About
78
Publications
11,797
Reads
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
4,927
Citations
Publications
Publications (78)
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 antimito...
Microtubules are polymeric filaments, constructed of α-β 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,...
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...
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...
Abstract 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 tu...
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 t...
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...
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 in order to identify the m...
The Toxoplasma gondii cytoskeleton is critically important to parasite motility, host cell invasion, tensile strength, subcellular organization, vesicle trafficking, nuclear division, formation of daughter cells, and microgamete motility. It consists of microtubules, actin filaments, intermediate filament–like proteins, centrins, kinesin, dynein an...
Over the last 40 years, the phenotypic consequences of point mutations to tubulin genes have been described in a wide variety of eukaryotes. We have assembled a publicly available web‐based catalogue of all published point mutations to tubulin. Each entry records a specific substitution to a discrete tubulin, the species where the mutation was desc...
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...
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 lac...
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 ev...
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. De...
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...
Toxoplasma gondii invasive forms or “zoites” are highly polarized cells with an elaborate cytoskeleton. Some components of the cytoskeleton, such as tubulin, are conserved throughout eukaryotes while others, such as the inner membrane complex proteins, are unique to the Apicomplexa or alveolates. The cytoskeletal protein complexes provide a structu...
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...
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...
Alkyl Grignard reagents that contain β-hydrogen atoms were used in a stereospecific nickel-catalyzed cross-coupling reaction to form C(sp 3)-C(sp3) 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 cell...
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 Toxop...
Schematic representation of the constructs engineered for obtain the iΔSFA3 and iΔSFA2 conditional knock out strains and PCR screens. (A) Cosmid PSBLE51 containing the entire TgSFA3 gene (TgME49_ 018880) and several kilo bases upstream of the start codon was modified by recombineering a mutagenic cassette positioning an inducible promoter immediate...
Confirmation of the insertion of a triple hemagglutinin tag at the endogenous locus of TgSFA2. (A) Southern blot of the parental strain, the polyclonal transfected population, and the SFA2-HA clone used in this study is shown. (B) A radioactively labeled probe complementary to the 3′ end of TgSFA2 was used for hybridization and is represented with...
The SFA fiber is dynamic and grows off the centrosome in dividing parasites. Compilation of time-lapse images of a single SFA3-YFP (green) parasites transiently co-expressing Centrin1-RFP (red). Centrin1 is a marker for the centrosome. Note that only one fiber and one centrosome appear in the focal plane. Images were taken every 10 min for 4 h. Rap...
Name and sequence of all primers used in this study.
(PDF)
SFAs form a dynamic structure and their expression is temporarily regulated. Compilation of time-lapse images of SFA3-YFP parasites. Two parasite vacuoles are shown. The uppermost vacuole contains four parasites, while the lowermost vacuole contains two. Parasites outlines were obtained from overlaying fluorescence images with DIC images prior to c...
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...
Toxoplasma gondii is an obligate intracellular parasite that infects all nucleated cell types in diverse warm-blooded organisms. Infection (which occurs by ingestion of tissue cysts or oocysts) is life-threatening to immunocompromised individuals. Parasite effector molecules transit through the Toxoplasma ER and Golgi to specialized membrane compar...
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 lac...
Human infection with the protozoan parasite Toxoplasma gondii can cause serious disease in immunocompromised individuals and the developing fetus. Human infection is characterized by the tachyzoite (acute infection) and bradyzoite (encysted form, latent infection) stages. Current therapies are ineffective against the bradyzoite stage of infection b...
Tubulin is a highly conserved, negatively charged protein that is found in essentially all eukaryotic cells. These properties ensure that isolation protocols successful in one system will likely work, with a few modifications, in most systems. Tubulin has been isolated most frequently from mammalian brain, and the main difference encountered in oth...
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 d...
Toxoplasma gondii is an obligate intracellular protozoan parasite that invades and replicates within most nucleated cells of warm-blooded animals.
The basis for this wide host cell tropism is unknown but could be because parasites invade host cells using distinct pathways
and/or repertoires of host factors. Using synchronized parasite invasion assa...
ISP ortholog groups. OrthoMCL DB (www.orthomcl.org) was utilized to identify ortholog groups for the ISP family. ISP1 and 2 belong to one OrthoMCL group (OG4_23348) (A) while ISP3 belongs to another group (OG4_34375) (B). The ISP1 and 2 group contains proteins from all apicomplexans available in the OrthoMCL DB while the ISP3 group contains only pr...
Mutation of ISP3 residues predicted for acylation results in ISP3 mistargeting. Mutations of residues predicted for myristoylation or palmitoylation were generated in an HA epitope-tagged copy of ISP3 and expressed in parasites under the control of the endogenous promoter. A severe targeting defect occurs in ISP3 (G2A) with the mutant protein dispe...
Mutation of ISP2 residues predicted for acylation results in ISP2 mistargeting. Mutations of residues predicted for myristoylation or palmitoylation were generated in an HA epitope-tagged copy of ISP2 and expressed in parasites under the control of the endogenous promoter. A severe targeting defect occurs in ISP2 (G2A) in which ISP2 signal is dispe...
Primers used in this study as discussed in text. Restriction sites and mutated bases are shown in lowercase.
(2.29 MB TIF)
ISP1 and 3 during Toxoplasma endodyogeny. Parasites stably expressing ISP3-HA were allowed to infect HFFs and grow 24 hrs before fixation and IFA analysis. Serial sections were acquired, deconvolved and projected as a three-dimensional image. Visualization of ISP1 and ISP3 during endodyogeny shows that ISP1 is present in the maternal IMC apical cap...
Disruption of ISP3. Western blot analysis using polyclonal anti-ISP3 confirms the loss of ISP3 in Δisp3 parasites. ROP1 serves as a loading control.
(0.49 MB TIF)
Antibody confirmation of sub-compartment localizations for endogenous ISP2 and ISP3. A. ISP2 antisera confirms the localization of endogenous ISP2 to the central IMC sub-compartment in a fashion identical to the HA epitope-tagged ISP2 shown in Figure 2B. Endogenous ISP2 is clearly absent from the apical cap (brackets) and basal portion of the IMC....
ISP1 early bud rings in oryzalin treated Toxoplasma. Parasites stably expressing ISP1-HA were allowed to infect HFFs in the presence of 0.5 µM oryzalin and grow 30 hours before fixation and IFA analysis. Serial sections were acquired, deconvolved and projected as a three-dimensional image. In the absence of cortical microtubules, a single mother pa...
In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the si...
Author Summary
Apicomplexans are the cause of important diseases in humans and animals including malaria (Plasmodium falciparum), which claims over a million human lives each year, and toxoplasmosis (Toxoplasma gondii), which causes birth defects and neurological disorders. These parasites possess a unique cortical system of membrane sacs arranged...
The asexually proliferating stages of apicomplexan parasites cause acute symptoms of diseases such as malaria, cryptosporidiosis and toxoplasmosis. These stages are characterized by the presence of two independent microtubule organizing centers (MTOCs). Centrioles are found at the poles of the intranuclear spindle. The apical polar ring (APR), a MT...
The synthesis and evaluation of 20 dinitroanilines and related compounds against the obligate intracellular parasite Toxoplasma gondii is reported. Using in vitro cultures of parasites in human fibroblasts, we determined that most of these compounds selectively disrupted Toxoplasma microtubules, and several displayed sub-micromolar potency against...
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...
In the century since the first description of Toxoplasma gondii history and circumstance have led scientists to define this organism in diverse contexts. From its discovery by researchers shaped by early 20th century versions of the germ theory to its more recent roles as an important globally distributed pathogen and a model apicomplexan, our defi...
The dinitroanilines are small molecules that selectively bind to plant and protozoan tubulin dimers and disrupt microtubules
in these organisms. Despite the high degree of sequence conservation among all tubulins, these compounds do not bind to tubulins
from fungi or vertebrates, nor are microtubules in these organisms disrupted by dinitroaniline t...
Fas-associated death domain protein (FADD) and caspase-8 (casp8) are vital intermediaries in apoptotic signaling induced by tumor necrosis factor family ligands. Paradoxically, lymphocytes lacking FADD or casp8 fail to undergo normal clonal expansion following antigen receptor cross-linking and succumb to caspase-independent cell death upon activat...
In the course of studies to identify novel treatment strategies against the pathogenic bacterium, Chlamydia, we tested the carrier peptide, Pep-1, for activity against an intracellular infection.
Using a cell culture model of Chlamydia trachomatis infection, the effect of Pep-1 was measured by incubating the peptide with extracellular chlamydiae pr...
Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing alpha1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relat...
FADD and caspase‐8 (casp8) transduce an apoptotic signal through tumor necrosis factor family members. Paradoxically, lymphocytes lacking FADD or casp8 function fail to undergo normal clonal expansion and instead succumb to caspase‐independent cell death. However, the exact nature of the survival defects of these T cells has remained elusive. We sh...
Protozoan microtubules are sensitive to disruption by dinitroanilines, compounds that kill intracellular Toxoplasma gondii parasites without affecting microtubules in vertebrate host cells. We previously isolated a number of resistant Toxoplasma lines that harbor mutations to the alpha1-tubulin gene. Some of the mutations are localized in or near t...
Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in alpha-tubul...
Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walke...
The Apicomplexa are a phylum of diverse obligate intracellular parasites including Plasmodium spp., the cause of malaria; Toxoplasma gondii and Cryptosporidium parvum, opportunistic pathogens of immunocompromised individuals; and Eimeria spp. and Theileria spp., parasites of considerable agricultural importance. These protozoan parasites share dist...
The tachyzoite stage of the protozoan parasite Toxoplasma gondii has two populations of microtubules: spindle microtubules and subpellicular microtubules. To determine how these two microtubule populations are regulated, we investigated microtubule behavior during the cell cycle following treatment with microtubule-disrupting drugs. Previous work h...
Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive immune response. Amphiphysin II participates in receptor-mediated endocytosis, in part, by recruiting the GTPase dynamin to the nascent endosome. We demonstrate here that a novel isoform of amphiphysin II associates with early phag...
In order to identify novel proteins associated with various stages of macrophage phagocytosis, we have generated monoclonal antibodies that recognize phagosomes. Purified Fc receptor-mediated phagosomes, isolated by feeding IgG-conjugated magnetic beads to LPS-primed murine peritoneal macrophages, were used as the immunogen. An immunofluorescence s...
Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on e...
In the spirit of the Leiden Conferences, the first Keystone Meeting on Macrophage Biology brought together scientists from a wide range of disciplines. Precisely because macrophages perform such diverse functions, this meeting had an extraordinarily broad scope. While this was exhilarating to experience, it is impossible to report in full. We have...
Antigens associated with the conoid and apical pellicle in Apicomplexan parasites have previously been defined by a panel of monoclonal antibodies that recognize distinct but overlapping protein subsets (Morrissette et al. 1994). To probe the association of these antigens with the apical membrane skeleton of Toxoplasma gondii, we examined their dis...
Application of Fourier analysis techniques to images of isolated, frozen-hydrated subpellicular microtubules from the protozoan parasite Toxoplasma gondii demonstrates a distinctive 32 nm periodicity along the length of the microtubules. A 32 nm longitudinal repeat is also observed in the double rows of intramembranous particles seen in freeze-frac...
Submicromolar concentrations of several dinitroaniline herbicides have been found to specifically inhibit intracellular replication of the protozoan parasite Toxoplasma gondii. IC50 concentrations for T. gondii survival were approximately 100 nM for ethalfluralin and oryzalin and approximately 300 nM for trifluralin. Primary human fibroblasts emplo...
The genetic structure of Toxoplasma gondii is notable chiefly for being relatively conventional— similar to that of its mammalian host cells with respect to gene organization, codon usage, and nucleotide bias. These observations have led several investigators to examine the feasibility of molecular transformation in this parasite. This chapter outl...
The protozoan parasite Toxoplasma gondii is a highly polarized and motile organism, but little is known of the structural basis for these essential properties. Invasion of host cells by this obligate intracellular pathogen is thought to be facilitated by a specialized complex of apical organelles. The apical cytoskeleton includes the conoid and twe...
We have isolated 26 monoclonal antibodies which specifically recognize the extreme apex of Toxoplasma gondii, a protozoan parasite which attaches to and invades host cells via its specialized apical end. The unique apical organelles which define the phylum Apicomplexa are thought to be involved in mechanical and enzymatic aspects of invasion. Immun...
