Stable endocytic structures navigate the complex pellicle of apicomplexan parasites.

Apicomplexan parasites have immense impacts on humanity, but their basic cellular processes are often poorly understood. Where endocytosis occurs in these cells, how conserved this process is with other eukaryotes, and what the functions of endocytosis are across this phylum are major unanswered questions. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. We identify that these endocytic structures correspond to the 'micropore' that has been observed throughout the Apicomplexa. Moreover, conserved molecular adaptation of this structure is seen in apicomplexans including the kelch-domain protein K13 that is central to malarial drug-resistance. We determine that a dominant function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

Endocytosis in Plasmodium and Toxoplasma Parasites.

Endocytosis is critical for many functions in eukaryotic cells. Uptake of host cell cytosol, an indispensable endocytic process in malaria blood-stage parasites, has been known for a long time. However, it is only recently that the proteins involved in this process have started to emerge. Unexpectedly, some of these proteins revealed a critical role for endocytosis in artemisinin resistance. More recently, endocytosis was discovered in both intracellular and extracellular Toxoplasma gondii parasites. Here, we review these findings, compare the endolysosomal systems of Toxoplasma and Plasmodium parasites, and present current knowledge about endocytic mechanisms in apicomplexans.

A highly dynamic F-actin network regulates transport and recycling of micronemes in Toxoplasma gondii vacuoles.

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.

An endocytic-secretory cycle participates in Toxoplasma gondii in motility.

Apicomplexan parasites invade host cells in an active process involving their ability to move by gliding motility. While the acto-myosin system of the parasite plays a crucial role in the formation and release of attachment sites during this process, there are still open questions regarding the involvement of other mechanisms in parasite motility. In many eukaryotes, a secretory-endocytic cycle leads to the recycling of receptors (integrins), necessary to form attachment sites, regulation of surface area during motility, and generation of retrograde membrane flow. Here, we demonstrate that endocytosis operates during gliding motility in Toxoplasma gondii and appears to be crucial for the establishment of retrograde membrane flow, because inhibition of endocytosis blocks retrograde flow and motility. We demonstrate that extracellular parasites can efficiently incorporate exogenous material, such as labelled phospholipids, nanogold particles (NGPs), antibodies, and Concanavalin A (ConA). Using labelled phospholipids, we observed that the endocytic and secretory pathways of the parasite converge, and endocytosed lipids are subsequently secreted, demonstrating the operation of an endocytic-secretory cycle. Together our data consolidate previous findings, and we propose an additional model, working in parallel to the acto-myosin motor, that reconciles parasite motility with observations in other eukaryotes: an apicomplexan fountain-flow-model for parasite motility.

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo.

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondii. Methods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2 causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

Toxoplasma gondii F-actin forms an extensive filamentous network required for material exchange and parasite maturation.

Apicomplexan actin is important during the parasite's life cycle. Its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. It has not been possible to study actin in vivo and so its physiological roles have remained obscure, leading to models distinct from conventional actin behaviour. Here a modified version of the commercially available actin-chromobody was tested as a novel tool for visualising F-actin dynamics in Toxoplasma gondii. Cb labels filamentous actin structures within the parasite cytosol and labels an extensive F-actin network that connects parasites within the parasitophorous vacuole and allows vesicles to be exchanged between parasites. In the absence of actin, parasites lack a residual body and inter-parasite connections and grow in an asynchronous and disorganized manner. Collectively, these data identify new roles for actin in the intracellular phase of the parasites lytic cycle and provide a robust new tool for imaging parasitic F-actin dynamics.

Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion.

BACKGROUND: Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite's actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement. RESULTS: In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites. CONCLUSION: We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.

Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development.

Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.

Eimeripain, a cathepsin B-like cysteine protease, expressed throughout sporulation of the apicomplexan parasite Eimeria tenella.

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.