Thursday, March 05, 2015

Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii

2015 Feb 28. pii: S1046-5928(15)00029-7. doi: 10.1016/j.pep.2015.02.017. [Epub ahead of print]
 
 
Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous E. coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural-functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS:

Crystallization; Drug discovery; MARS complex; tRNA synthetase
PMID:
25736594
[PubMed - as supplied by publisher]

Wednesday, March 04, 2015

A Novel Bipartite Centrosome Coordinates the Apicomplexan Cell Cycle

 2015 Mar 3;13(3):e1002093. doi: 10.1371/journal.pbio.1002093. eCollection 2015.

Abstract

Apicomplexan parasites can change fundamental features of cell division during their life cycles, suspending cytokinesis when needed and changing proliferative scale in different hosts and tissues. The structural and molecular basis for this remarkable cell cycle flexibility is not fully understood, although the centrosome serves a key role in determining when and how much replication will occur. Here we describe the discovery of multiple replicating core complexes with distinct protein composition and function in the centrosome of Toxoplasma gondii. An outer core complex distal from the nucleus contains the TgCentrin1/TgSfi1 protein pair, along with the cartwheel protein TgSas-6 and a novel Aurora-related kinase, while an inner core closely aligned with the unique spindle pole (centrocone) holds distant orthologs of the CEP250/C-Nap protein family. This outer/inner spatial relationship of centrosome cores is maintained throughout the cell cycle. When in metaphase, the duplicated cores align to opposite sides of the kinetochores in a linear array. As parasites transition into S phase, the cores sequentially duplicate, outer core first and inner core second, ensuring that each daughter parasite inherits one copy of each type of centrosome core. A key serine/threonine kinase distantly related to the MAPK family is localized to the centrosome, where it restricts core duplication to once per cycle and ensures the proper formation of new daughter parasites. Genetic analysis of the outer core in a temperature-sensitive mutant demonstrated this core functions primarily in cytokinesis. An inhibition of ts-TgSfi1 function at high temperature caused the loss of outer cores and a severe block to budding, while at the same time the inner core amplified along with the unique spindle pole, indicating the inner core and spindle pole are independent and co-regulated. The discovery of a novel bipartite organization in the parasite centrosome that segregates the functions of karyokinesis and cytokinesis provides an explanation for how cell cycle flexibility is achieved in apicomplexan life cycles.
PMID:
 
25734885
 
[PubMed - as supplied by publisher] 

Tuesday, March 03, 2015

Neurobiological studies on the relationship between toxoplasmosis and neuropsychiatric diseases

2015 Feb 21. pii: S0022-510X(15)00095-7. doi: 10.1016/j.jns.2015.02.028. [Epub ahead of print]
 
 
Toxoplasma gondii is a widespread protozoan parasite infecting approximately one third of the world population. After proliferation of tachyzoites during the acute stage, the parasite forms tissue cysts in various anatomical sites including the Central Nervous tissue, and establishes a chronic infection. Clinical spectrum normally ranges from a completely asymptomatic infection to severe multi-organ involvement. Many studies have suggested T. gondii infection as a risk factor for the development of some neuropsychiatric disorders, particularly schizophrenia. During the last years, a potential link with other neurobiological diseases such as Parkinson disease and Alzheimer disease has also been suggested. This review will focus on neurobiological and epidemiological data relating infection with T. gondii to neuropsychiatric diseases.
Copyright © 2015. Published by Elsevier B.V.

KEYWORDS:

Immune response; Neurobiology; Neurological diseases; Neurotransmitter pathway; Schizophrenia spectrum disorders; T. gondii
PMID:
25725931
[PubMed - as supplied by publisher]

Monday, March 02, 2015

Secreted Toxoplasma gondii molecules interfere with expression of MHC-II in interferon gamma-activated macrophages

 2015 Feb 23. pii: S0020-7519(15)00024-7. doi: 10.1016/j.ijpara.2015.01.003. [Epub ahead of print]

Abstract

The obligate intracellular protozoan parasite Toxoplasma gondii interferes with major histocompatibility complex (MHC)-II antigen presentation to dampen host CD4+ T cell responses. While it is known that T. gondii inhibits MHC-II gene transcription and expression in infected host cells, the mechanism of this host manipulation is unknown. Here, we show that soluble parasite proteins inhibit IFNγ-induced expression of MHC-II on the surface of the infected cell in a dose-dependent response that was abolished by protease treatment. Subcellular fractionation of T. gondii tachyzoites revealed that the MHC-II inhibitory activity co-partitioned with rhoptries (ROP) and/or dense granules (GRA). However, parasite mutants deleted for single ROP or GRA genes (ROP1, 4/7, 14, 16 and 18 or GRA 2 - 9 and 12 knock-out strains) retained the ability to inhibit expression of MHC-II. In addition, excreted/secreted antigens (ESA) released by extracellular tachyzoites displayed immunomodulatory activity characterized by an inhibition of MHC-II expression, and reduced expression and release of TNFα by macrophages. Tandem MS analysis of parasite ESA generated a list of T. gondii secreted proteins that may participate in MHC-II inhibition and the modulation of host immune functions.
Copyright © 2015. Published by Elsevier Ltd.

KEYWORDS: 

Antigen presentation; Excreted/secreted antigens; Immunomodulation; Major histocompatibility complex II; Secretory organelles; Toxoplasma gondii
PMID:
 
25720921
 
[PubMed - as supplied by publisher] 

Thursday, February 26, 2015

Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination

2015 Feb 24;6(2). pii: e02280-14. doi: 10.1128/mBio.02280-14.
 
 
The intracellular parasite Toxoplasma gondii infects a wide variety of vertebrate species globally. Infection in most hosts causes a lifelong chronic infection and generates immunological memory responses that protect the host against new infections. In regions where the organism is endemic, multiple exposures to T. gondii likely occur with great frequency, yet little is known about the interaction between a chronically infected host and the parasite strains from these areas. A widely used model to explore secondary infection entails challenge of chronically infected or vaccinated mice with the highly virulent type I RH strain. Here, we show that although vaccinated or chronically infected C57BL/6 mice are protected against the type I RH strain, they are not protected against challenge with most strains prevalent in South America or another type I strain, GT1. Genetic and genomic analyses implicated the parasite-secreted rhoptry effectors ROP5 and ROP18, which antagonize the host's gamma interferon-induced immunity-regulated GTPases (IRGs), as primary requirements for virulence during secondary infection. ROP5 and ROP18 promoted parasite superinfection in the brains of challenged survivors. We hypothesize that superinfection may be an important mechanism to generate T. gondii strain diversity, simply because two parasite strains would be present in a single meal consumed by the feline definitive host. Superinfection may drive the genetic diversity of Toxoplasma strains in South America, where most isolates are IRG resistant, compared to North America, where most strains are IRG susceptible and are derived from a few clonal lineages. In summary, ROP5 and ROP18 promote Toxoplasma virulence during reinfection.

IMPORTANCE:

Toxoplasma gondii is a widespread parasite of warm-blooded animals and currently infects one-third of the human population. A long-standing assumption in the field is that prior exposure to this parasite protects the host from subsequent reexposure, due to the generation of protective immunological memory. However, this assumption is based on clinical data and mouse models that analyze infections with strains common to Europe infections with strains common to Europe and North America. In contrast, we found that the majority of strains sampled from around the world, in particular those from South America, were able to kill or reinfect the brains of hosts previously exposed to T. gondii. The T. gondii virulence factors ROP5 and ROP18, which inhibit key host effectors that mediate parasite killing, were required for these phenotypes. We speculate that these results underpin clinical observations that pregnant women previously exposed to Toxoplasma can develop congenital infection upon reexposure to South American strains.
Copyright © 2015 Jensen et al.
PMID:
25714710
[PubMed - as supplied by publisher]

Ly6Chigh Monocytes Control Cerebral Toxoplasmosis

2015 Feb 20. pii: 1402037. [Epub ahead of print]
 
 
Cerebral infection with the parasite Toxoplasma gondii is followed by activation of resident cells and recruitment of immune cells from the periphery to the CNS. In this study, we show that a subset of myeloid cells, namely Ly6ChighCCR2+ inflammatory monocytes that infiltrate the brain upon chronic T. gondii infection, plays a decisive role in host defense. Depletion of this monocyte subset resulted in elevated parasite load and decreased survival of infected mice, suggesting their crucial role. Notably, Ly6ChighCCR2+ monocytes governed parasite control due to production of proinflammatory mediators, such as IL-1α, IL-1β, IL-6, inducible NO synthase, TNF, and reactive oxygen intermediate. Interestingly, Ly6ChighCCR2+ monocytes were also able to produce the regulatory cytokine IL-10, revealing their dual feature. Moreover, we confirmed by adoptive transfer that the recruited monocytes further develop into two distinct subpopulations contributing to parasite control and profound host defense. The differentiated Ly6CintCCR2+F4/80int subset upregulated MHC I and MHC II molecules, suggesting dendritic cell properties such as interaction with T cells, whereas the Ly6CnegF4/80high cell subset displayed elevated phagocytic capacity while upregulating triggering receptor expressed on myeloid cells-2. Finally, we have shown that the recruitment of Ly6Chigh monocytes to the CNS is regulated by P-selectin glycoprotein ligand-1. These results indicate the critical importance of recruited Ly6Chigh monocytes upon cerebral toxoplasmosis and reveal the behavior of further differentiated myeloid-derived mononuclear cell subsets in parasite control and immune regulation of the CNS.
Copyright © 2015 by The American Association of Immunologists, Inc.
PMID:
25710908
[PubMed - as supplied by publisher]

Toxoplasma gondii: biochemical and biophysical characterization of recombinant soluble dense granule proteins GRA2 and GRA6

2015 Feb 21. pii: S0006-291X(15)00306-X. doi: 10.1016/j.bbrc.2015.02.078. [Epub ahead of print]
 
The most prominent structural feature of the parasitophorous vacuole (PV) in which the intracellular parasite Toxoplasma gondii proliferates is a membranous nanotubular network (MNN), which interconnects the parasites and the PV membrane. The MNN function remains unclear. The GRA2 and GRA6 proteins secreted from the parasite dense granules into the PV have been implicated in the MNN biogenesis. Amphipathic alpha-helices (AAHs) predicted in GRA2 and an alpha-helical hydrophobic domain predicted in GRA6 have been proposed to be responsible for their membrane association, thereby potentially molding the MMN in its structure. Here we report an analysis of the recombinant proteins (expressed in detergent-free conditions) by circular dichroism, which showed that full length GRA2 displays an alpha-helical secondary structure while recombinant GRA6 and GRA2 truncated of its AAHs are mainly random coiled. Dynamic light scattering and transmission electron microscopy showed that recombinant GRA6 and truncated GRA2 constitute a homogenous population of small particles (6-8 nm in diameter) while recombinant GRA2 corresponds to 2 populations of particles (∼8-15 nm and up to 40 nm in diameter, respectively). The unusual properties of GRA2 due to its AAHs are discussed.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS:

Amphipathic alpha-helices; Toxoplasma gondii; circular dichroism; dense granule proteins (GRA); dynamic light scattering; transmission electron microscopy
PMID:
25712518
[PubMed - as supplied by publisher]

Friday, February 20, 2015

IL-33/ST2 involves the immunopathology of ocular toxoplasmosis in murine model

2015 Feb 20. [Epub ahead of print]
 
 
Ocular toxoplasmosis (OT) is the major cause of infective uveitis. Since the eye is a special organ protected by immune privilege, its immune response is different from general organs with Toxoplasma gondii infection. Here, we used Kunming outbred mice to establish OT by intravitreal injection of T. gondii RH strain tachyzoites, IL-33 expression in the eyes was localized by immunostaining, the levels of interleukin (IL)-33 and ST2 (IL-33 receptor) and T-helper (Th)1 and Th2-associated cytokines in the eye and cervical lymph nodes (CLNs) of infected mice were measured, and their correlations were analyzed. Our results showed that the pathologies of the eye and CLN tissues and the IL-33 positive cells in the eye tissues of ocular T. gondii-infected mice were all increased at days 2, 6, and 9 postinfection (p.i.), accompanied with significantly increased transcript levels of IL-33, ST2, IL-1β, IFN-γ, IL-12p40, IL-10, and IL-13 in both the eyes and CLNs, and increased IL-4 expressions in the eyes of T. gondii-infected mice. There were significant correlations between the levels of IFN-γ and ST2, IL-4 and ST2, and IL-13 and ST2 in the eye tissues (P < 0.001), significant correlations between the levels of IFN-γ and ST2 (P < 0.001) as well as between IL-13 and ST2 (P < 0.05) in the CLNs, and significant correlations between the levels of IL-1β and IL-33 in the eyes (P < 0.05) and between IL-1β and IL-33/ST2 in the CLNs (P < 0.001 and P < 0.01, respectively). Our data indicated that IL-33/ST2 may involve the regulation of ocular immunopathology induced by T. gondii infection.
PMID:
25693767
[PubMed - as supplied by publisher]

Toxoplasmosis and Epilepsy - Systematic Review and Meta Analysis

2015 Feb 19;9(2):e0003525. doi: 10.1371/journal.pntd.0003525. eCollection 2015.
 

BACKGROUND:

Toxoplasmosis is an important, widespread, parasitic infection caused by Toxoplasma gondii. The chronic infection in immunocompetent patients, usually considered as asymptomatic, is now suspected to be a risk factor for various neurological disorders, including epilepsy. We aimed to conduct a systematic review and meta-analysis of the available literature to estimate the risk of epilepsy due to toxoplasmosis.

METHODS:

A systematic literature search was conducted of several databases and journals to identify studies published in English or French, without date restriction, which looked at toxoplasmosis (as exposure) and epilepsy (as disease) and met certain other inclusion criteria. The search was based on keywords and suitable combinations in English and French. Fixed and random effects models were used to determine odds ratios, and statistical significance was set at 5.0%.

PRINCIPAL FINDINGS:

Six studies were identified, with an estimated total of 2888 subjects, of whom 1280 had epilepsy (477 positive for toxoplasmosis) and 1608 did not (503 positive for toxoplasmosis). The common odds ratio (calculated) by random effects model was 2.25 (95% CI 1.27-3.9), p = 0.005.

CONCLUSIONS:

Despite the limited number of studies, and a lack of high-quality data, toxoplasmosis should continue to be regarded as an epilepsy risk factor. More and better studies are needed to determine the real impact of this parasite on the occurrence of epilepsy.
PMID:
25695802
[PubMed - as supplied by publisher]

Thursday, February 19, 2015

Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

2015 Feb 17;6(1). pii: e02357-14. doi: 10.1128/mBio.02357-14.
 
 
The inner membrane complex (IMC) of Toxoplasma gondii is a peripheral membrane system that is composed of flattened alveolar sacs that underlie the plasma membrane, coupled to a supporting cytoskeletal network. The IMC plays important roles in parasite replication, motility, and host cell invasion. Despite these central roles in the biology of the parasite, the proteins that constitute the IMC are largely unknown. In this study, we have adapted a technique named proximity-dependent biotin identification (BioID) for use in T. gondii to identify novel components of the IMC. Using IMC proteins in both the alveoli and the cytoskeletal network as bait, we have uncovered a total of 19 new IMC proteins in both of these suborganellar compartments, two of which we functionally evaluate by gene knockout. Importantly, labeling of IMC proteins using this approach has revealed a group of proteins that localize to the sutures of the alveolar sacs that have been seen in their entirety in Toxoplasma species only by freeze fracture electron microscopy. Collectively, our study greatly expands the repertoire of known proteins in the IMC and experimentally validates BioID as a strategy for discovering novel constituents of specific cellular compartments of T. gondii.

IMPORTANCE:

The identification of binding partners is critical for determining protein function within cellular compartments. However, discovery of protein-protein interactions within membrane or cytoskeletal compartments is challenging, particularly for transient or unstable interactions that are often disrupted by experimental manipulation of these compartments. To circumvent these problems, we adapted an in vivo biotinylation technique called BioID for Toxoplasma species to identify binding partners and proximal proteins within native cellular environments. We used BioID to identify 19 novel proteins in the parasite IMC, an organelle consisting of fused membrane sacs and an underlying cytoskeleton, whose protein composition is largely unknown. We also demonstrate the power of BioID for targeted discovery of proteins within specific compartments, such as the IMC cytoskeleton. In addition, we uncovered a new group of proteins localizing to the alveolar sutures of the IMC. BioID promises to reveal new insights on protein constituents and interactions within cellular compartments of Toxoplasma.
Copyright © 2015 Chen et al.
PMID:
25691595
[PubMed - in process]