Genetic and pathologic data suggest that amyloid beta (Aβ), produced by processing of the amyloid precursor protein, is a major initiator of Alzheimer's disease (AD). To gain new insights into Aβ modulation, we sought to harness the power of the coevolution between the neurotropic parasite Toxoplasma gondii and the mammalian brain. Two prior studies attributed Toxoplasma-associated protection against Aβ to increases in anti-inflammatory cytokines (TGF-β and IL-10) and infiltrating phagocytic monocytes. These studies only used one Toxoplasma strain making it difficult to determine if the noted changes were associated with Aβ protection or simply infection. To address this limitation, we infected a third human amyloid precursor protein AD mouse model (J20) with each of the genetically distinct, canonical strains of Toxoplasma (Type I, Type II, or Type III). We then evaluated the central nervous system (CNS) for Aβ deposition, immune cell responses, global cytokine environment, and parasite burden. We found that only Type II infection was protective against Aβ deposition despite both Type II and Type III strains establishing a chronic CNS infection and inflammatory response. Compared with uninfected and Type I-infected mice, both Type II- and Type III-infected mice showed increased numbers of CNS T cells and microglia and elevated pro-inflammatory cytokines, but neither group showed a >2-fold elevation of TGF-β or IL-10. These data suggest that we can now use our identification of protective (Type II) and nonprotective (Type III) Toxoplasma strains to determine what parasite and host factors are linked to decreased Aβ burden rather than simply with infection.
Very little is known about concordance of Toxoplasma gondii (T. gondii) infection markers among couples. Through a cross-sectional study, we sought to determine the correlation of T. gondii infection in a sample of 119 heterosexual couples in Durango State, Mexico.
Participants were examined for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunoassays. IgG and IgM seropositive couples were further analyzed for the presence of T. gondii DNA by using polymerase chain reaction (PCR).
Anti-T. gondii IgG antibodies were found in 71 (59.7%) men and in 63 (52.9%) women (odds ratio (OR) = 1.31; 95% confidence interval (CI): 0.78 - 2.19; P = 0.29). Of the 71 seropositive men, 40 (56.3%) had a seropositive couple; in contrast, of the 63 seropositive women, 40 (63.5%) had a seropositive couple (OR = 0.74; 95% CI: 0.37 - 1.48; P = 0.39). In total, 65 (54.6%) couples had concordant results (both IgG positive or both IgG negative), and 54 (45.4%) had discordant results (a seropositive man with a seronegative woman, or a seropositive woman with a seronegative man) (kappa index = 0.08; 95% CI: -0.09 - 0.26). With respect to high (> 150 IU/mL) levels of anti-T. gondii IgG antibodies, 79 (66.4%) of the 119 couples had concordant results and 40 (33.6%) had discordant results (kappa index = -0.15; 95% CI: -0.03 - 0.33). Forty couples were positive for anti-T. gondii IgM antibodies. Of them, 21 (52.5%) had concordant results, and 19 (47.5%) had discordant results (kappa index = -0.01; 95% CI: -0.39 - 0.28). Concerning PCR, eight (50%) of 16 couples were positive for T. gondii DNA. Of them, 11 (68.8%) had concordant results, and five (31.2%) had discordant results (kappa index = 0.31; 95% CI: -0.17 - 0.79).
Results suggest a poor concordance of serological and molecular markers of T. gondii infection among heterosexual couples. Further studies to confirm our results should be conducted.
As an indispensable molecular machine universal in all living organisms, the ribosome has been selected by evolution to be the natural target of many antibiotics and small-molecule inhibitors. High-resolution structures of pathogen ribosomes are crucial for understanding the general and unique aspects of translation control in disease-causing microbes. With cryo-electron microscopy technique, we have determined structures of the cytosolic ribosomes from two human parasites, Trichomonas vaginalis and Toxoplasma gondii, at resolution of 3.2-3.4 Å. Although the ribosomal proteins from both pathogens are typical members of eukaryotic families, with a co-evolution pattern between certain species-specific insertions/extensions and neighboring ribosomal RNA (rRNA) expansion segments, the sizes of their rRNAs are sharply different. Very interestingly, rRNAs of T. vaginalis are in size comparable to prokaryotic counterparts, with nearly all the eukaryote-specific rRNA expansion segments missing. These structures facilitate the dissection of evolution path for ribosomal proteins and RNAs, and may aid in design of novel translation inhibitors.Cell Research advance online publication 15 August 2017; doi:10.1038/cr.2017.104.
Toxoplasma gondii (T. gondii) is one of the most common parasite that can infect almost any warm-blooded animals including humans. The cyclic nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii. Here, T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was constructed. The growth of the TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains (TgPKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of TgPKAC in Vero cells by laser scanning microscope. We found that TgPKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro, and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in TgPKAC-H89 parasite compared to RH△Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in TgPKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we hypothesized that inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. TgPKAC plays an important role in parasite virulence in vivo, and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii.
Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T. gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-treatment of infected trophoblasts. The infection index/replication and the impact of the antibiotic therapy on the cytokine milieu were characterized. It was observed that TgChBrUD2 infection induced lower infection index/replication as compared to TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to control the trophoblast infection with both genotypes when compared to conventional antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of azithromycin to control the TgChBrUD2-infection was not associated with the IL-12 production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and the azithromycin treatment showed to be more effective than conventional antibiotics to control the T. gondii infection/replication regardless the parasite genotype.
Toxoplasma gondii and Neospora caninum are closely related intracellular protozoan parasites and tissue cyst-forming Coccidia of the phylum Apicomplexa. There are remarkable similarities between the morphology, genomes and transcriptomes of both parasites. Toxoplasma is zoonotic, with a wide host range and is mainly transmitted horizontally between its definitive host, the cat, and its intermediate hosts. Neospora causes disease within a narrow host range and with reduced virulence potential to the hosts. The dog is the definitive host of Neospora and its epidemiology in cattle mainly depends on vertical transmission. What causes these biological differences is not well understood. Since these parasites secrete an array of secretory proteins, including kinases, during infection to manipulate host cell responses. Host-parasite interactions due to phosphorylation of host cell proteins by T. gondii kinases enhance virulence and maintenance of infection. In this study, proteome-wide phosphorylation events of host cell proteins were investigated in response to infection with T. gondii and N. caninum using phosphoproteomic analyses, followed by pathway analysis on host signalling pathways. A few interesting differences in host responses at both the qualitative and quantitative levels were identified between the two infections; about one third of the phosphoproteomes, approximately 21% of the phospho-motifs and several pathways such as glycolysis/gluconeogenesis and mTOR pathways of the host cell were found differentially enriched between infection with these parasites. Identifying the differences in host-parasite interactions represents a promising step forward for uncovering the biological dissimilarities between both parasites.
Biological differences; Neospora caninum; Phosphopeptides enrichment; Proteome-wide phosphorylation; Tandem mass spectrometry; Toxoplasma gondii
The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.
Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this studywas to assess the activity of rosuvastatin in HeLa cells infectedwiththe RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significantto promote further studiesandthe search for alternative treatment for toxoplasmosis.
We recently developed a yeast two-hybrid (Y2H) library with Hybrigenics to facilitate the discovery of protein-protein interactions. The library was made from Toxoplasma type I RH strain tachyzoites and is ready for use! --Bill Sullivan
Please find below a summary of Hybrigenics differentiating
features regarding our Y2H screening services:
1. Bait design: Investigators
have an option of expressing their “bait” either as a N- or C-terminal fusion
protein relative to the DNA-binding domain (DBD).
2. Bait testing: An
initial test screen will be performed to evaluate whether your “bait”
autoactivates a HIS3 gene reporter. The “bait” will be tested in the
absence or presence of a dose-range of 3-aminotriazol (3-AT), which is used to
minimize background levels. If your “bait” displays some degree of
autoactivation, then the amount of 3-AT will be adjusted accordingly.
the “bait” exhibits some degree of toxicity in yeast, then your cDNA insert
will be transferred into an inducible vector.
The use of domains
is one of the key features of our libraries. Why is this important? When
expressed in yeast, full-length proteins, especially if they are large, can be
misfolded, mislocalized or toxic. Construction of highly complex random-primed
libraries overcomes these barriers. Random oligonucleotide priming generates
overlapping fragments of a given protein domain thereby increasing the chances
of proper folding. Moreover, overlapping fragments span the entire length of
the protein sequence, which increases the likelihood of detecting a protein
interaction. Importantly, fragments of membrane, secreted and toxic proteins
are well represented in our libraries and are routinely identified in our Y2H
screens. Lastly, the identification of several independent fragments in a Y2H
screen allows us to easily determine a minimal interacting domain of a prey
screening: Large-scale Y2H screens are performed using our LexA and
Gal4 systems. We use a patented cell-to-cell yeast-mating assay in order to
obtain and subsequently test on average 83 million interactions, which
corresponds to a 8-fold in library coverage.
5. Turnaround time: Approximately
analysis: Included in our Y2H results package is a comprehensive
bioinformatics analysis of protein interactors. Hybrigenics’ bioinformatics
algorithms compute a numerical rank for each protein interaction, which is then
assigned a statistical confidence score. This information helps guide
investigators to identify the most relevant protein binding partners as well as
to exclude false positives.
Results from your ULTImate Y2H screen are reported on 3 separate files:
a) Results Summary File:
Includes the technical parameters of your screen, a Predicted Biological Score
(PBS), which is computed to assess the reliability of each interaction and prey
b) DomSight file: Compares the
bait fragment and the Selected Interaction Domain (SID) of the prey proteins
with the functional and structural domains (PFAM, SMART, TMHMM, SignalP, Coil
algorithms) of these proteins.
c) Excel worksheet file:
Contains raw data, in particular 5’ and 3’ experimental sequences of the preys
and bioinformatics analysis.
8. Expertise and
scientific assistance: You will be assigned a dedicated scientific
project leader, who will be in charge of your project and will be working with
you throughout the course of your Y2H screen. They will help you with the
design of your “bait”, provide regular updates on your Y2H screen and
thoroughly review all results with you.
For more information about getting Hybrigenics to complete your Y2H screen, contact Brent Passer.
Toxoplasma gondii is one of the world's most successful parasites, in part because of its ability to infect and persist in most warm-blooded animals. A unique characteristic of T. gondii is its ability to persist in the central nervous system (CNS) of a variety of hosts, including humans and rodents. How, what, and why T. gondii encysts in the CNS has been the topic of study for decades. In this review, we will discuss recent work on how T. gondii is able to traverse the unique barrier surrounding the CNS, what cells of the CNS play host to T. gondii, and finally, how T. gondii infection may influence global and cellular physiology of the CNS.
Toxoplasma gondii is considered as one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host-cell types of essentially any warm-blooded organism. Our earlier work has demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not been resolved yet. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for the tachyzoite survival. TgPEPCKnet can also be ablated in glycolysis-deficient mutant, whereas TgPEPCKmt is refractory to deletion. In accord, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function of TgPEPCKmt in cohesive operation of glycolysis and TCA cycle under normal glucose-replete milieu. Conversely, we found that otherwise-integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.
The unicellular parasite Toxoplasma gondii infects warm-blooded animals and humans, and it is highly prevalent throughout the world. Infection of immunocompetent hosts is usually asymptomatic or benign but leads to long-term parasite persistence mainly within neural and muscular tissues. The transition from acute primary infection towards chronic toxoplasmosis is accompanied by a developmental switch from fast replicating and metabolically highly active tachyzoites to slow replicating and largely dormant bradyzoites within tissue cysts. Such developmental differentiation is critical for T. gondii in order to complete its life cycle and for pathogenesis. Herein, we summarize accumulating evidence indicating a major impact of the host cell physiology on stage conversion between the tachyzoite and the bradyzoite stage of the parasite. Withdrawal from cell cycle progression, proinflammatory responses, reduced availability of nutrients and extracellular adenosine can indeed induce tachyzoite-to-bradyzoite differentiation and tissue cyst formation. In contrast, high glycolytic activity as indicated by increased lactate secretion can inhibit bradyzoite formation. These examples argue for the intriguing possibility that after dissemination within its host, T. gondii can sense its cellular microenvironment to initiate the developmental program towards the bradyzoite stage in distinct cells. This may also explain the predominant localization of T. gondii in neural and muscular tissues during chronic toxoplasmosis.