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.


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.
[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.
[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.


Amphipathic alpha-helices; Toxoplasma gondii; circular dichroism; dense granule proteins (GRA); dynamic light scattering; transmission electron microscopy
[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.
[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.


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.


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%.


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.


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.
[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.


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.
[PubMed - in process]

Friday, February 13, 2015

Common Parasite Could Manipulate Our Behavior

Toxoplasma gondii, a parasite inhabiting the brains of an estimated three billion people, could tweak its host’s behavior
Feb 12, 2015 |By Gustavo Arrizabalaga and Bill Sullivan
Scientific American MIND
Imagine a world without fear. It might be empowering to go about your daily life uninhibited by everyday distresses. You could cross highways with confidence, take on all kinds of daredevilry and watch horror flicks without flinching. Yet consider the prospect a little more deeply, and the possibilities become darker, even deadly. Our fears, after all, can protect us.
The basic aversion that a mouse has for a cat, for instance, keeps the rodent out of death's jaws. But unfortunately for mice everywhere, there is a second enemy with which to contend, one that may prevent them from experiencing that fear in the first place. A unicellular organism (a protozoan), Toxoplasma gondii, can override a rodent's most basic survival instincts. The result is a rodent that does not race away from a cat but is instead strangely attracted to it.

Saturday, February 07, 2015

Secreted effectors in Toxoplasma gondii and related species: determinants of host range and pathogenesis?

 2014 Dec 8. doi: 10.1111/pim.12166. [Epub ahead of print]


Recent years have witnessed the discovery of a number of secreted proteins in Toxoplasma gondii that play important roles in host-pathogen interactions and parasite virulence, particularly in the mouse model. However, the role that these proteins play in driving the unique features of T. gondii compared to some of its nearest apicomplexan relatives (Hammondia hammondi and Neospora caninum) is unknown. These unique feature include distinct dissemination characteristics in vivo and a vast host range. In this review we comprehensively survey what is known about disease outcome, the host response, and host range for T. gondii, H. hammondi and N. caninum. We then review what is presently known about recently identified secreted virulence effectors in these three genetically-related, but phenotypically distinct, species. Finally we exploit the existence of genome sequences for these three organisms and discuss what is known about the presence, and functionality, of key T. gondii effectors in these three species. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.


comparative genomics; host range expansion; virulence
[PubMed - as supplied by publisher]

Tuesday, February 03, 2015

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles

 2015 Feb 2. doi: 10.1111/cmi.12426. [Epub ahead of print]


Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome-like-compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin-like receptor and syntaxin-6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab-GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps-C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi-endosomal-related-compartments, the vacuole and immature apical secretory organelles. Conditional knock-down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock-down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite-specific functions in the biogenesis of their unique secretory organelles.
This article is protected by copyright. All rights reserved.


Apicomplexa; Dense granules; Endosomal compartment; Golgi; Membrane fusion; Micronemes; Rab-GTPase; Rhoptries; Secretory organelle biogenesis; Tet-inducible system; Tethering; Toxoplasma gondii; Traffic; Vacuolar Protein Sorting
[PubMed - as supplied by publisher]