Thursday, October 20, 2016

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

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


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.


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

Thursday, October 13, 2016

Toxoplasma gondii as a Parasite in Food: Analysis and Control

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


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.

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

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

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="">


HIV; Latent toxoplasmosis; Neurocognitive impairment; Young adults

Tuesday, October 11, 2016

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

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

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.


Toxoplasma gondii ; Parasite; disease burden; neglected disease; neglected zoonosis; public health; toxoplasmosis
[PubMed - as supplied by publisher]

Friday, October 07, 2016

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



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.


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

Wednesday, October 05, 2016

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

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


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.


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

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

 2016 Sep 22;1(15):e85955.


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 Tgondii. 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.

Friday, September 30, 2016

Characterization of aspartyl aminopeptidase from Toxoplasma gondii

 2016 Sep 28;6:34448. doi: 10.1038/srep34448.


Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii.

Wednesday, September 28, 2016

Developmental change in translation initiation alters the localization of a common microbial protein necessary for Toxoplasma chronic infection

2016 Sep 27. doi: 10.1111/mmi.13538. [Epub ahead of print]

The Toxoplasma gondii cyst stage is resistant to drug therapy. To identify potential targets for new therapeutics, we screened insertional mutants of T. gondii for a reduced ability to form cysts in the brains of mice. In one of these mutants, named 38C3, the mutagenesis plasmid inserted into the mRNA of a protein that is highly conserved in microbes but is not present in humans. The mutation in 38C3 causes reduced brain cyst production during chronic infection, but does not affect acute virulence, so the disrupted gene and protein are called T. gondii Brain Colonization Protein 1 (TgBCP1). TgBCP1 has three potential in frame start codons that produce either 51, 33 or 25 kDa proteins. In rapidly replicating tachyzoites, translation initiates at the third methionine, producing the 25 kDa form that is conserved in many bacteria and protozoans. Brain cysts exclusively express the 51 kDa form of TgBCP1, which is secreted from the parasites and localizes to the cyst wall. Only expression of the long form of TgBCP1 restored cyst formation in the 38C3 mutant. TgBCP1 is essential for cyst formation and is the first example of a developmental regulation in translation initiation site preference for a T. gondii protein. This article is protected by copyright. All rights reserved.
© 2016 John Wiley & Sons Ltd.


Apicomplexa; Toxoplasma; cyst development; parasite; translation initiation