Depletion of a Toxoplasma porin leads to defects in mitochondrial morphology and contacts with the endoplasmic reticulum.

The voltage-dependent anion channel (VDAC) is a ubiquitous channel in the outer membrane of the mitochondrion with multiple roles in protein, metabolite and small molecule transport. In mammalian cells, VDAC protein, as part of a larger complex including the inositol triphosphate receptor, has been shown to have a role in mediating contacts between the mitochondria and endoplasmic reticulum (ER). We identify VDAC of the pathogenic apicomplexan Toxoplasma gondii and demonstrate its importance for parasite growth. We show that VDAC is involved in protein import and metabolite transfer to mitochondria. Further, depletion of VDAC resulted in significant morphological changes in the mitochondrion and ER, suggesting a role in mediating contacts between these organelles in T. gondii. This article has an associated First Person interview with the first author of the paper.

Orchestrating a heist: uptake and storage of metals by apicomplexan parasites.

The acquisition and storage of metals has been a preoccupation of life for millennia. Transition metals, in particular iron, copper and zinc, have vital roles within cells. However, metals also make dangerous cargos; inappropriate uptake or storage of transition metals leads to cell death. This paradox has led to cells developing elegant and frequently redundant mechanisms for fine-tuning local metal concentrations. In the context of infection, pathogens must overcome further hurdles, as hosts act to weaponize metal availability to prevent pathogen colonization and spread. Here, we detail the methods used by the Apicomplexa, a large family of eukaryotic parasites, to obtain and store essential metals.

Gliding Associated Proteins Play Essential Roles during the Formation of the Inner Membrane Complex of Toxoplasma gondii.

The inner membrane complex (IMC) of apicomplexan parasites is a specialised structure localised beneath the parasite's plasma membrane, and is important for parasite stability and intracellular replication. Furthermore, it serves as an anchor for the myosin A motor complex, termed the glideosome. While the role of this protein complex in parasite motility and host cell invasion has been well described, additional roles during the asexual life cycle are unknown. Here, we demonstrate that core elements of the glideosome, the gliding associated proteins GAP40 and GAP50 as well as members of the GAPM family, have critical roles in the biogenesis of the IMC during intracellular replication. Deletion or disruption of these genes resulted in the rapid collapse of developing parasites after initiation of the cell cycle and led to redistribution of other glideosome components.

The inner membrane complex through development of Toxoplasma gondii and Plasmodium.

Plasmodium spp. and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualization of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarizes recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa.

The Legionella pneumophila Dot/Icm-secreted effector PlcC/CegC1 together with PlcA and PlcB promotes virulence and belongs to a novel zinc metallophospholipase C family present in bacteria and fungi.

Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn(2+) ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn(2+)-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

LtpD is a novel Legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1.

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide-binding L. pneumophila effector that has a role in intracellular bacterial replication.

The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice.

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

Investigation of new ferrocenyl-artesunate derivatives as antiparasitics.

Artesunate (Ars) is a semisynthetic antimalarial drug and is a part of the artemisinin-based combination therapy arsenal employed for malaria treatment. The drug functions mainly by activation of its endoperoxide bridge leading to increased oxidative stress in malaria parasites. The purpose of this study was to ascertain the antiparasitic effects of combining ferrocene and Arsvia short or long chain ester or amide linkages (C1-C4). The compounds were evaluated for growth inhibition activity on the apicomplexan parasites, Plasmodium falciparum (P. falciparum) and Toxoplasma gondii (T. gondii). All the complexes demonstrated good activity against T. gondii with IC50 values in the low micromolar range (0.28-1.2 µM) and good to excellent antimalarial activity against a chloroquine sensitive strain of P. falciparum (NF54). Further investigations on T. gondii revealed that the likely mode of action (MoA) is through the generation of reactive oxygen species. Additionally, immunofluorescence microscopy suggested a novel change in the morphology of the parasite by complex C3, an artesunate-ferrocenyl ethyl amide complex. The complexes were not cytotoxic or showed low cytotoxicity to two normal cell lines tested.

The vacuolar iron transporter mediates iron detoxification in Toxoplasma gondii.

Iron is essential to cells as a cofactor in enzymes of respiration and replication, however without correct storage, iron leads to the formation of dangerous oxygen radicals. In yeast and plants, iron is transported into a membrane-bound vacuole by the vacuolar iron transporter (VIT). This transporter is conserved in the apicomplexan family of obligate intracellular parasites, including in Toxoplasma gondii. Here, we assess the role of VIT and iron storage in T. gondii. By deleting VIT, we find a slight growth defect in vitro, and iron hypersensitivity, confirming its essential role in parasite iron detoxification, which can be rescued by scavenging of oxygen radicals. We show VIT expression is regulated by iron at transcript and protein levels, and by altering VIT localization. In the absence of VIT, T. gondii responds by altering expression of iron metabolism genes and by increasing antioxidant protein catalase activity. We also show that iron detoxification has an important role both in parasite survival within macrophages and in virulence in a mouse model. Together, by demonstrating a critical role for VIT during iron detoxification in T. gondii, we reveal the importance of iron storage in the parasite and provide the first insight into the machinery involved.

Legionella pneumophila pathogenesis in the Galleria mellonella infection model.

Legionella pneumophila is a facultative intracellular human pathogen and the etiological agent of severe pneumonia known as Legionnaires' disease. Its virulence depends on protein secretion systems, in particular, the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication-permissive vacuole in macrophages. The analysis of the role of these systems and their substrates for pathogenesis requires easy-to-use models which approximate human infection. We examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for L. pneumophila infection. We found that the L. pneumophila strains 130b, Paris, and JR32 caused mortality of the G. mellonella larvae that was strain, infectious dose, growth phase, and T4SS dependent. Wild-type L. pneumophila persisted and replicated within the larvae, whereas T4SS mutants were rapidly cleared. L. pneumophila strain Lp02, which is attenuated in the absence of thymidine but has a functional T4SS, resisted clearance in G. mellonella up to 18 h postinfection without inducing mortality. Immunofluorescence and transmission electron microscopy revealed that L. pneumophila resided within insect hemocytes in a vacuole that ultrastructurally resembled the Legionella-containing vacuole (LCV) observed in macrophages. The vacuole was decorated with the T4SS effector and LCV marker SidC. Infection caused severe damage to the insect organs and triggered immune responses, including activation of the phenoloxidase cascade leading to melanization, nodule formation, and upregulation of antimicrobial peptides. Taken together, these results suggest that G. mellonella provides an effective model to investigate the interaction between L. pneumophila and the host.

mSphere of Influence: an Army Marching on Its Stomach-Malaria Parasites Sense and Subvert Host Nutrition.

Clare Harding works on the metal biology of the parasite Toxoplasma gondii In this mSphere of Influence article, she reflects on how two papers from the laboratory of Maria Mota, "Host-mediated regulation of superinfection in malaria" by Portugal et al. (S. Portugal, C. Carret, M. Recker, A. E. Armitage, et al., Nat Med 17:732-737, 2011, https://doi.org/10.1038/nm.2368) and "Nutrient sensing modulates malaria parasite virulence" by Mancio-Silva et al. (L. Mancio-Silva, K. Slavic, M. T. Grilo Ruivo, A. R. Grosso, et al., Nature 547:213-216, 2017, https://doi.org/10.1038/nature23009), made an impact on her understanding of host-pathogen interactions by examining the complex interplay between parasites and their hosts' nutritional status.

Supply and demand-heme synthesis, salvage and utilization by Apicomplexa.

The Apicomplexa phylum groups important human and animal pathogens that cause severe diseases, encompassing malaria, toxoplasmosis, and cryptosporidiosis. In common with most organisms, apicomplexans rely on heme as cofactor for several enzymes, including cytochromes of the electron transport chain. This heme derives from de novo synthesis and/or the development of uptake mechanisms to scavenge heme from their host. Recent studies have revealed that heme synthesis is essential for Toxoplasma gondii tachyzoites, as well as for the mosquito and liver stages of Plasmodium spp. In contrast, the erythrocytic stages of the malaria parasites rely on scavenging heme from the host red blood cell. The unusual heme synthesis pathway in Apicomplexa spans three cellular compartments and comprises enzymes of distinct ancestral origin, providing promising drug targets. Remarkably given the requirement for heme, T. gondii can tolerate the loss of several heme synthesis enzymes at a high fitness cost, while the ferrochelatase is essential for survival. These findings indicate that T. gondii is capable of salvaging heme precursors from its host. Furthermore, heme is implicated in the activation of the key antimalarial drug artemisinin. Recent findings established that a reduction in heme availability corresponds to decreased sensitivity to artemisinin in T. gondii and Plasmodium falciparum, providing insights into the possible development of combination therapies to tackle apicomplexan parasites. This review describes the microeconomics of heme in Apicomplexa, from supply, either from de novo synthesis or scavenging, to demand by metabolic pathways, including the electron transport chain.

Legionella pneumophila strain 130b possesses a unique combination of type IV secretion systems and novel Dot/Icm secretion system effector proteins.

Legionella pneumophila is a ubiquitous inhabitant of environmental water reservoirs. The bacteria infect a wide variety of protozoa and, after accidental inhalation, human alveolar macrophages, which can lead to severe pneumonia. The capability to thrive in phagocytic hosts is dependent on the Dot/Icm type IV secretion system (T4SS), which translocates multiple effector proteins into the host cell. In this study, we determined the draft genome sequence of L. pneumophila strain 130b (Wadsworth). We found that the 130b genome encodes a unique set of T4SSs, namely, the Dot/Icm T4SS, a Trb-1-like T4SS, and two Lvh T4SS gene clusters. Sequence analysis substantiated that a core set of 107 Dot/Icm T4SS effectors was conserved among the sequenced L. pneumophila strains Philadelphia-1, Lens, Paris, Corby, Alcoy, and 130b. We also identified new effector candidates and validated the translocation of 10 novel Dot/Icm T4SS effectors that are not present in L. pneumophila strain Philadelphia-1. We examined the prevalence of the new effector genes among 87 environmental and clinical L. pneumophila isolates. Five of the new effectors were identified in 34 to 62% of the isolates, while less than 15% of the strains tested positive for the other five genes. Collectively, our data show that the core set of conserved Dot/Icm T4SS effector proteins is supplemented by a variable repertoire of accessory effectors that may partly account for differences in the virulences and prevalences of particular L. pneumophila strains.

The Riveting Cellular Structures of Apicomplexan Parasites.

Parasitic protozoa of the phylum Apicomplexa cause a range of human and animal diseases. Their complex life cycles - often heteroxenous with sexual and asexual phases in different hosts - rely on elaborate cytoskeletal structures to enable morphogenesis and motility, organize cell division, and withstand diverse environmental forces. This review primarily focuses on studies using Toxoplasma gondii and Plasmodium spp. as the best studied apicomplexans; however, many cytoskeletal adaptations are broadly conserved and predate the emergence of the parasitic phylum. After decades cataloguing the constituents of such structures, a dynamic picture is emerging of the assembly and maintenance of apicomplexan cytoskeletons, illuminating how they template and orient critical processes during infection. These observations impact our view of eukaryotic diversity and offer future challenges for cell biology.

Genetic screens reveal a central role for heme metabolism in artemisinin susceptibility.

Artemisinins have revolutionized the treatment of Plasmodium falciparum malaria; however, resistance threatens to undermine global control efforts. To broadly explore artemisinin susceptibility in apicomplexan parasites, we employ genome-scale CRISPR screens recently developed for Toxoplasma gondii to discover sensitizing and desensitizing mutations. Using a sublethal concentration of dihydroartemisinin (DHA), we uncover the putative transporter Tmem14c whose disruption increases DHA susceptibility. Screens performed under high doses of DHA provide evidence that mitochondrial metabolism can modulate resistance. We show that disrupting a top candidate from the screens, the mitochondrial protease DegP2, lowers porphyrin levels and decreases DHA susceptibility, without significantly altering parasite fitness in culture. Deleting the homologous gene in P. falciparum, PfDegP, similarly lowers heme levels and DHA susceptibility. These results expose the vulnerability of heme metabolism to genetic perturbations that can lead to increased survival in the presence of DHA.

Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii.

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depolymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites.

Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Endocytosis and Sphingolipid Scavenging in Leishmania mexicana Amastigotes.

Leishmania species are the causative agents of the leishmaniases, a spectrum of neglected tropical diseases. Amastigote stage parasites exist within macrophages and scavenge host factors for survival, for example, Leishmania species utilise host sphingolipid for synthesis of complex sphingolipid. In this study L. mexicana endocytosis was shown to be significantly upregulated in amastigotes, indicating that sphingolipid scavenging may be enhanced. However, inhibition of host sphingolipid biosynthesis had no significant effect on amastigote proliferation within a macrophage cell line. In addition, infection itself did not directly influence host biosynthesis. Notably, in contrast to L. major, L. mexicana amastigotes are indicated to possess a complete biosynthetic pathway suggesting that scavenged sphingolipids may be nonessential for proliferation. This suggested that Old and New World species differ in their interactions with the macrophage host. This will need to be considered when targeting the Leishmania sphingolipid biosynthetic pathway with novel therapeutics.