Analysis of Ca2+ mediated signalling regulating Toxoplasma infectivity reveals complex relationships between key molecules

Cell Microbiol. 2016 Oct 26. doi: 10.1111/cmi.12685. [Epub ahead of print]

Stewart RJ1,2, Whitehead L1,2, Nijagal B3, Sleebs BE1,2, Lessene G1,2,4, McConville MJ5, Rogers KL1,2, Tonkin CJ6,7.

Author information

Abstract

Host cell invasion, exit and parasite dissemination is critical to the pathogenesis of apicomplexan parasites such as Toxoplasma gondii and Plasmodium spp. These processes are regulated by intracellular Ca2+ signalling although the temporal dynamics of Ca2+fluxes and down-stream second messenger pathways are poorly understood. Here we use a genetically encoded biosensor, GFP-Calmodulin-M13-6 (GCaMP6), to capture Ca2+ flux in live Toxoplasma and investigate the role of Ca2+ signalling in egress and motility. Our analysis determines how environmental cues and signal activation influence intracellular Ca2+ flux, allowing placement of effector molecules within this pathway. Importantly, we have identified key interrelationships between cGMP and Ca2+ signalling that are required for activation of egress and motility. Furthermore, we extend this analysis to show that the Ca2+ Dependent Protein Kinases - TgCDPK1 and TgCDPK3 - play a role in signal quenching before egress. This work highlights the interrelationships of second messenger pathways of Toxoplasma in space and time, which is likely required for pathogenesis of all apicomplexan species.

This article is protected by copyright. All rights reserved.

PMID:

 

27781359

 

DOI:

 

10.1111/cmi.12685

Migratory activation of parasitized dendritic cells by the protozoan Toxoplasma gondii 14-3-3 protein

Cell Microbiol. 2016 Nov;18(11):1537-1550. doi: 10.1111/cmi.12595. Epub 2016 May 6.

Weidner JM1,2, Kanatani S1,2, Uchtenhagen H3, Varas-Godoy M4,5, Schulte T3, Engelberg K6, Gubbels MJ6, Sun HS7, Harrison RE7, Achour A3, Barragan A8,9.

Author information

Abstract

The obligate intracellular parasite Toxoplasma gondii exploits cells of the immune system to disseminate. Upon infection, parasitized dendritic cells (DCs) and microglia exhibit a hypermigratory phenotype in vitro that has been associated with enhancing parasite dissemination in vivo in mice. One unresolved question is how parasites commandeer parasitized cells to achieve systemic dissemination by a 'Trojan-horse' mechanism. By chromatography and mass spectrometry analyses, we identified an orthologue of the 14-3-3 protein family, T. gondii 14-3-3 (Tg14-3-3), as mediator of DC hypermotility. We demonstrate that parasite-derived polypeptide fractions enriched for Tg14-3-3 or recombinant Tg14-3-3 are sufficient to induce the hypermotile phenotype when introduced by protein transfection into murine DCs, human DCs or microglia. Further, gene transfer of Tg14-3-3 by lentiviral transduction induced hypermotility in primary human DCs. In parasites expressing Tg14-3-3 in a ligand-regulatable fashion, overexpression of Tg14-3-3 was correlated with induction of hypermotility in parasitized DCs. Localization studies in infected DCs identified Tg14-3-3 within the parasitophorous vacuolar space and a rapid recruitment of host cell 14-3-3 to the parasitophorous vacuole membrane. The present work identifies a determinant role for Tg14-3-3 in the induction of the migratory activation of immune cells by T. gondii. Collectively, the findings reveal Tg14-3-3 as a novel target for an intracellular pathogen that acts by hijacking the host cell's migratory properties to disseminate.

© 2016 John Wiley & Sons Ltd.

PMID:

 

27018989

 

PMCID:

 

PMC5040621

 [Available on 2017-09-28]

 

DOI:

 

10.1111/cmi.12595

The apicoplast: now you see it, now you don't

Int J Parasitol. 2016 Oct 20. pii: S0020-7519(16)30230-2. doi: 10.1016/j.ijpara.2016.08.005. [Epub ahead of print]

Ian McFadden G¹, Yeh E².

Parasites such as Plasmodium and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of algae and plants. The plastid (known as the apicoplast; for apicomplexan plastid) is non-photosynthetic and very much reduced, but has clear endosymbiotic ancestry including a circular genome that encodes RNAs and proteins and a suite of bacterial biosynthetic pathways. Here we review the initial discovery of the apicoplast, and recount the major new insights into apicoplast origin, biogenesis and function. We conclude by examining how the apicoplast can be removed from malaria parasites in vitro, ultimately completing its reduction by chemical supplementation.

Copyright © 2016. Published by Elsevier Ltd.

KEYWORDS:

Apicoplast; Endosymbiosis; Malaria; Plasmodium; Plastid; Toxoplasma

PMID:

27773518

DOI:

10.1016/j.ijpara.2016.08.005

[PubMed - as supplied by publisher]

The role of ticks in transmission cycle of Toxoplasma gondii

Ann Parasitol. 2016 Oct 1;62(3):185–191. doi: 10.17420/ap6203.52.

Skotarczak BI¹, et al.

Toxoplasmosis is globally distributed, water- and food borne zoonosis caused by the single protozoan Toxoplasma gondii and probably one-third of the world’s human population is infected with this pathogen. Domestic and wild felids are definitive hosts of this pathogen and intermediate hosts for great variety of other homoeothermic animals. Human as other of the intermediate hosts may become infected in the main route of infection; it is the ingestion of parasite oocysts in contaminated water or soil and undercooked meat. However, the mechanism which this parasite uses to so large spread is not fully understood, because oral transmission does not explain the common event of this parasite in a variety of hosts, such as herbivorous animals or rodents and birds, as well as routes of spread to domestic hosts. Such a wide circle of hosts suggests a possibility of other paths of transmission and a role of ticks, the bloodseeking arthropods was considered in the transmission of T. gondii.

KEYWORDS:

Toxoplasma gondii; genotypes; transmission ways; role of ticks

PMID:

27770758

Exosomes in the context of Toxoplasma gondii – host communication

Ann Parasitol. 2016 Oct 1;62(3):169–174. doi: 10.17420/ap6203.50.

Długońska H¹, Gatkowska J¹, et al.

Extracellular vesicles – EV’s, including exosomes, are known to be essential tools of intercellular communication, enabling the exchange of information without direct contact between cells. Exosomes are secreted both in vitro and in vivo by single- and multi-cellular organisms, regardless of their type, and play an essential role in cell-to-cell communication. EV’s may carry various materials and ongoing studies have provided a new insight into their potential participation in various critical biological processes, including carcinogenesis, protein trafficking, immunostimulation and pathogenesis of infectious diseases. Although knowledge of the contribution of exosomes in Toxoplasma invasion is still very limited, the present article discusses aspects of their involvement in the interactions between host and T. gondii.

KEYWORDS:

exosomes; Toxoplasma gondii; hosts

PMID:

27770756

Toxoplasma gondii mitogen-activated protein kinases are associated with inflammasome activation in infected mice

Microbes Infect. 2016 Nov;18(11):696-700. doi: 10.1016/j.micinf.2016.07.004. Epub 2016 Jul 27.

Wang S¹, Wang Z¹, Gu Y¹, Li Z¹, Li Z¹, Wei F², Liu Q³.

Toxoplasma gondii can activate the nucleotide-binding domain and leucine-rich repeat-containing proteins NLRP1/3 inflammasomes, which mediate host resistance to the infection. Here we showed that deletion of mitogen-activated protein kinases MAPK1 and MAPK2 of type I parasite decreases acute virulence in mice, characterized by low levels of interleukin (IL)-18, NLRP1/3, ASC, and caspase-1, and high levels of IL-10 and interferon (IFN)-β transcripts. Additionally, the mutants increased phosphorylation of STAT1, and decreased phosphorylation of STAT3. These findings suggest that MAPKs are associated with inflammasome activation in T. gondii-infected mice, which may contribute to new insight into the pathogenesis of T. gondii infection.

Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

KEYWORDS:

IFN-β; IL-18; Inflammasome; Mitogen-activated protein kinase; Toxoplasma gondii

PMID:

27475900

DOI:

10.1016/j.micinf.2016.07.004

Cdk7 mediates RPB1-driven mRNA synthesis in Toxoplasma gondii

Sci Rep. 2016 Oct 19;6:35288. doi: 10.1038/srep35288.

Deshmukh AS1, Mitra P2, Maruthi M3.

Author information

Abstract

Cyclin-dependent kinase 7 in conjunction with CyclinH and Mat1 activates cell cycle CDKs and is a part of the general transcription factor TFIIH. Role of Cdk7 is well characterized in model eukaryotes however its relevance in protozoan parasites has not been investigated. This important regulator of key processes warrants closer examination particularly in this parasite given its unique cell cycle progression and flexible mode of replication. We report functional characterization of TgCdk7 and its partners TgCyclinH and TgMat1. Recombinant Cdk7 displays kinase activity upon binding its cyclin partner and this activity is further enhanced in presence of Mat1. The activated kinase phosphorylates C-terminal domain of TgRPB1 suggesting its role in parasite transcription. Therefore, the function of Cdk7 in CTD phosphorylation and RPB1 mediated transcription was investigated using Cdk7 inhibitor. Unphosphorylated CTD binds promoter DNA while phosphorylation by Cdk7 triggers its dissociation from DNA with implications for transcription initiation. Inhibition of Cdk7 in the parasite led to strong reduction in Serine 5 phosphorylation of TgRPB1-CTD at the promoters of constitutively expressed actin1 and sag1 genes with concomitant reduction of both nascent RNA synthesis and 5'-capped transcripts. Therefore, we provide compelling evidence for crucial role of TgCdk7 kinase activity in mRNA synthesis.

PMID:

 

27759017

 

DOI:

 

10.1038/srep35288

Is chronic toxoplasmosis a risk factor for diabetes mellitus? A systematic review and meta-analysis of case-control studies

Braz J Infect Dis. 2016 Oct 18. pii: S1413-8670(16)30334-8. doi: 10.1016/j.bjid.2016.09.002. [Epub ahead of print]

Majidiani H1, Dalvand S2, Daryani A3, Galvan-Ramirez ML4, Foroutan-Rad M5.

Author information

Abstract

INTRODUCTION: 

The global protozoan parasite, Toxoplasma gondii, infects many warm-blooded animals and humans by employing different transmission routes. There have been some recent studies on the probable relevance of infectious agents and diabetes. Therefore, we conducted a systematic review and meta-analysis to identify the possible association between chronic toxoplasmosis and diabetes mellitus.

METHODS: 

This study was conducted following the general methodology recommended for systematic reviews and meta-analysis. Nine English literature databases (Google scholar, PubMed, Scopus, Web of science, Science Direct, Ovid, ProQuest, IngentaConnect, and Wiley Online Library) were searched, up to January 2016. Random effects model was used to determine odds ratios and their 95% confidence intervals.

RESULTS: 

Our review resulted in a total of seven publications meeting the inclusion criteria. Because of significant heterogeneity, we estimated a common OR by a random effects model at 1.10 (95% CI=0.13-9.57) with p=0.929 and 2.39 (95% CI=1.20-4.75) with p=0.013 for type 1 and type 2 diabetes mellitus, respectively.

CONCLUSION: 

Despite the limitations such as low number of studies, this meta-analysis suggests chronic toxoplasmosis as a possible risk factor for type 2 DM. However, based on random effects model no statistically significant association was observed between T. gondii and type 1 DM. It is highly recommended for researchers to carry out more accurate studies aiming to better understand this association.

Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

KEYWORDS: 

Diabetes mellitus; Meta-analysis; Systematic review; Toxoplasma gondii

PMID:

 

27768900

 

DOI:

 

10.1016/j.bjid.2016.09.002

Reduced ERPs and theta oscillations underlie working memory deficits in Toxoplasma gondii infected seniors

Biol Psychol. 2016 Oct;120:35-45. doi: 10.1016/j.biopsycho.2016.08.002. Epub 2016 Aug 8.

Gajewski PD1, Falkenstein M2, Hengstler JG2, Golka K2.

Author information

Abstract

Toxoplasma gondii is one of the most widespread infections in humans. Recent studies give evidence for memory deficits in infected older adults. To investigate working memory dysfunction in infected elderly, a double-blinded electrophysiological study was conducted. 84 persons derived from a sample of 131 healthy participants with the mean age of 70 years were assigned to two groups of 42 non-infected and 42 infected individuals. The outcome measures were behavioral performance, target and response-related ERPs, and time-frequency wavelets during performance in a n-back working-memory task. The infected individuals showed a reduced rate of detected targets and diminished P3b amplitude both in target-locked as well as response-locked data compared to the non-infected group. Time-frequency decomposition of the EEG-signals revealed lower evoked power in the theta frequency range in the target-locked as well as in the response-locked data in infected individuals. The reported effects were comparable with differences between healthy young and old adults described previously. Taking together, the reduced working-memory performance accompanied by an attenuated P3b and frontal theta activity may suggest neurotransmitter imbalance like dopamine and norepinephrine in T. gondii infected individuals. In face of a high prevalence of T. gondii infection and the increasing ratio of older population their accelerated memory decline may have substantial socioeconomic consequences.

Copyright © 2016 Elsevier B.V. All rights reserved.

KEYWORDS: 

Aging; Dopamine; Event-related potentials (ERPs); Norepinephrine; Response-locked ERPs; Stimulus-locked ERPs; Theta; Toxoplasma gondii; Working memory

PMID:

 

27516127

 

DOI:

 

10.1016/j.biopsycho.2016.08.002

Toxoplasma gondii as a Parasite in Food: Analysis and Control

Microbiol Spectr. 2016 Aug;4(4). doi: 10.1128/microbiolspec.PFS-0011-2015.

Hill DE, Dubey JP.

Abstract

Foodborne infections are a significant cause of morbidity and mortality worldwide, and foodborne parasitic diseases, though not as widespread as bacterial and viral infections, are common on all continents and in most ecosystems, including arctic, temperate, and tropical regions. Outbreaks of disease resulting from foodstuffs contaminated by parasitic protozoa have become increasingly recognized as a problem in the United States and globally. Increased international trade in food products has made movement of these organisms across national boundaries more frequent, and the risks associated with infections have become apparent in nations with well-developed food safety apparatus in place.

PMID:

27726776

DOI:

10.1128/microbiolspec.PFS-0011-2015

Latent toxoplasmosis is associated with neurocognitive impairment in young adults with and without chronic HIV infection

J Neuroimmunol. 2016 Oct 15;299:1-7. doi: 10.1016/j.jneuroim.2016.08.003. Epub 2016 Aug 4.

Ene L¹, Marcotte TD², Umlauf A², Grancea C³, Temereanca A³, Bharti A², Achim CL², Letendre S², Ruta SM³.

We evaluated the impact of latent toxoplasmosis (LT) on neurocognitive (NC) and neurobehavioural functioning in young adults with and without chronic HIV infection, using a standardised NC test battery, self-reported Beck Depression Inventory, Frontal System Behavior Scale, MINI-International Neuropsychiatric Interview and risk-assessment battery. 194 young adults (median age 24years, 48.2% males) with chronic HIV infection (HIV+) since childhood and 51 HIV seronegative (HIV-) participants were included. HIV+ individuals had good current immunological status (median CD4: 479 cells/μl) despite a low CD4 nadir (median: 93 cells/μl). LT (positive anti-Toxoplasma IgG antibodies) was present in one third of participants. The impairment rates in the HIV- with and without Toxo were not significantly different (p=0.17). However, we observed an increasing trend (p<0 .001="" a="" abstracttext="" adults="" also="" analysis="" and="" antibody="" any="" associated="" behaviors="" chronic="" cognition="" concentration="" contribute="" depressive="" domains.="" dysfunction="" effects="" entire="" for="" frontal="" gds="" globally="" group="" groups.="" had="" higher="" hiv-="" hiv="" impairment="" in="" including="" infection.="" information="" latent="" learning="" lower="" lt.="" lt="" main="" may="" memory="" multivariable="" nc="" not="" of="" on="" or="" p="0.01)" participants="" performance="" processing="" rates="" risk="" scores.="" speed="" status:="" study="" symptoms="" systems="" t="" the="" there="" those="" to="" toxoplasma="" toxoplasmosis="" using="" values="" verbal="" was="" were="" with="" within="" without="" worse="" young="">

Copyright © 2016 Elsevier B.V. All rights reserved.

KEYWORDS:

HIV; Latent toxoplasmosis; Neurocognitive impairment; Young adults

PMID:

27725106

DOI:

10.1016/j.jneuroim.2016.08.003

Impaired health status and increased incidence of diseases in Toxoplasma-seropositive subjects - an explorative cross-sectional study

Parasitology. 2016 Oct 10:1-16. [Epub ahead of print]

Flegr J¹, Escudero DQ¹.

The global seroprevalence of latent toxoplasmosis is estimated to be higher than 30%. The presence of slowly dividing parasites in tissue cysts located mainly in immunoprivileged organs was long considered asymptomatic. Recently, many studies have shown that latent Toxoplasma infections could have serious impacts on human health. Here we ran a cross-sectional study in a population of 1486 volunteers. The results showed that 333 infected subjects scored worse than 1153 controls in 28 of 29 health-related variables. Similarly, they reported higher rates of 77 of a list of 134 disorders reported by at least 10 participants of the study. Toxoplasmosis was associated most strongly with musculoskeletal (τ = 0·107, P < 0·0005), followed by neurological (τ = 0·088, P < 0·0005), immune (τ = 0·085, p < 0·0005), metabolic (τ = 0·079, P < 0·0005), respiratory (τ = 0·068, P = 0·0001), allergic (τ = 0·053, P = 0·004), digestive system (τ = 0·052, P = 0·004) and mental health disorders (τ = 0·050, P = 0·008). Results of the present cohort study, along with the previous data from many case-control studies or ecological studies suggest that latent toxoplasmosis represents a large and so far underrated public health problem.

KEYWORDS:

Toxoplasma gondii ; Parasite; disease burden; neglected disease; neglected zoonosis; public health; toxoplasmosis

PMID:

27719690

DOI:

10.1017/S0031182016001785

[PubMed - as supplied by publisher]

Development of CRISPR/Cas9 for Efficient Genome Editing in Toxoplasma gondii

Methods Mol Biol. 2017;1498:79-103.

Shen B1, Brown K2, Long S2, Sibley LD3.

Author information

Abstract

Efficient and site-specific alteration of the genome is key to decoding and altering the genomic information of an organism. Over the last couple of years, the RNA-guided Cas9 nucleases derived from the prokaryotic type 2 CRISPR (clustered regularly interspaced short palindromic repeats) systems have drastically improved our ability to engineer the genomes of a variety of organisms including Toxoplasma gondii. In this chapter, we describe detailed protocols for using the CRISPR/Cas9 system adapted from Streptococcus pyogenes to perform efficient genetic manipulations in T. gondii such as gene disruption, gene tagging and genetic complementation. The technical details of the strategy, including CRISPR plasmid construction, target construct generation, parasite transfection and positive clone identification are also provided. These methods are easy to customize to any gene of interest (GOI) and will greatly accelerate studies on this important pathogen.

KEYWORDS: 

CRISPR/Cas9; Gene editing; Genetic transformation; Protozoan parasites; Selectable markers

PMID:

 

27709570

 

DOI:

 

10.1007/978-1-4939-6472-7_6

Novel insights into the composition and function of the Toxoplasma IMC sutures

Cell Microbiol. 2016 Oct 1. doi: 10.1111/cmi.12678. [Epub ahead of print]

Chen AL1, Moon AS1, Bell HN1, Huang AS1, Vashisht AA2, Toh JY1, Lin AH1, Nadipuram SM1, Kim EW1,3, Choi CP1,3, Wohlschlegel JA2,3, Bradley PJ4,5.

Author information

Abstract

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named ISCs. Here we have used proximity-dependent biotin identification (BioID) at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures which we named TSCs, demonstrating that components of the IMC sutures consist of two groups, those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

This article is protected by copyright. All rights reserved.

KEYWORDS: 

BioID; IMC; Inner membrane complex; Sutures; Toxoplasma; choline transporter-like protein

PMID:

 

27696623

 

DOI:

 

10.1111/cmi.12678

Adjuvanted multi-epitope vaccines protect HLA-A*11:01 transgenic mice against Toxoplasma gondii

JCI Insight. 2016 Sep 22;1(15):e85955.

El Bissati K1, Chentoufi AA2, Krishack PA3, Zhou Y1, Woods S4, Dubey JP5, Vang L6, Lykins J1, Broderick KE7, Mui E1, Suzuki Y8, Sa Q8, Bi S1, Cardona N1, Verma SK5, Frazeck L1, Reardon CA3, Sidney J9, Alexander J6, Sette A9, Vedvick T10, Fox C10, Guderian JA10, Reed S10, Roberts CW4, McLeod R1.

Author information

Abstract

We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8+ T cell-eliciting epitopes, a universal CD4+ helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8+ T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8+ T cell-eliciting epitopes in a vaccine that prevents toxoplasmosis.

PMID:

 

27699241

 

DOI:

 

10.1172/jci.insight.85955