Human Parasite Toxoplasma Gondii Research Articles

The initiation and early development of apical-basal polarity in Toxoplasma gondii.

The human parasite Toxoplasma gondii has a distinctive body plan with a well-defined polarity. In the apical complex, the minus ends of the 22 cortical microtubules are anchored to the apical polar ring, a putative microtubule-organizing center. The basal complex caps and constricts the parasite posterior end, and is critical for cytokinesis. How this apical-basal polarity axis is initiated was unknown. Here we examined the development of the apical polar ring and the basal complex in nascent daughters using expansion microscopy. We found that different substructures in the apical polar ring have different sensitivity to stress. In addition, apical-basal differentiation is already established upon nucleation of the cortical microtubule array: arc forms of the apical polar ring and basal complex associate with opposite ends of the microtubules. As the construction of the daughter framework progresses towards the centrioles, the apical and the basal arcs co-develop in striking synchrony ahead of the microtubule array, and close into a ring-form before all the microtubules are nucleated. We also found that two apical polar ring components, APR2 and KinesinA, act synergistically. The removal of each protein individually has modest to no impact on the lytic cycle. However, the loss of both results in abnormalities in the microtubule array and highly reduced plaquing and invasion efficiency.

The initiation and early development of the tubulin-containing cytoskeleton in the human parasite Toxoplasma gondii.

The tubulin-containing cytoskeleton of the human parasite Toxoplasma gondii includes several distinct structures: the conoid, formed of 14 ribbon-like tubulin polymers, and the array of 22 cortical microtubules (MTs) rooted in the apical polar ring. Here we analyze the structure of developing daughter parasites using both 3D-SIM and expansion microscopy. Cortical MTs and the conoid start to develop almost simultaneously, but from distinct precursors near the centrioles. Cortical MTs are initiated in a fixed sequence, starting around the periphery of a short arc that extends to become a complete circle. The conoid also develops from an open arc into a full circle, with a fixed spatial relationship to the centrioles. The patterning of the MT array starts from a "blueprint" with ∼five-fold symmetry, switching to 22-fold rotational symmetry in the final product, revealing a major structural rearrangement during daughter growth. The number of MT is essentially invariant in the wild-type array, but is perturbed by the loss of some structural components of the apical polar ring. This study provides insights into the development of tubulin-containing structures that diverge from conventional models, insights that are critical for understanding the evolutionary paths leading to construction and divergence of cytoskeletal frameworks.

Contact sites between host organelles and pathogens: boon or bane?

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.

Conoid extrusion regulates glideosome assembly to control motility and invasion in Apicomplexa.

Members of Apicomplexa are defined by apical cytoskeletal structures and secretory organelles, tailored for motility, invasion and egress. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In the human parasite Toxoplasma gondii, the conoid, composed of tubulin fibres and preconoidal rings (PCRs), is a dynamic organelle of undefined function. Here, using ultrastructure expansion microscopy, we established that PCRs serve as a hub for glideosome components including Formin1. We also identified components of the PCRs conserved in Apicomplexa, Pcr4 and Pcr5, that contain B-box zinc-finger domains, assemble in heterodimer and are essential for the formation of the structure. The fitness conferring Pcr6 tethers the PCRs to the cone of tubulin fibres. F-actin produced by Formin1 is used by Myosin H to generate the force for conoid extrusion which directs the flux of F-actin to the pellicular space, serving as gatekeeper to control parasite motility.

An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large “SPOTs” (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

IFN-γ mediates Paneth cell death via suppression of mTOR.

Paneth cells constitutively produce antimicrobial peptides and growth factors that allow for intestinal homeostasis, host protection, and intestinal stem cell replication. Paneth cells rely heavily on the glycolytic metabolic program, which is in part controlled by the kinase complex Mechanistic target of rapamycin (mTORC1). Yet, little is known about mTOR importance in Paneth cell integrity under steady-state and inflammatory conditions. Our results demonstrate that IFN-γ, a crucial mediator of the intestinal inflammation, acts directly on murine Paneth cells to alter their mitochondrial integrity and membrane potential, resulting in an TORC1-dependent cell death mechanism distinct from canonical cell death pathways including apoptosis, necroptosis, and pyroptosis. These results were established with the purified cytokine and a physiologically relevant common Th1-inducing human parasite Toxoplasma gondii. Given the crucial role for IFN-γ, which is a cytokine frequently associated with the development of inflammatory bowel disease and compromised Paneth cell functions, the identified mechanisms underlying mTORC1-dependent Paneth cell death downstream of IFN-γ may provide promising novel approaches for treating intestinal inflammation.

Cryo-EM structure of cortical microtubules from human parasite Toxoplasma gondii identifies their microtubule inner proteins

In living cells, microtubules (MTs) play pleiotropic roles, which require very different mechanical properties. Unlike the dynamic MTs found in the cytoplasm of metazoan cells, the specialized cortical MTs from Toxoplasma gondii, a prevalent human pathogen, are extraordinarily stable and resistant to detergent and cold treatments. Using single-particle cryo-EM, we determine their ex vivo structure and identify three proteins (TrxL1, TrxL2 and SPM1) as bona fide microtubule inner proteins (MIPs). These three MIPs form a mesh on the luminal surface and simultaneously stabilize the tubulin lattice in both longitudinal and lateral directions. Consistent with previous observations, deletion of the identified MIPs compromises MT stability and integrity under challenges by chemical treatments. We also visualize a small molecule like density at the Taxol-binding site of β-tubulin. Our results provide the structural basis to understand the stability of cortical MTs and suggest an evolutionarily conserved mechanism of MT stabilization from the inside.

Centrin2 from the human parasite Toxoplasma gondii is required for its invasion and intracellular replication.

Centrins are EF-hand containing proteins ubiquitously found in eukaryotes and are key components of centrioles/basal bodies as well as certain contractile fibers. We previously identified three centrins in the human parasite Toxoplasma gondii, all of which localized to the centrioles. However, one of them, T. gondii (Tg) Centrin2 (CEN2), is also targeted to structures at the apical and basal ends of the parasite, as well as to annuli at the base of the apical cap of the membrane cortex. The role(s) that CEN2 play in these locations were unknown. Here, we report the functional characterization of CEN2 using a conditional knockdown method that combines transcriptional and protein stability control. The knockdown resulted in an ordered loss of CEN2 from its four compartments, due to differences in incorporation kinetics and structural inheritance over successive generations. This was correlated with a major invasion deficiency at early stages of CEN2 knockdown, and replication defects at later stages. These results indicate that CEN2 is incorporated into multiple cytoskeletal structures to serve distinct functions that are required for parasite survival.

Structure of a Novel Dimeric SET Domain Methyltransferase that Regulates Cell Motility

Lysine methyltransferases (KMTs) were initially associated with transcriptional control through their methylation of histones and other nuclear proteins, but have since been found to regulate many other cellular activities. The apical complex lysine (K) methyltransferase (AKMT) of the human parasite Toxoplasma gondii was recently shown to play a critical role in regulating cellular motility. Here we report a 2.1-Å resolution crystal structure of the conserved and functional C-terminal portion (aa289–709) of T. gondii AKMT. AKMT dimerizes via a unique intermolecular interface mediated by the C-terminal tetratricopeptide repeat-like domain together with a specific zinc-binding motif that is absent from all other KMTs. Disruption of AKMT dimerization impaired both its enzyme activity and parasite egress from infected host cells in vivo. Structural comparisons reveal that AKMT is related to the KMTs in the SMYD family, with, however, a number of distinct structural features in addition to the unusual dimerization interface. These features are conserved among the apicomplexan parasites and their free-living relatives, but not found in any known KMTs in animals. AKMT therefore is the founding member of a new subclass of KMT that has important implications for the evolution of the apicomplexans.

An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy.

The ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assemblies in vivo by measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report, the construction, properties and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasite Toxoplasma gondii are described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.

In Vitro Effect of the Synthetic cal14.1a Conotoxin, Derived from Conus californicus, on the Human Parasite Toxoplasma gondii.

Toxins that are secreted by cone snails are small peptides that are used to treat several diseases. However, their effects on parasites with human and veterinary significance are unknown. Toxoplasma gondii is an opportunistic parasite that affects approximately 30% of the world’s population and can be lethal in immunologically compromised individuals. The conventional treatment for this parasitic infection has remained the same since the 1950s, and its efficacy is limited to the acute phase of infection. These findings have necessitated the search for new drugs that specifically target T. gondii. We examined the effects of the synthetic toxin cal14.1a (s-cal14.1a) from C. californicus on the tachyzoite form of T. gondii. Our results indicate that, at micromolar concentrations, s-cal14.1a lowers viability and inhibits host cell invasion (by 50% and 61%, respectively) on exposure to extracellular parasites. Further, intracellular replication decreased significantly while viability of the host cell was unaffected. Our study is the first report on the antiparasitic activity of a synthetic toxin of C. californicus.

Understanding the Signaling Architecture of Calcium‐Dependent Protein Kinases in Apicomplexan Parasites

Identifying the substrates of different protein kinases remains a major obstacle to understanding the signaling pathways that regulate eukaryotic cells. The complexity and plasticity of these pathways makes it difficult to distinguish the contribution of individual kinases in global studies, and yet in vitro assays frequently lack many of the cellular features that dictate kinase‐substrate specificity. By engineering kinases that accommodate bio‐orthogonal ATP analogues, others have previously demonstrated the feasibility of monitoring the activity of individual kinases in complex lysates. We have improved on these methods using pore‐forming toxins to semi‐permeabilize cells and quantitative phosphoproteomics to identify the substrates of two related kinases, CDPK1 and CDPK3, in the human parasite Toxoplasma gondii. We have previously demonstrated that both calcium‐dependent protein kinases (CDPKs) contribute to the regulation of parasite motility and are essential for the infectious cycle. By preserving cellular ultrastructure, we are able to determine the contribution of subcellular localization to kinase specificity. Furthermore, analysis of the substrates has extended our understanding of the cellular processes regulated by these kinases. Beyond its relevance to human health, T. gondii serves as a model for other apicomplexan parasites, including Plasmodium spp., the causative agents of malaria. We hope that our work will serve as the basis for comparing calcium‐regulated signaling within the phylum, and as a model for understanding the role of subcellular localization in the evolution of kinase families.

Structure of Prolyl-tRNA Synthetase-Halofuginone Complex Provides Basis for Development of Drugs against Malaria and Toxoplasmosis

The Chinese herb Dichroa febrifuga has traditionally treated malaria-associated fever. Its active component febrifugine (FF) and derivatives such as halofuginone (HF) are potent anti-malarials. Here, we show that FF-based derivatives arrest parasite growth by direct interaction with and inhibition of the protein translation enzyme prolyl-tRNA synthetase (PRS). Dual administration of inhibitors that target different tRNA synthetases suggests high utility of these drug targets. We reveal the ternary complex structure of PRS-HF and adenosine 5'-(β,γ-imido)triphosphate where the latter facilitates HF integration into the PRS active site. Structural analyses also highlight spaces within the PRS architecture for HF derivatization ofits quinazolinone, but not piperidine, moiety. We also show a remarkable ability of HF to kill the related human parasite Toxoplasma gondii, suggesting wider HF efficacy against parasitic PRSs. Hence, our cell-, enzyme-, and structure-based data on FF-based inhibitors strengthen the case for their inclusion in anti-malarial and anti-toxoplasmosis drug development efforts.

α-Tubulin Mutations Alter Oryzalin Affinity and Microtubule Assembly Properties To Confer Dinitroaniline Resistance

Plant and protozoan microtubules are selectively sensitive to dinitroanilines, which do not disrupt vertebrate or fungal microtubules. Tetrahymena thermophila is an abundant source of dinitroaniline-sensitive tubulin, and we have modified the single T. thermophila α-tubulin gene to create strains that solely express mutant α-tubulin in functional dimers. Previous research identified multiple α-tubulin mutations that confer dinitroaniline resistance in the human parasite Toxoplasma gondii, and when two of these mutations (L136F and I252L) were introduced into T. thermophila, they conferred resistance in these free-living ciliates. Purified tubulin heterodimers composed of L136F or I252L α-tubulin display decreased affinity for the dinitroaniline oryzalin relative to wild-type T. thermophila tubulin. Moreover, the L136F substitution dramatically reduces the critical concentration for microtubule assembly relative to the properties of wild-type T. thermophila tubulin. Our data provide additional support for the proposed dinitroaniline binding site on α-tubulin and validate the use of T. thermophila for expression of genetically homogeneous populations of mutant tubulins for biochemical characterization.

A complex small RNA repertoire is generated by a plant/fungal-like machinery and effected by a metazoan-like Argonaute in the single-cell human parasite Toxoplasma gondii.

In RNA silencing, small RNAs produced by the RNase-III Dicer guide Argonaute-like proteins as part of RNA-induced silencing complexes (RISC) to regulate gene expression transcriptionally or post-transcriptionally. Here, we have characterized the RNA silencing machinery and exhaustive small RNAome of Toxoplasma gondii, member of the Apicomplexa, a phylum of animal- and human-infecting parasites that cause extensive health and economic damages to human populations worldwide. Remarkably, the small RNA-generating machinery of Toxoplasma is phylogenetically and functionally related to that of plants and fungi, and accounts for an exceptionally diverse array of small RNAs. This array includes conspicuous populations of repeat-associated small interfering RNA (siRNA), which, as in plants, likely generate and maintain heterochromatin at DNA repeats and satellites. Toxoplasma small RNAs also include many microRNAs with clear metazoan-like features whose accumulation is sometimes extremely high and dynamic, an unexpected finding given that Toxoplasma is a unicellular protist. Both plant-like heterochromatic small RNAs and metazoan-like microRNAs bind to a single Argonaute protein, Tg-AGO. Toxoplasma miRNAs co-sediment with polyribosomes, and thus, are likely to act as translational regulators, consistent with the lack of catalytic residues in Tg-AGO. Mass spectrometric analyses of the Tg-AGO protein complex revealed a common set of virtually all known RISC components so far characterized in human and Drosophila, as well as novel proteins involved in RNA metabolism. In agreement with its loading with heterochromatic small RNAs, Tg-AGO also associates substoichiometrically with components of known chromatin-repressing complexes. Thus, a puzzling patchwork of silencing processor and effector proteins from plant, fungal and metazoan origin accounts for the production and action of an unsuspected variety of small RNAs in the single-cell parasite Toxoplasma and possibly in other apicomplexans. This study establishes Toxoplasma as a unique model system for studying the evolution and molecular mechanisms of RNA silencing among eukaryotes.

Dinitroaniline Activity inToxoplasma gondiiExpressing Wild-Type or Mutant α-Tubulin

The human parasite Toxoplasma gondii is sensitive to dinitroaniline compounds which selectively disrupt microtubules in diverse protozoa but which have no detectable effect on vertebrate host cell microtubules or other functions. Replication of wild-type T. gondii is inhibited by 0.5 to 2.5 microM oryzalin, but mutant parasites harboring amino acid substitutions in the predicted dinitroaniline binding site confer resistance up to 40 microM oryzalin. However, the precise interaction between dinitroanilines and the binding site in alpha-tubulin remains unclear. We have investigated the activity of 12 dinitroanilines and the related compound amiprophos methyl on wild-type and dinitroaniline-resistant parasite lines that contain proposed binding site mutations. These data indicate that dinitramine is the most effective dinitroaniline to inhibit Toxoplasma growth in wild-type parasites and most resistant lines. Dinitramine has an amine group at the meta position not present in any of the other dinitroanilines tested here that is predicted to form hydrogen bonds with residues Arg2 and Gln133 according to docking data. Remarkably, although the binding site mutation Ile235Val confers increased resistance to most dinitroanilines, it confers increased sensitivity to GB-II-5, a compound optimized for activity against kinetoplastid tubulin. Kinetoplastid parasites have a valine at position 235 of alpha-tubulin, whereas apicomplexan parasites have an isoleucine at this site. We suggest that this heterogeneity in binding site environment influences relative dinitroaniline sensitivity in distinct protozoan lineages and hypothesize that a mutation that makes the apicomplexan dinitroaniline binding site more like the kinetoplastid site increases sensitivity to a dinitroaniline optimized for activity in the latter parasites.

The urban house mouse (Mus domesticus) as a reservoir of infection for the human parasite Toxoplasma gondii: an unrecognised public health issue?

Toxoplasma gondii is a protozoan parasite capable of infecting almost all warm-blooded animals. The cat is the definitive host and becomes infected by consuming contaminated meat or infected prey. Humans can act as intermediate hosts and in healthy individuals the infection is mild and self-limiting. In pregnant women it can cause spontaneous abortions and foetal abnormalities and is capable of inducing serious illness in immuno-compromised patients. In infested dwellings, mice could act as intermediate hosts and play a role in the persistence/propagation of the disease. A total of 200 mice were trapped alive in 27 infested properties in Manchester, UK, and screened for Toxoplasma infection; 59% tested positive. Evidence of vertical transmission from infected dams to foetus was found, possibly maintaining the infection in urban areas. These findings have important implications when considering approaches to rodent control.

Protozoan Parasite Shares a Hormone with Plants

Research by Nagamune et al . has revealed that the plant hormone abscisic acid (ABA) acts in the human parasite Toxoplasma gondii . In the protozoan parasite T. gondii , calcium release from intracellular stores (which has been implicated in various processes that involve calcium-mediated protein secretion) is regulated by the second messenger cyclic ADP ribose (cADPR). Noting that, in plants, ABA regulates calcium release from intracellular stores by means of cADPR, Nagamune et al . exposed T. gondii to ABA and found that it increased both their cADPR content and the calcium-dependent secretion of the adhesin MIC2 (microneme protein 2). Biochemical analyses indicated that ABA was present in T. gondii lysates, and enzyme-linked immunosorbent assay indicated that its concentration was decreased by treatment with the herbicide fluridone (which inhibits ABA synthesis in plants). There was a spike in the intracellular concentration of ABA before parasite egress from infected cells, and, whereas fluridone failed to prevent invasion of host cells, it inhibited T. gondii exit from infected human foreskin fibroblasts. Instead, it promoted differentiation of the parasites into tissue cysts. Exogenous ABA stimulated early egress of parasites from host cells and enabled egress in the presence of fluridone. Fluridone also decreased parasite burden in infected mice and markedly improved survival of mice inoculated with T. gondii . The authors thus conclude that a plantlike ABA-mediated calcium-signaling pathway is present in the protozoan T. gondii and that disruption of ABA signaling may provide a therapeutic approach against toxoplasmosis. K. Nagamune, L. M. Hicks, B. Fux, F. Brossier, E. N. Chini, L. D. Sibley, Abscisic acid controls calcium-dependent egress and development in Toxoplasma gondii . Nature 451 , 207-210 (2008). [PubMed]

Substrate Specificity of Rhomboid Intramembrane Proteases Is Governed by Helix-Breaking Residues in the Substrate Transmembrane Domain

Rhomboid intramembrane proteases initiate cell signaling during Drosophila development and Providencia bacterial growth by cleaving transmembrane ligand precursors. We have determined how specificity is achieved: Drosophila Rhomboid-1 is a site-specific protease that recognizes its substrate Spitz by a small region of the Spitz transmembrane domain (TMD). This substrate motif is necessary and sufficient for cleavage and is composed of residues known to disrupt helices. Rhomboids from diverse organisms including bacteria and vertebrates recognize the same substrate motif, suggesting that they use a universal targeting strategy. We used this information to search for other rhomboid substrates and identified a family of adhesion proteins from the human parasite Toxoplasma gondii, the TMDs of which were efficient substrates for rhomboid proteases. Intramembrane cleavage of these proteins is required for host cell invasion. These results provide an explanation of how rhomboid proteases achieve specificity, and allow some rhomboid substrates to be predicted from sequence information.