Toxoplasma replication is inhibited by MMV676477 without development of resistance.

Protozoan parasites cause life-threatening infections in both humans and animals, including agriculturally significant livestock. Available treatments are typically narrow spectrum and are complicated by drug toxicity and the development of resistant parasites. Protozoan tubulin is an attractive target for the development of broad-spectrum antimitotic agents. The Medicines for Malaria Pathogen Box compound MMV676477 was previously shown to inhibit replication of kinetoplastid parasites, such as Leishmania amazonensis and Trypanosoma brucei, and the apicomplexan parasite Plasmodium falciparum by selectively stabilizing protozoan microtubules. In this report, we show that MMV676477 inhibits intracellular growth of the human apicomplexan pathogen Toxoplasma gondii with an EC50 value of ~50 nM. MMV676477 does not stabilize vertebrate microtubules or cause other toxic effects in human fibroblasts. The availability of tools for genetic studies makes Toxoplasma a useful model for studies of the cytoskeleton. We conducted a forward genetics screen for MMV676477 resistance, anticipating that missense mutations would delineate the binding site on protozoan tubulin. Unfortunately, we were unable to use genetics to dissect target interactions because no resistant parasites emerged. This outcome suggests that future drugs based on the MMV676477 scaffold would be less likely to be undermined by the emergence of drug resistance.

The tubulin database: Linking mutations, modifications, ligands and local interactions.

Microtubules are polymeric filaments, constructed of α-ß tubulin heterodimers that underlie critical subcellular structures in eukaryotic organisms. Four homologous proteins (γ-, δ-, ε- and ζ-tubulin) additionally contribute to specialized microtubule functions. Although there is an immense volume of publicly available data pertaining to tubulins, it is difficult to assimilate all potentially relevant information across diverse organisms, isotypes, and categories of data. We previously assembled an extensive web-based catalogue of published missense mutations to tubulins with >1,500 entries that each document a specific substitution to a discrete tubulin, the species where the mutation was described and the associated phenotype with hyperlinks to the amino acid sequence and citation(s) for research. This report describes a significant update and expansion of our online resource (TubulinDB.bio.uci.edu) to nearly 18,000 entries. It now encompasses a cross-referenced catalog of post-translational modifications (PTMs) to tubulin drawn from public datasets, primary literature, and predictive algorithms. In addition, tubulin protein structures were used to define local interactions with bound ligands (GTP, GDP and diverse microtubule-targeting agents) and amino acids at the intradimer interface, within the microtubule lattice and with associated proteins. To effectively cross-reference these datasets, we established a universal tubulin numbering system to map entries into a common framework that accommodates specific insertions and deletions to tubulins. Indexing and cross-referencing permitted us to discern previously unappreciated patterns. We describe previously unlinked observations of loss of PTM sites in the context of cancer cells and tubulinopathies. Similarly, we expanded the set of clinical substitutions that may compromise MAP or microtubule-motor interactions by collecting tubulin missense mutations that alter amino acids at the interface with dynein and doublecortin. By expanding the database as a curated resource, we hope to relate model organism data to clinical findings of pathogenic tubulin variants. Ultimately, we aim to aid researchers in hypothesis generation and design of studies to dissect tubulin function.

The Tubulin Superfamily in Apicomplexan Parasites.

Microtubules and specialized microtubule-containing structures are assembled from tubulins, an ancient superfamily of essential eukaryotic proteins. Here, we use bioinformatic approaches to analyze features of tubulins in organisms from the phylum Apicomplexa. Apicomplexans are protozoan parasites that cause a variety of human and animal infectious diseases. Individual species harbor one to four genes each for α- and ß-tubulin isotypes. These may specify highly similar proteins, suggesting functional redundancy, or exhibit key differences, consistent with specialized roles. Some, but not all apicomplexans harbor genes for δ- and ε-tubulins, which are found in organisms that construct appendage-containing basal bodies. Critical roles for apicomplexan δ- and ε-tubulin are likely to be limited to microgametes, consistent with a restricted requirement for flagella in a single developmental stage. Sequence divergence or the loss of δ- and ε-tubulin genes in other apicomplexans appears to be associated with diminished requirements for centrioles, basal bodies, and axonemes. Finally, because spindle microtubules and flagellar structures have been proposed as targets for anti-parasitic therapies and transmission-blocking strategies, we discuss these ideas in the context of tubulin-based structures and tubulin superfamily properties.

Inhibiting parasite proliferation using a rationally designed anti-tubulin agent.

Infectious diseases caused by apicomplexan parasites remain a global public health threat. The presence of multiple ligand-binding sites in tubulin makes this protein an attractive target for anti-parasite drug discovery. However, despite remarkable successes as anti-cancer agents, the rational development of protozoan parasite-specific tubulin drugs has been hindered by a lack of structural and biochemical information on protozoan tubulins. Here, we present atomic structures for a protozoan tubulin and microtubule and delineate the architectures of apicomplexan tubulin drug-binding sites. Based on this information, we rationally designed the parasite-specific tubulin inhibitor parabulin and show that it inhibits growth of parasites while displaying no effects on human cells. Our work presents for the first time the rational design of a species-specific tubulin drug providing a framework to exploit structural differences between human and protozoa tubulin variants enabling the development of much-needed, novel parasite inhibitors.

Auranofin Resistance in Toxoplasma gondii Decreases the Accumulation of Reactive Oxygen Species but Does Not Target Parasite Thioredoxin Reductase.

Auranofin, a reprofiled FDA-approved drug originally designed to treat rheumatoid arthritis, has emerged as a promising anti-parasitic drug. It induces the accumulation of reactive oxygen species (ROS) in parasites, including Toxoplasma gondii. We generated auranofin resistant T. gondii lines through chemical mutagenesis to identify the molecular target of this drug. Resistant clones were confirmed with a competition assay using wild-type T. gondii expressing yellow fluorescence protein (YFP) as a reference strain. The predicted auranofin target, thioredoxin reductase, was not mutated in any of our resistant lines. Subsequent whole genomic sequencing analysis (WGS) did not reveal a consensus resistance locus, although many have point mutations in genes encoding redox-relevant proteins such as superoxide dismutase (TgSOD2) and ribonucleotide reductase. We investigated the SOD2 L201P mutation and found that it was not sufficient to confer resistance when introduced into wild-type parasites. Resistant clones accumulated less ROS than their wild type counterparts. Our results demonstrate that resistance to auranofin in T. gondii enhances its ability to abate oxidative stress through diverse mechanisms. This evidence supports a hypothesized mechanism of auranofin anti-parasitic activity as disruption of redox homeostasis.

Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent.

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

A Toxoplasma gondii patatin-like phospholipase contributes to host cell invasion.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of any warm-blooded animal. In a previous screen to identify virulence determinants, disruption of gene TgME49_305140 generated a T. gondii mutant that could not establish a chronic infection in mice. The protein product of TgME49_305140, here named TgPL3, is a 277 kDa protein with a patatin-like phospholipase (PLP) domain and a microtubule binding domain. Antibodies generated against TgPL3 show that it is localized to the apical cap. Using a rapid selection FACS-based CRISPR/Cas-9 method, a TgPL3 deletion strain (ΔTgPL3) was generated. ΔTgPL3 parasites have defects in host cell invasion, which may be caused by reduced rhoptry secretion. We generated complementation clones with either wild type TgPL3 or an active site mutation in the PLP domain by converting the catalytic serine to an alanine, ΔTgPL3::TgPL3S1409A (S1409A). Complementation of ΔTgPL3 with wild type TgPL3 restored all phenotypes, while S1409A did not, suggesting that phospholipase activity is necessary for these phenotypes. ΔTgPL3 and S1409A parasites are also virtually avirulent in vivo but induce a robust antibody response. Vaccination with ΔTgPL3 and S1409A parasites protected mice against subsequent challenge with a lethal dose of Type I T. gondii parasites, making ΔTgPL3 a compelling vaccine candidate. These results demonstrate that TgPL3 has a role in rhoptry secretion, host cell invasion and survival of T. gondii during acute mouse infection.

The tubulin mutation database: A resource for the cytoskeleton community.

Over the last 40 years, the phenotypic consequences of point mutations to tubulin genes have been described in a wide variety of eukaryotes. A publicly available web-based catalog of all published point mutations to tubulin was assembled. Each entry records a specific substitution to a discrete tubulin, the species where the mutation was described, the associated phenotype, and provides hyperlinks to the parental amino acid sequence and citation(s) for the original research. The data is represented in individual tables for the universal tubulin families (α-, ß-, and γ-tubulins) with the smaller datasets for point mutations to δ-, ε-, and ζ-tubulins individually appended to the γ-tubulin mutation table. Because tubulins are highly conserved proteins, the benefit of organizing the database tables in order of amino acid position is that comparison between equivalent residues in different isotypes or species is straightforward. For example, it was shown that seven substitutions which are associated with human brain malformations known as tubulinopathies were previously identified in other contexts that suggest that they influence microtubule stability. It was anticipated that this resource will simplify evaluation of the role of specific amino acids or domains in microtubule function.

From B to A: making an essential cofactor in a human parasite.

Trypanosomatids are parasitic eukaryotic organisms that cause human disease. These organisms have complex lifestyles; cycling between vertebrate and insect hosts and alternating between two morphologies; a replicating form and an infective, nonreplicating one. Because trypanosomatids are one of the few organisms that do not synthesize the essential cofactor, heme, these parasites sequester the most common form, heme B, from their hosts. Once acquired, the parasites derivatize heme B to heme A by two sequential enzyme reactions. Although heme C is found in many cytochrome c and c1 proteins, heme A is the cofactor of only one known protein, cytochrome c oxidase (CcO). In a recent issue of the Biochemical Journal, Merli et al. [Biochem. J. (2017) 474, 2315-2332] demonstrate that the final step in the synthesis of heme A by heme A synthase (TcCox15) and the subsequent activity of CcO are essential for infectivity and replication of Trypanosoma cruzi.

An ensemble of specifically targeted proteins stabilizes cortical microtubules in the human parasite Toxoplasma gondii.

Although all microtubules within a single cell are polymerized from virtually identical subunits, different microtubule populations carry out specialized and diverse functions, including directional transport, force generation, and cellular morphogenesis. Functional differentiation requires specific targeting of associated proteins to subsets or even subregions of these polymers. The cytoskeleton of Toxoplasma gondii, an important human parasite, contains at least five distinct tubulin-based structures. In this work, we define the differential localization of proteins along the cortical microtubules of T. gondii, established during daughter biogenesis and regulated by protein expression and exchange. These proteins distinguish cortical from mitotic spindle microtubules, even though the assembly of these subsets is contemporaneous during cell division. Finally, proteins associated with cortical microtubules collectively protect the stability of the polymers with a remarkable degree of functional redundancy.

Extracellular Toxoplasma gondii tachyzoites metabolize and incorporate unnatural sugars into cellular proteins.

Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Many of the surface antigens and effector molecules secreted by the parasite during invasion and intracellular growth are modified by glycans. Glycosylated proteins in the nucleus and cytoplasm have also been reported. Despite their prevalence, the complete inventory and biological significance of glycosylated proteins in Toxoplasma remain unknown. In this study, we aimed to globally profile parasite glycoproteins using a bioorthogonal chemical reporter strategy. This strategy involves the metabolic incorporation of unnatural functional groups (i.e., "chemical reporters") into Toxoplasma glycans, followed by covalent labeling with visual probes or affinity tags. The two-step approach enables the visualization and identification of newly biosynthesized glycoconjugates in the parasite. Using a buffer that mimics intracellular conditions, extracellular Toxoplasma tachyzoites were found to metabolize and incorporate unnatural sugars (equipped with bioorthogonal functional groups) into diverse proteins. Covalent chemistries were used to visualize and retrieve these labeled structures. Subsequent mass spectrometry analysis revealed 89 unique proteins. This survey identified novel proteins as well as previously characterized proteins from lectin affinity analyses.

Compartmentalized Toxoplasma EB1 bundles spindle microtubules to secure accurate chromosome segregation.

Toxoplasma gondii replicates asexually by a unique internal budding process characterized by interwoven closed mitosis and cytokinesis. Although it is known that the centrosome coordinates these processes, the spatiotemporal organization of mitosis remains poorly defined. Here we demonstrate that centrosome positioning around the nucleus may signal spindle assembly: spindle microtubules (MTs) are first assembled when the centrosome moves to the basal side and become extensively acetylated after the duplicated centrosomes reposition to the apical side. We also tracked the spindle MTs using the MT plus end-binding protein TgEB1. Endowed by a C-terminal NLS, TgEB1 resides in the nucleoplasm in interphase and associates with the spindle MTs during mitosis. TgEB1 also associates with the subpellicular MTs at the growing end of daughter buds toward the completion of karyokinesis. Depletion of TgEB1 results in escalated disintegration of kinetochore clustering. Furthermore, we show that TgEB1's MT association in Toxoplasma and in a heterologous system (Xenopus) is based on the same principles. Finally, overexpression of a high-MT-affinity TgEB1 mutant promotes the formation of overstabilized MT bundles, resulting in avulsion of otherwise tightly clustered kinetochores. Overall we conclude that centrosome position controls spindle activity and that TgEB1 is critical for mitotic integrity.

Targeting Toxoplasma tubules: tubulin, microtubules, and associated proteins in a human pathogen.

Toxoplasma gondii is an obligate intracellular parasite that causes serious opportunistic infections, birth defects, and blindness in humans. Microtubules are critically important components of diverse structures that are used throughout the Toxoplasma life cycle. As in other eukaryotes, spindle microtubules are required for chromosome segregation during replication. Additionally, a set of membrane-associated microtubules is essential for the elongated shape of invasive "zoites," and motility follows a spiral trajectory that reflects the path of these microtubules. Toxoplasma zoites also construct an intricate, tubulin-based apical structure, termed the conoid, which is important for host cell invasion and associates with proteins typically found in the flagellar apparatus. Last, microgametes specifically construct a microtubule-containing flagellar axoneme in order to fertilize macrogametes, permitting genetic recombination. The specialized roles of these microtubule populations are mediated by distinct sets of associated proteins. This review summarizes our current understanding of the role of tubulin, microtubule populations, and associated proteins in Toxoplasma; these components are used for both novel and broadly conserved processes that are essential for parasite survival.

Basal body structure and composition in the apicomplexans Toxoplasma and Plasmodium.

The phylum Apicomplexa encompasses numerous important human and animal disease-causing parasites, including the Plasmodium species, and Toxoplasma gondii, causative agents of malaria and toxoplasmosis, respectively. Apicomplexans proliferate by asexual replication and can also undergo sexual recombination. Most life cycle stages of the parasite lack flagella; these structures only appear on male gametes. Although male gametes (microgametes) assemble a typical 9+2 axoneme, the structure of the templating basal body is poorly defined. Moreover, the relationship between asexual stage centrioles and microgamete basal bodies remains unclear. While asexual stages of Plasmodium lack defined centriole structures, the asexual stages of Toxoplasma and closely related coccidian apicomplexans contain centrioles that consist of nine singlet microtubules and a central tubule. There are relatively few ultra-structural images of Toxoplasma microgametes, which only develop in cat intestinal epithelium. Only a subset of these include sections through the basal body: to date, none have unambiguously captured organization of the basal body structure. Moreover, it is unclear whether this basal body is derived from pre-existing asexual stage centrioles or is synthesized de novo. Basal bodies in Plasmodium microgametes are thought to be synthesized de novo, and their assembly remains ill-defined. Apicomplexan genomes harbor genes encoding δ- and ε-tubulin homologs, potentially enabling these parasites to assemble a typical triplet basal body structure. Moreover, the UNIMOD components (SAS6, SAS4/CPAP, and BLD10/CEP135) are conserved in these organisms. However, other widely conserved basal body and flagellar biogenesis elements are missing from apicomplexan genomes. These differences may indicate variations in flagellar biogenesis pathways and in basal body arrangement within the phylum. As apicomplexan basal bodies are distinct from their metazoan counterparts, it may be possible to selectively target parasite structures in order to inhibit microgamete motility which drives generation of genetic diversity in Toxoplasma and transmission for Plasmodium.

Diaryl and heteroaryl sulfides: synthesis via sulfenyl chlorides and evaluation as selective anti-breast-cancer agents.

A mild protocol for the synthesis of diaryl and heteroaryl sulfides is described. In a one-pot procedure, thiols are converted to sulfenyl chlorides and reacted with arylzinc reagents. This method tolerates functional groups including aryl fluorides and chlorides, ketones, as well as N-heterocycles including pyrimidines, imidazoles, tetrazoles, and oxadiazoles. Two compounds synthesized by this method exhibited selective activity against the MCF-7 breast cancer cell line in the micromolar range.

Stereospecific nickel-catalyzed cross-coupling reactions of alkyl Grignard reagents and identification of selective anti-breast-cancer agents.

Alkyl Grignard reagents that contain ß-hydrogen atoms were used in a stereospecific nickel-catalyzed cross-coupling reaction to form C(sp(3))-C(sp(3)) bonds. Aryl Grignard reagents were also utilized to synthesize 1,1-diarylalkanes. Several compounds synthesized by this method exhibited selective inhibition of proliferation of MCF-7 breast cancer cells.

A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved from flagellar components.

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.

Cell division in Apicomplexan parasites is organized by a homolog of the striated rootlet fiber of algal flagella.

Apicomplexa are intracellular parasites that cause important human diseases including malaria and toxoplasmosis. During host cell infection new parasites are formed through a budding process that parcels out nuclei and organelles into multiple daughters. Budding is remarkably flexible in output and can produce two to thousands of progeny cells. How genomes and daughters are counted and coordinated is unknown. Apicomplexa evolved from single celled flagellated algae, but with the exception of the gametes, lack flagella. Here we demonstrate that a structure that in the algal ancestor served as the rootlet of the flagellar basal bodies is required for parasite cell division. Parasite striated fiber assemblins (SFA) polymerize into a dynamic fiber that emerges from the centrosomes immediately after their duplication. The fiber grows in a polarized fashion and daughter cells form at its distal tip. As the daughter cell is further elaborated it remains physically tethered at its apical end, the conoid and polar ring. Genetic experiments in Toxoplasma gondii demonstrate two essential components of the fiber, TgSFA2 and 3. In the absence of either of these proteins cytokinesis is blocked at its earliest point, the initiation of the daughter microtubule organizing center (MTOC). Mitosis remains unimpeded and mutant cells accumulate numerous nuclei but fail to form daughter cells. The SFA fiber provides a robust spatial and temporal organizer of parasite cell division, a process that appears hard-wired to the centrosome by multiple tethers. Our findings have broader evolutionary implications. We propose that Apicomplexa abandoned flagella for most stages yet retained the organizing principle of the flagellar MTOC. Instead of ensuring appropriate numbers of flagella, the system now positions the apical invasion complexes. This suggests that elements of the invasion apparatus may be derived from flagella or flagellum associated structures.

SPM1 stabilizes subpellicular microtubules in Toxoplasma gondii.

We have identified two novel proteins that colocalize with the subpellicular microtubules in the protozoan parasite Toxoplasma gondii and named these proteins SPM1 and SPM2. These proteins have basic isoelectric points and both have homologs in other apicomplexan parasites. SPM1 contains six tandem copies of a 32-amino-acid repeat, whereas SPM2 lacks defined protein signatures. Alignment of Toxoplasma SPM2 with apparent Plasmodium SPM2 homologs indicates that the greatest degree of conservation lies in the carboxy-terminal half of the protein. Analysis of Plasmodium homologs of SPM1 indicates that while the central 32-amino-acid repeats have expanded to different degrees (7, 8, 9, 12, or 13 repeats), the amino- and carboxy-terminal regions remain conserved. In contrast, although the Cryptosporidium SPM1 homolog has a conserved carboxy tail, the five repeats are considerably diverged, and it has a smaller amino-terminal domain. SPM1 is localized along the full length of the subpellicular microtubules but does not associate with the conoid or spindle microtubules. SPM2 has a restricted localization along the middle region of the subpellicular microtubules. Domain deletion analysis indicates that four or more copies of the SPM1 repeat are required for localization to microtubules, and the amino-terminal 63 residues of SPM2 are required for localization to the subpellicular microtubules. Gene deletion studies indicate that neither SPM1 nor SPM2 is essential for tachyzoite viability. However, loss of SPM1 decreases overall parasite fitness and eliminates the stability of subpellicular microtubules to detergent extraction.