Thursday, October 23, 2014

Functional Analysis of Rhomboid Proteases during Toxoplasma Invasion

2014 Oct 21;5(5). pii: e01795-14. doi: 10.1128/mBio.01795-14.
 
 
Host cell invasion by Toxoplasma gondii and other apicomplexan parasites requires transmembrane adhesins that mediate binding to receptors on the substrate and host cell to facilitate motility and invasion. Rhomboid proteases (ROMs) are thought to cleave adhesins within their transmembrane segments, thus allowing the parasite to disengage from receptors and completely enter the host cell. To examine the specific roles of individual ROMs during invasion, we generated single, double, and triple knockouts for the three ROMs expressed in T. gondii tachyzoites. Analysis of these mutants demonstrated that ROM4 is the primary protease involved in adhesin processing and host cell invasion, whereas ROM1 or ROM5 plays negligible roles in these processes. Deletion of ROM4 blocked the shedding of adhesins such as MIC2 (microneme protein 2), causing them to accumulate on the surface of extracellular parasites. Increased surface adhesins led to nonproductive attachment, altered gliding motility, impaired moving junction formation, and reduced invasion efficiency. Despite the importance of ROM4 for efficient invasion, mutants lacking all three ROMs were viable and MIC2 was still efficiently removed from the surface of invaded mutant parasites, implying the existence of ROM-independent mechanisms for adhesin removal during invasion. Collectively, these results suggest that although ROM processing of adhesins is not absolutely essential, it is important for efficient host cell invasion by T. gondii.

IMPORTANCE:

Apicomplexan parasites such as Toxoplasma gondii express surface proteins that bind host cell receptors to aid invasion. Many of these adhesins are subject to cleavage by rhomboid proteases (ROMs) within their transmembrane segments during invasion. Previous studies have demonstrated the importance of adhesin cleavage for parasite invasion and proposed that the ROMs responsible for processing would be essential for parasite survival. In T. gondii, ROM5 was thought to be the critical ROM for adhesin shedding due to its robust protease activity in vitro and posterior localization on the parasite surface. Here, we knocked out all three ROMs in T. gondii tachyzoites and found that ROM4, but not ROM5, was key for adhesin cleavage. However, none of the ROMs individually or in combination was essential for cell entry, further emphasizing that essential pathways such as invasion typically rely on redundant pathways to ensure survival.
Copyright © 2014 Shen et al.
PMID:
25336455
[PubMed - in process]

Wednesday, October 22, 2014

Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii

2014 Oct 20. doi: 10.1111/cmi.12383. [Epub ahead of print]
 
 
Phosphoinositides regulate numerous cellular processes, by recruiting cytosolic effector proteins and acting as membrane signaling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3-Kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiontic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P binding proteins and identified in total six PX and FYVE-domain containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2 . While depletion of putative PI(3)P binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon the loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking-down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2 . Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species which lack an apicoplast, supporting a direct role of PI(3,5)P2 in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.
This article is protected by copyright. All rights reserved.

KEYWORDS:

Apicomplexa; Apicoplast biogenesis; Delayed death phenotype; Lipid modifications; Phosphoinositides; Tet-inducible system; Toxoplasma gondii; Traffic
PMID:
25329540
[PubMed - as supplied by publisher]

Thursday, October 16, 2014

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

2014 Sep;109(6):767-774. Epub 2014 Aug 13.
 
 
Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.
PMID:
25317704
[PubMed - as supplied by publisher]

Wednesday, October 15, 2014

Toxoplasmosis complications and novel therapeutic synergism combination of diclazuril plus atovaquone

2014 Sep 15;5:484. doi: 10.3389/fmicb.2014.00484. eCollection 2014.
 

Abstract

Toxoplasmosis is a major cause of foodborne disease, congenital complication, and morbidity. There is an urgent need for safe and effective therapies to encounter congenital and persisting toxoplasmosis. The hypothesis was: combination diclazuril plus atovaquone to exert a novel therapeutic synergy to prevent toxoplasmosis syndromes.

METHODS:

Pregnant dams were treated with diclazuril and atovaquone monotherapy or combination therapy and infected i.p with Toxoplasma tachyzoites.

RESULTS:

Infected dams developed severe toxoplasmosis associated syndrome with increases in the abdominal adiposity surrounding uteri, gansterointestinal and other internal organs and excessive weight gain. Numerous organisms along with infiltration of inflammatory cells were detected scattered into adipose tissues. Combination therapy (p < 0.01) and to a lesser extent diclazuril (p < 0.05) protected dams from inflammatory fat and excess weight gains. This was consistent with pancreatitis development in infected dams (versus normal p < 0.05) with infiltration of inflammatory cells, degeneration and necrosis of pancreatic cells followed by the degeneration and loss of islets. Combination and monotherapy protected dams from these inflammatory and pathological aspects of pancreatitis. Infected dams exhibited severe colitis, and colonic tissues significantly shortened in length. Brush border epithelial cells were replaced with infiltration of lymphocytes, granulocytes, and microabscess formations into cryptic microstructures. Combination therapy synergistically preserved colonic structure and normalized pathological damages (p < 0.001) and to a lesser degree diclazuril monotherapy protected dams from colitis (p < 0.05) and gastrointestinal toxoplasmosis. Other complications included severe splenitis (p < 0.001) and hepatitis (p < 0.001) which were normalized with combination therapy.

CONCLUSION:

Combination diclazuril plus atovaquone was safe and with a novel therapeutic synergism protected dams and fetuses from toxoplasmosis.

KEYWORDS:

Toxoplasma; atovaquone; combination; diclazuril; gastroenteritis; obesity; synergism; toxoplasmosis
PMID:
25309522
[PubMed]

The urban risk and migration risk factors for schizophrenia: Are cats the answer?

2014 Oct 9. pii: S0920-9964(14)00519-2. doi: 10.1016/j.schres.2014.09.027. [Epub ahead of print]
 
 
Being born in and/or raised in an urban area is a proven risk factor for developing schizophrenia. Migrating from countries such as Jamaica or Morocco to countries such as England or the Netherlands is also a proven risk factor for developing schizophrenia. The transmission of Toxoplasma gondii oocysts to children is reviewed and proposed as a partial explanation for both of these risk factors.
Copyright © 2014. Published by Elsevier B.V.

KEYWORDS:

Oocysts; Toxoplasma gondii; Toxoplasmosis; Urban risk
PMID:
25308833

Tuesday, October 14, 2014

"Latent" infection with Toxoplasma gondii: Association with trait aggression and impulsivity in healthy adults

2014 Sep 28. pii: S0022-3956(14)00286-6. doi: 10.1016/j.jpsychires.2014.09.019. [Epub ahead of print]
 

BACKGROUND:

Latent chronic infection with Toxoplasma gondii (T. gondii), a common neurotropic pathogen, has been previously linked with suicidal self-directed violence (SSDV). We sought to determine if latent infection with T. gondii is associated with trait aggression and impulsivity, intermediate phenotypes for suicidal behavior, in psychiatrically healthy adults.

METHODS:

Traits of aggression and impulsivity were analyzed in relationship to IgG antibody seropositivity for T. gondii and two other latent neurotropic infections, herpes simplex virus 1 (HSV1) and cytomegalovirus (CMV). One thousand community-residing adults residing in the Munich metropolitan area with no Axis I or II conditions by SCID for DSM-IV (510 men, 490 women, mean age 53.6 ± 15.8, range 20-74). Plasma samples were tested for IgG antibodies to T. gondii, HSV-1 and CMV by ELISA. Self-reported ratings of trait aggression scores (Questionnaire for Measuring Factors of Aggression [FAF]) and trait impulsivity (Sensation-Seeking Scale-V [SSS-V]) were analyzed using linear multivariate methods.

RESULTS:

T. gondii IgG seropositivity was significantly associated with higher trait reactive aggression scores among women (p < .01), but not among men. T. gondii-positivity was also associated with higher impulsive sensation-seeking (SSS-V Disinhibition) among younger men (p < .01) aged 20-59 years old (median age = 60). All associations with HSV-1 and CMV were not significant.

CONCLUSIONS:

Aggression and impulsivity, personality traits considered as endophenotypes for SSDV, are associated with latent T. gondii infection in a gender and age-specific manner, and could be further investigated as prognostic and treatment targets in T. gondii-positive individuals at risk for SSDV.
Published by Elsevier Ltd.

KEYWORDS:

Aggression; Impulsivity; Personality; Toxoplasma gondii
PMID:
25306262
[PubMed - as supplied by publisher]

Saturday, October 11, 2014

Toxoplasma gondii Is Dependent on Glutamine and Alters Migratory Profile of Infected Host Bone Marrow Derived Immune Cells through SNAT2 and CXCR4 Pathways

 2014 Oct 9;9(10):e109803. doi: 10.1371/journal.pone.0109803. eCollection 2014.

Abstract

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both "hypermotility" and "enhanced migration" to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility. 
PMID:
 
25299045
 
[PubMed - in process] 

Aquatic polymers can drive pathogen transmission in coastal ecosystems

 2014 Nov 22;281(1795). pii: 20141287. doi: 10.1098/rspb.2014.1287.

Abstract

Gelatinous polymers including extracellular polymeric substances (EPSs) are fundamental to biophysical processes in aquatic habitats, including mediating aggregation processes and functioning as the matrix of biofilms. Yet insight into the impact of these sticky molecules on the environmental transmission of pathogens in the ocean is limited. We used the zoonotic parasite Toxoplasma gondii as a model to evaluate polymer-mediated mechanisms that promote transmission of terrestrially derived pathogens to marine fauna and humans. We show that transparent exopolymer particles, a particulate form of EPS, enhance T. gondii association with marine aggregates, material consumed by organisms otherwise unable to access micrometre-sized particles. Adhesion to EPS biofilms on macroalgae also captures T. gondii from the water, enabling uptake of pathogens by invertebrates that feed on kelp surfaces. We demonstrate the acquisition, concentration and retention of T. gondii by kelp-grazing snails, which can transmit T. gondii to threatened California sea otters. Results highlight novel mechanisms whereby aquatic polymers facilitate incorporation of pathogens into food webs via association with particle aggregates and biofilms. Identifying the critical role of invisible polymers in transmission of pathogens in the ocean represents a fundamental advance in understanding and mitigating the health impacts of coastal habitat pollution with contaminated runoff. 

KEYWORDS: 

Toxoplasma gondii; extracellular polymeric substances; marine transmission; sea otter; transparent exopolymer particles; zoonotic pathogen
PMID:
 
25297861
 
[PubMed - in process] 

Thursday, October 09, 2014

Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

2014 Oct 3. pii: S0166-6851(14)00139-X. doi: 10.1016/j.molbiopara.2014.09.009. [Epub ahead of print]
 
 
Histone H2Ba of Toxoplasma gondii was expressed as recombinant protein (rH2Ba) and used to generate antibody in mouse that is highly specific. Antibody recognizing rH2Ba detects a single band in tachyzoite lysate of the expected molecular weight (12kDa). By indirect immunofluorescence (IFA) in in vitro grown tachyzoites and bradyzoites, the signal was detected only in the parasite nucleus. The nucleosome composition of H2Ba was determined through co-immunoprecipitation assays. H2Ba was detected in the same immunocomplex as H2A.X, but not with H2A.Z. Through chromatin immunoprecipitation (ChIP) assays and qPCR, it was observed that H2Ba is preferentially located at promoters of inactive genes and silent regions, accompanying H2A.X and opposed to H2A.Z/H2B.Z dimers.
Copyright © 2014. Published by Elsevier B.V.

KEYWORDS:

ChIP; Toxoplasma; canonical histones; chromatin; epigenetics; nucleosome
PMID:
25286383
[PubMed - as supplied by publisher]

Structural and functional divergence of the aldolase fold in Toxoplasma gondii

2014 Oct 2. pii: S0022-2836(14)00518-X. doi: 10.1016/j.jmb.2014.09.019. [Epub ahead of print
 
 
Parasites of the phylum Apicomplexa are highly successful pathogens of humans and animals world-wide. As obligate intracellular parasites, they have significant energy requirements for invasion and gliding motility that are supplied by various metabolic pathways. Aldolases have emerged as key enzymes involved in these pathways, and all apicomplexans express one or both of fructose 1,6-bisphosphate (F16BP) aldolase and 2-deoxyribose 5-phosphate (dR5P) aldolase (DERA). Intriguingly, Toxoplasma gondii, a highly successful apicomplexan parasite, expresses F16BP aldolase (TgALD1), d5RP aldolase (TgDERA), and a divergent dR5P aldolase-like protein (TgDPA) exclusively in the latent bradyzoite stage. While the importance of TgALD1 in glycolysis is well established and TgDERA is also likely to be involved in parasite metabolism, the detailed function of TgDPA remains elusive. To gain mechanistic insight into the function of different T. gondii aldolases, we first determined the crystal structures of TgALD1 and TgDPA. Structural analysis revealed that both aldolases adopt a TIM barrel fold accessorized with divergent secondary structure elements. Structural comparison of TgALD1 and TgDPA with members of their respective enzyme families revealed that while the active site residues are conserved in TgALD1, key catalytic residues are absent in TgDPA. Consistent with this observation, biochemical assays showed that while TgALD1 was active on F16BP, TgDPA was inactive on dR5P. Intriguingly, both aldolases are competent to bind polymerized actin in vitro. Altogether, structural and biochemical analyses of T. gondii aldolase and aldolase-like proteins reveal diverse functionalization of the classic TIM barrel aldolase fold.
Copyright © 2014. Published by Elsevier Ltd.

KEYWORDS:

Apicomplexa; F16BP aldolase; X-ray crystallography; dR5P aldolase; pseudoaldolase
PMID:
25284756
[PubMed - as supplied by publisher]

Triazole-based compound as the candidate to develop a novel medicines to treat toxoplasmosis

2014 Oct 6. pii: AAC.03832-14. [Epub ahead of print]
 
 
This letter reports anti-Toxoplasma gondii activity of 3-(thiophen-2-yl)-1,2,4-triazole-5-thione. The compound displayed significant and reproducible anti-parasitic effects at non-toxic concentrations for the host cells, with experimentally determined IC50 value at least thirty time better than the one of known chemotherapeutic sulfadiazine. Purine nucleoside phosphorylase was defined as probable target for anti-Toxoplasma activity of tested compound. These results provide the foundation for the future work to develop a new class of medicines to better treat toxoplasmosis.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
25288090
[PubMed - as supplied by publisher]