plasma membrane to daughter cells in order to complete cell division.

Functional expression of ribozymes in Apicomplexa: towards exogenous control of gene expression by inducible RNA-cleavage.

The ability to control expression of a specific gene is a prerequisite to understand the function of essential genes. Many gene regulation systems operate on the transcriptional level by employing heterologous cis- and trans-acting elements. Recently, novel approaches employing autocatalytic RNA have been reported for different organisms. Here we show specific downregulation of gene expression in the apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum, employing self-cleaving ribozymes integrated into the transcriptional unit of different genes. Moreover, we demonstrate the potential to specifically upregulate reporter gene expression by employment of the recently identified ribozyme inhibitor toyocamycin. At the RNA-level, we were able to significantly stabilise the mRNA in T. gondii. Furthermore we show that the adenosine analogue toyocamycin needs to be phosphorylated by adenosine kinase in order to act as an inhibitor for hammerhead ribozymes, since neither upregulation of reporter gene expression nor a toxic effect of toyocamycin can be detected in parasites that do not express the enzyme adenosine kinase.

Rapid control of protein level in the apicomplexan Toxoplasma gondii.

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.

Molecular tools for analysis of gene function in parasitic microorganisms.

With the completion of several genome sequences for parasitic protozoa, research in molecular parasitology entered the "post-genomic" era. Accompanied by global transcriptome and proteome analysis, huge datasets have been generated that have added many novel candidates to the list of drug and vaccine targets. The challenge is now to validate these factors and to bring science back to the bench to perform a detailed characterization. In some parasites, like Trypanosoma brucei, high-throughput genetic screens have been established using RNA interference [for a detailed review, see Motyka and Englund (2004)]. In most protozoan parasites, however, more time-consuming approaches have to be employed to identify and characterize the function of promising candidates in detail. This review aims to summarize the status of molecular genetic tools available for a variety of protozoan pathogens and discuss how they can be implemented to advance our understanding of parasite biology.