Monday, June 30, 2014

Efficient Genome Engineering of Toxoplasma gondii Using CRISPR/Cas9

2014 Jun 27;9(6):e100450. doi: 10.1371/journal.pone.0100450. eCollection 2014.
 
Toxoplasma gondii is a parasite of humans and animals, and a model for other apicomplexans including Plasmodium spp., the causative agents of malaria. Despite many advances, manipulating the T. gondii genome remains labor intensive, and is often restricted to lab-adapted strains or lines carrying mutations that enable selection. Here, we use the RNA-guided Cas9 nuclease to efficiently generate knockouts without selection, and to introduce point mutations and epitope tags into the T. gondii genome. These methods will streamline the functional analysis of parasite genes and enable high-throughput engineering of their genomes.
PMID: 24971596
 

Total synthesis of the cyanobacterial metabolite nostodione A: discovery of its antiparasitic activity against Toxoplasma gondii

2014 Jun 27. [Epub ahead of print]
 
A total synthesis of the cyanobacterial natural product nostodione A is reported involving a convergent, diversity-oriented route. A small assemblage of structural analogues were prepared and their cytotoxicity and anti-invasion activity against the protozoal parasite Toxoplasma gondii is reported for the first time.
PMID: 24970332
 

Thursday, June 26, 2014

Gene Set Enrichment Analysis (GSEA) of Toxoplasma gondii expression datasets links cell cycle progression and the bradyzoite developmental program


2014 Jun 24;15(1):515. [Epub ahead of print]
 

BACKGROUND:

Large amounts of microarray expression data have been generated for the Apicomplexan parasite Toxoplasma gondii in an effort to identify genes critical for virulence or developmental transitions. However, researchers' ability to analyze this data is limited by the large number of unannotated genes, including many that appear to be conserved hypothetical proteins restricted to Apicomplexa. Further, differential expression of individual genes is not always informative and often relies on investigators to draw big-picture inferences without the benefit of context. We hypothesized that customization of gene set enrichment analysis (GSEA) to T. gondii would enable us to rigorously test whether groups of genes serving a common biological function are co-regulated during the developmental transition to the latent bradyzoite form.

RESULTS:

Using publicly available T. gondii expression microarray data, we created Toxoplasma gene sets related to bradyzoite differentiation, oocyst sporulation, and the cell cycle. We supplemented these with lists of genes derived from community annotation efforts that identified contents of the parasite-specific organelles, rhoptries, micronemes, dense granules, and the apicoplast. Finally, we created gene sets based on metabolic pathways annotated in the KEGG database and Gene Ontology terms associated with gene annotations available at www.toxodb.org. These gene sets were used to perform GSEA analysis using two sets of published T. gondii expression data that characterized T. gondii stress response and differentiation to the latent bradyzoite form.

CONCLUSIONS:

GSEA provides evidence that cell cycle regulation and bradyzoite differentiation are coupled. Deltagcn5A mutants unable to induce bradyzoite-associated genes in response to alkaline stress have different patterns of cell cycle and bradyzoite gene expression from stressed wild-type parasites. Extracellular tachyzoites resemble a transitional state that differs in gene expression from both replicating intracellular tachyzoites and in vitro bradyzoites by expressing genes that are enriched in bradyzoites as well as genes that are associated with the G1 phase of the cell cycle. The gene sets we have created are readily modified to reflect ongoing research and will aid researchers' ability to use a knowledge-based approach to data analysis facilitating the development of new insights into the intricate biology of Toxoplasma gondii.
PMID: 24962434
 

Monday, June 23, 2014

Toxoplasma Development of Its Replicative Niche: In Its Host Cell and Beyond

2014 Jun 20. pii: EC.00081-14. [Epub ahead of print]

Toxoplasma Development of Its Replicative Niche: In Its Host Cell and Beyond

 
Intracellular pathogens can only replicate efficiently after they manipulate and modify their host cells to create an environment conducive for its replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cytoskeletal architecture, and cell death are the three primary cellular processes that are modified by infections. Toxoplasma gondii is an obligate intracellular protozoan that infects ∼30% of the world's population and causes severe and life-threatening disease in developing fetuses, immune-comprised patients, and in certain otherwise healthy individuals who are primarily in South America. The high prevalence of Toxoplasma in humans is in large part a result of its ability to modulate these three host cell processes. Here, we will highlight recent work defining the mechanisms by which Toxoplasma interacts with the processes. In addition, we will hypothesize why some processes are modified not only in the infected host cell but also in neighboring uninfected cells.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
24951442
[PubMed - as supplied by publisher]

Toxoplasmosis as a travel risk

2014 Jun 7. pii: S1477-8939(14)00115-X. doi: 10.1016/j.tmaid.2014.05.007. [Epub ahead of print]

Toxoplasmosis as a travel risk

 

Toxoplasma gondii is a protozoan parasite with worldwide distribution that infects more than one third of the global population. Primary infection in immunocompetent individuals is usually asymptomatic; however, different organs can be affected in immunocompromised individuals leading to the development of encephalitis, myocarditis or pneumonitis. The prevalence of infection with Toxoplasma as well as its genetic structure varies geographically and for that reason travel may be considered as a risk factor to acquire the infection. As toxoplasmosis is a foodborne disease, health care providers should give health education on prevention measures to all prospective travelers in order to decrease the risk of infection in endemic areas. This review presents an overview of the infection with T. gondii with some considerations for travelers to and from endemic zones.
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS:

Clinical presentation; Prevention; Toxoplasmosis; Travelers; Treatment
PMID:
24951321
[PubMed - as supplied by publisher]

Influence of toxoplasmosis on acetylcholinesterase activity, nitric oxide levels and cellular lesion on the brain of mice

2014 May 16. pii: S0344-0338(14)00136-8. doi: 10.1016/j.prp.2014.04.025. [Epub ahead of print]

Influence of toxoplasmosis on acetylcholinesterase activity, nitric oxide levels and cellular lesion on the brain of mice

 

The objective of this study was to investigate the activity of acetylcholinesterase (AChE), nitrite/nitrate (NOx) levels, as well as the biomarkers of cellular damage in the brain of mice experimentally infected with Toxoplasma gondii. Sixty mice were divided into two experiments: in experiment I the mice were infected with T. gondii/RH strain, while in experiment II they were infected with T. gondii, strains VEG and ME-49. Our evaluations were carried out on brain homogenized samples, assessing the AChE and glutathione reductase (GR) activities, and NOx, TBARS and AOPP levels in all the infected animals, compared with the control group. In both experiments, I and II, it was observed an increase in the activity of AChE and GR, as well as in the levels of NOx in the brain of infected mice with T. gondii. TBARS levels were increased in mice infected with the three different strains, RH, ME-49, and VEG. AOPP concentration was increased only in mice infected with the RH strain. Animals infected with the strains VEG and ME-49 showed histological lesions, associated with the presence of the parasite in the brain. Therefore, the infection by T. gondii is able to interfere in cholinesterase activity and NO levels, in association with oxidative stress and histological lesion.
Copyright © 2014 Elsevier GmbH. All rights reserved.

KEYWORDS:

ME-49; RH; Toxoplasmosis; VEG
PMID:
24951243
[PubMed - as supplied by publisher]

Saturday, June 21, 2014

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

 2014 Jun 19;10(6):e1004197. doi: 10.1371/journal.ppat.1004197. eCollection 2014.

The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis

Abstract

Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.
PMID:
 
24945436
 
[PubMed - in process]

Toxoplasma gondii Profilin Promotes Recruitment of Ly6Chi CCR2+ Inflammatory Monocytes That Can Confer Resistance to Bacterial Infection

 2014 Jun 12;10(6):e1004203. doi: 10.1371/journal.ppat.1004203. eCollection 2014.

Toxoplasma gondii Profilin Promotes Recruitment of Ly6Chi CCR2+ Inflammatory Monocytes That Can Confer Resistance to Bacterial Infection

Abstract

Ly6C+ inflammatory monocytes are essential to host defense against Toxoplasma gondii, Listeria monocytogenes and other infections. During T. gondii infection impaired inflammatory monocyte emigration results in severe inflammation and failure to control parasite replication. However, the T. gondii factors that elicit these monocytes are unknown. Early studies from the Remington laboratory showed that mice with a chronic T. gondii infection survive lethal co-infections with unrelated pathogens, including L. monocytogenes, but a mechanistic analysis was not performed. Here we report that this enhanced survival against L. monocytogenes is due to early reduction of bacterial burdens and elicitation of Ly6C+ inflammatory monocytes. We demonstrate that a single TLR11/TLR12 ligand profilin (TgPRF) was sufficient to reduce bacterial burdens similar to T. gondii chronic infection. Stimulation with TgPRF was also sufficient to enhance animal survival when administered either pre- or post-Listeria infection. The ability of TgPRF to reduce L. monocytogenes burdens was dependent on TLR11 and required IFN-γ but was not dependent on IL-12 signaling. TgPRF induced rapid production of MCP-1 and resulted in trafficking of Ly6Chi CCR2+ inflammatory monocytes and Ly6G+ neutrophils into the blood and spleen. Stimulation with TgPRF reduced L. monocytogenes burdens in mice depleted with the Ly6G specific MAb 1A8, but not in Ly6C/Ly6G specific RB6-8C5 depleted or CCR2-/- mice, indicating that only inflammatory monocytes are required for TgPRF-induced reduction in bacterial burdens. These results demonstrate that stimulation of TLR11 by TgPRF is a mechanism to promote the emigration of Ly6Chi CCR2+ monocytes, and that TgPRF recruited inflammatory monocytes can provide an immunological benefit against an unrelated pathogen.
PMID:
 
24945711
 
[PubMed - in process]

Forward Genetic Screening Identifies a Small Molecule That Blocks Toxoplasma gondii Growth by Inhibiting Both Host- and Parasite-Encoded Kinases

 2014 Jun 12;10(6):e1004180. doi: 10.1371/journal.ppat.1004180. eCollection 2014.

Forward Genetic Screening Identifies a Small Molecule That Blocks Toxoplasma gondii Growth by Inhibiting Both Host- and Parasite-Encoded Kinases

Abstract

The simultaneous targeting of host and pathogen processes represents an untapped approach for the treatment of intracellular infections. Hypoxia-inducible factor-1 (HIF-1) is a host cell transcription factor that is activated by and required for the growth of the intracellular protozoan parasite Toxoplasma gondii at physiological oxygen levels. Parasite activation of HIF-1 is blocked by inhibiting the family of closely related Activin-Like Kinase (ALK) host cell receptors ALK4, ALK5, and ALK7, which was determined in part by use of an ALK4,5,7 inhibitor named SB505124. Besides inhibiting HIF-1 activation, SB505124 also potently blocks parasite replication under normoxic conditions. To determine whether SB505124 inhibition of parasite growth was exclusively due to inhibition of ALK4,5,7 or because the drug inhibited a second kinase, SB505124-resistant parasites were isolated by chemical mutagenesis. Whole-genome sequencing of these mutants revealed mutations in the Toxoplasma MAP kinase, TgMAPK1. Allelic replacement of mutant TgMAPK1 alleles into wild-type parasites was sufficient to confer SB505124 resistance. SB505124 independently impacts TgMAPK1 and ALK4,5,7 signaling since drug resistant parasites could not activate HIF-1 in the presence of SB505124 or grow in HIF-1 deficient cells. In addition, TgMAPK1 kinase activity is inhibited by SB505124. Finally, mice treated with SB505124 had significantly lower tissue burdens following Toxoplasma infection. These data therefore identify SB505124 as a novel small molecule inhibitor that acts by inhibiting two distinct targets, host HIF-1 and TgMAPK1.
PMID:
 
24945800
 
[PubMed - in process]

Thursday, June 19, 2014

Toxoplasma gondii Decreases the Reproductive Fitness in Mice

 2014 Jun 18;9(6):e96770. doi: 10.1371/journal.pone.0096770. eCollection 2014.

Toxoplasma gondii Decreases the Reproductive Fitness in Mice

Abstract

Toxoplasma gondii is a common protozoan parasite that infects warm-blooded animals throughout the world, including mice and humans. During infection, both, the parasite and the host, utilize various mechanisms to maximize their own reproductive success. Mice and humans are both the intermediate hosts for Toxoplasma gondii, which forms specialized vacuoles containing reproductive cysts in the formers' tissue. As half of the human population is infected, developing a disease called toxoplasmosis, along with an ever-growing number of couples suffering with idiopathic infertility, it is therefore surprising that there is a lack of research on how Toxoplasma gondii can alter reproductive parameters. In this study, a detailed histometric screening of the testicular function along with the levels of the pituitary luteinizing hormone (LH) were analysed in infected mice. Data on relative testis and epididymis weight, and sperm count were also collected. Based on the results obtained, the level of LH in the urine of Toxoplasma gondii infected mice was lower compared to the control. In direct correlation with the hormone level, testicular function and sperm production was also significantly lower in Toxoplasma gondii positive group using sperm count and histometric analysis as a marker. Not only were the number of leptotene primary spermatocytes and spermatids lowered, but the number of Sertoli cells and the tubule diameter were elevated. In parallel, a pilot epigenetic study on global testicular methylation, and specific methylation of Crem, Creb1 and Hspa1genes essential for successfully ongoing spermatogenesis was performed. Global methylation was elevated in Toxoplasma infected mice, and differences in the DNA methylation of selected genes were detected between the Toxoplasma positive and control group. These findings demonstrate a direct relation between Toxoplasma gondii infection and the decrease of male reproductive fitness in mice, which may contribute to an increase of idiopathic infertility in humans.
PMID:
 
24940596
 
[PubMed - as supplied by publisher]

Wednesday, June 18, 2014

Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites

2014 Jun 17;5:4098. doi: 10.1038/ncomms5098.

Plasticity and redundancy among AMA-RON pairs ensure host cell entry of Toxoplasma parasites

 
Malaria and toxoplasmosis are infectious diseases caused by the apicomplexan parasites Plasmodium and Toxoplasma gondii, respectively. These parasites have developed an invasion mechanism involving the formation of a moving junction (MJ) that anchors the parasite to the host cell and forms a ring through which the parasite penetrates. The composition and the assembly of the MJ, and in particular the presence of protein AMA1 and its interaction with protein RON2 at the MJ, have been the subject of intense controversy. Here, using reverse genetics, we show that AMA1, a vaccine candidate, interacts with RON2 to maintain the MJ structural integrity in T. gondii and is subsequently required for parasite internalization. Moreover, we show that disruption of the AMA1 gene results in upregulation of AMA1 and RON2 homologues that cooperate to support residual invasion. Our study highlights a considerable complexity and molecular plasticity in the architecture of the MJ.
PMID:
24934579
[PubMed - in process]

Transcript maturation in apicomplexan parasites

2014 Jun 13;20C:82-87. doi: 10.1016/j.mib.2014.05.012. [Epub ahead of print]

Transcript maturation in apicomplexan parasites

 
The complex life cycles of apicomplexan parasites are associated with dynamic changes of protein repertoire. In Toxoplasma gondii, global analysis of gene expression demonstrates that dynamic changes in mRNA levels unfold in a serial cascade during asexual replication and up to 50% of encoded genes are unequally expressed in development. Recent studies indicate transcription and mRNA processing have important roles in fulfilling the 'just-in-time' delivery of proteins to parasite growth and development. The prominence of post-transcriptional mechanisms in the Apicomplexa was demonstrated by mechanistic studies of the critical RNA-binding proteins and regulatory kinases. However, it is still early in our understanding of how transcription and post-transcriptional mechanisms are balanced to produce adequate numbers of specialized forms that is required to complete the parasite life cycle.
Copyright © 2014 Elsevier Ltd. All rights reserved.
PMID:
24934558
[PubMed - as supplied by publisher]

Tuesday, June 17, 2014

Parasitophorous Vacuole Membrane of Toxoplasma Is Targeted for Disruption by Ubiquitin-like Conjugation Systems of Autophagy

2014 Jun 11. pii: S1074-7613(14)00186-1. doi: 10.1016/j.immuni.2014.05.006. [Epub ahead of print]

The Parasitophorous Vacuole Membrane of Toxoplasma gondii Is Targeted for Disruption by Ubiquitin-like Conjugation Systems of Autophagy

 
Autophagy is a lysosomal degradation pathway that is important in cellular homeostasis. Prior work showed a key role for the autophagy related 5 (Atg5) in resistance to Toxoplasma gondii. Here we show that the cassette of autophagy proteins involved in the conjugation of microtubule-associated protein 1 light chain 3 (LC3) to phosphatidylethanolamine, including Atg7, Atg3, and the Atg12-Atg5-Atg16L1 complex play crucial roles in the control of T. gondii in vitro and in vivo. In contrast, pharmacologic modulation of the degradative autophagy pathway or genetic deletion of other essential autophagy genes had no substantial effects. Rather the conjugation system was required for targeting of LC3 and interferon-γ effectors onto the vacuolar membrane of T. gondii and its consequent disruption. These data suggest that the ubiquitin-like conjugation systems that reorganize intracellular membranes during canonical autophagy are necessary for proper targeting of immune effectors to the intracellular vacuole membranes utilized by pathogens.
Copyright © 2014 Elsevier Inc. All rights reserved.
PMID:
24931121
[PubMed - as supplied by publisher]

The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma

2014 Jun 6;6:19. doi: 10.1186/1757-4749-6-19. eCollection 2014.

The impact of Toll-like-receptor-9 on intestinal microbiota composition and extra-intestinal sequelae in experimental Toxoplasma gondii induced ileitis

BACKGROUND:

Following peroral Toxoplasma (T.) gondii infection, susceptible mice develop acute ileitis due to a microbiota-dependent Th1 type immunopathology. Toll-like-receptor (TLR)-9 is known to recognize bacterial DNA and mediates intestinal inflammation, but its impact on intestinal microbiota composition and extra-intestinal sequelae following T. gondii infection has not yet been elucidated.

METHODS AND RESULTS:

Seven days following peroral infection (p.i.) with 100 cysts of T. gondii ME49 strain, TLR-9(-/-) and wildtype (WT) mice suffered from comparable ileitis, whereas ileal parasitic loads as well as IFN-γ and nitric oxide levels were higher in TLR-9(-/-) compared to WT mice. Locally, TLR-9(-/-) mice exhibited increased ileal CD3+, but not FOXP3+ cell numbers at day 7 p.i.; in mesenteric lymph nodes IFN-γ-producing CD4+ cell numbers and TNF-α and IFN-γ concentrations were also increased in TLR-9(-/-) compared to WT mice. T. gondii DNA levels, however, did not differ in mice of either genotype. Differences in intestinal microbiota were rather subtle except for bifidobacteria that were virtually absent in both, naïve and T. gondii infected TLR-9(-/-), but not WT mice. Extra-intestinally, TLR-9(-/-) mice displayed less distinct systemic immune responses as indicated by lower serum IL-6, and splenic TNF-α and IFN-γ levels as compared to WT mice despite higher translocation rates of intestinal bacteria to extra-intestinal compartments such as liver, spleen, kidney, and cardiac blood. Most importantly, brains were also affected in this inflammatory scenario as early as day 7 p.i. Remarkably, TLR-9(-/-) mice exhibited more pronounced inflammatory infiltrates with higher numbers of F4/80+ macrophages and microglia in the cortex and meninges as compared to WT mice, whereas T. gondii DNA levels did not differ.

CONCLUSION:

We here show that TLR-9 is not required for the development of T. gondii induced ileitis but mediates distinct inflammatory changes in intestinal and extra-intestinal compartments including the brain.

KEYWORDS:

Acute ileitis; Bacterial translocation; Bifidobacteria; Extra-intestinal immune responses; FOXP3; Gut-brain-axis; Intestinal microbiota composition; Intracerebral inflammation; Pro-inflammatory cytokines; Regulatory T cells; Systemic inflammatory response; TLR-9; Th1-type immunopathology; Toxoplasma gondii
PMID:
24932221
[PubMed]

Monday, June 16, 2014

Dynamics of the Toxoplasma gondii inner membrane complex

2014 Jun 13. pii: jcs.147736. [Epub ahead of print]

Dynamics of the Toxoplasma gondii inner membrane complex

Abstract

Unlike most cells, protozoa in the phylum apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony, endodyogeny), using scaffolding known as the Inner Membrane Complex. The 'IMC' underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. While the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears - is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live cell imaging, fluorescence photobleaching-recovery, and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during Toxoplasma gondii tachyzoite replication. These studies reveal that formation of the T. gondii IMC involves two distinct steps: de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.
PMID:
24928899
[PubMed - as supplied by publisher]

Müller cell activation and photoreceptor depletion in a mice model of congenital ocular toxoplasmosis

2014 Jun 11. pii: S0014-4894(14)00144-1. doi: 10.1016/j.exppara.2014.06.006. [Epub ahead of print]

Müller cell activation and photoreceptor depletion in a mice model of congenital ocular toxoplasmosis

 
Müller glial cells are critically involved in retinal inflammatory processes. Here, we investigate the activation of Müller cells in a model of congenital ocular toxoplasmosis (OT). Four weeks after infection, retinal sections were studied immunohistochemically using the markers glial fibrillary acidic protein (GFAP) and vimentin. Müller cells showed strong up-regulation of both markers, as well as a deteriorated morphology in all infected retinas. Moreover, cell density and color intensity of the outer nuclear layer (ONL) of photoreceptors were decreased. Our results indicate, for the first time, that the severe retinal damage and loss of vision observed in human OT is not directly caused by infection but rather mediated by infection induced reactive gliosis.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

GFAP; Müller cells; Photoreceptors; Toxoplasma gondii; Vimentin
PMID:
24929147
[PubMed - as supplied by publisher]

Saturday, June 14, 2014

ROP18 Is a Key Factor Responsible for Virulence Difference between Toxoplasma gondii and Neospora caninum

 2014 Jun 13;9(6):e99744. doi: 10.1371/journal.pone.0099744. eCollection 2014.

ROP18 Is a Key Factor Responsible for Virulence Difference between Toxoplasma gondii and Neospora caninum

Abstract

Toxoplasma gondii (T. gondii) and Neospora caninum (N. caninum) are both obligate intracellular protozoan parasites and share many common morphological and biological features. Despite these similarities the two parasites differ dramatically in virulence in mice, but the factors involved in virulence differences between the two parasites remain unknown. A secreted serine-threonine kinase called rhoptry protein 18 (ROP18) was identified to play a crucial role on virulence differences among different T. gondii clonal lineages. Intriguingly, we found that ROP18 in Nc1 strain of N. caninum (NcROP18) is a pseudogene due to several interrupting stop codons in the sequence in our previous studies. We assume that the difference of ROP18 leads to virulence difference between T. gondii and N. caninum. We constructed a transgenic N. caninum Nc1 stain by transfecting the TgROP18 from the T. gondii RH strain. Phenotype and virulence assays showed that the expression of TgROP18 in N. caninum did not affect the motility and cell invasion, but resulted in a significant increase in intracellular parasite proliferation and virulence in mice. Immunity-Related GTPase (IRG) phosphorylation assay showed that the transgenic parasite Nc1-TgROP18 was able to phosphorylate IRGs as T. gondii did. The present study indicated that the ROP18 plays a crucial role in virulence of the closely related parasites T. gondii and N. caninum and it is indeed a key factor responsible for the virulence difference between T. gondii and N. caninum.
PMID:
 
24927100
 
[PubMed - as supplied by publisher]

Calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites

 2014 Jun 13:1-11. [Epub ahead of print]

The gatekeeper residue and beyond: homologous calcium-dependent protein kinases as drug development targets for veterinarian Apicomplexa parasites

Abstract

SUMMARY Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.
PMID:
 
24927073
 
[PubMed - as supplied by publisher]

Abstract

SUMMARY Specific roles of individual CDPKs vary, but in general they mediate essential biological functions necessary for parasite survival. A comparative analysis of the structure-activity relationships (SAR) of Neospora caninum, Eimeria tenella and Babesia bovis calcium-dependent protein kinases (CDPKs) together with those of Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii was performed by screening against 333 bumped kinase inhibitors (BKIs). Structural modelling and experimental data revealed that residues other than the gatekeeper influence compound-protein interactions resulting in distinct sensitivity profiles. We subsequently defined potential amino-acid structural influences within the ATP-binding cavity for each orthologue necessary for consideration in the development of broad-spectrum apicomplexan CDPK inhibitors. Although the BKI library was developed for specific inhibition of glycine gatekeeper CDPKs combined with low inhibition of threonine gatekeeper human SRC kinase, some library compounds exhibit activity against serine- or threonine-containing CDPKs. Divergent BKI sensitivity of CDPK homologues could be explained on the basis of differences in the size and orientation of the hydrophobic pocket and specific variation at other amino-acid positions within the ATP-binding cavity. In particular, BbCDPK4 and PfCDPK1 are sensitive to a larger fraction of compounds than EtCDPK1 despite the presence of a threonine gatekeeper in all three CDPKs.
PMID:
 
24927073
 
[PubMed - as supplied by publisher]

Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma

 2014 Jun 13:1-9. [Epub ahead of print]

Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii

Abstract

SUMMARY The dissection of apicomplexan biology has been highly influenced by the genetic tools available for manipulation of parasite DNA. Here, we describe different techniques available for the generation of conditional mutants. Comparison of the advantages and disadvantages of the three most commonly used regulation systems: the tetracycline inducible system, the regulation of protein stability and site-specific recombination are discussed. Using some previously described examples we explore some of the pitfalls involved in gene-function analysis using these systems that can lead to wrong or over-interpretation of phenotypes. We will also mention different options to standardize the application of these techniques for the characterization of gene function in high-throughput.
PMID:
 
24926834
 
[PubMed - as supplied by publisher]

Thursday, June 12, 2014

A Survey of Innovation through Duplication in the Reduced Genomes of Twelve Parasites

2014 Jun 11;9(6):e99213. doi: 10.1371/journal.pone.0099213. eCollection 2014.

A Survey of Innovation through Duplication in the Reduced Genomes of Twelve Parasites

Abstract

We characterize the prevalence, distribution, divergence, and putative functions of detectable two-copy paralogs and segmental duplications in the Apicomplexa, a phylum of parasitic protists. Apicomplexans are mostly obligate intracellular parasites responsible for human and animal diseases (e.g. malaria and toxoplasmosis). Gene loss is a major force in the phylum. Genomes are small and protein-encoding gene repertoires are reduced. Despite this genomic streamlining, duplications and gene family amplifications are present. The potential for innovation introduced by duplications is of particular interest. We compared genomes of twelve apicomplexans across four lineages and used orthology and genome cartography to map distributions of duplications against genome architectures. Segmental duplications appear limited to five species. Where present, they correspond to regions enriched for multi-copy and species-specific genes, pointing toward roles in adaptation and innovation. We found a phylum-wide association of duplications with dynamic chromosome regions and syntenic breakpoints. Trends in the distribution of duplicated genes indicate that recent, species-specific duplicates are often tandem while most others have been dispersed by genome rearrangements. These trends show a relationship between genome architecture and gene duplication. Functional analysis reveals: proteases, which are vital to a parasitic lifecycle, to be prominent in putative recent duplications; a pair of paralogous genes in Toxoplasma gondii previously shown to produce the rate-limiting step in dopamine synthesis in mammalian cells, a possible link to the modification of host behavior; and phylum-wide differences in expression and subcellular localization, indicative of modes of divergence. We have uncovered trends in multiple modes of duplicate divergence including sequence, intron content, expression, subcellular localization, and functions of putative recent duplicates that highlight the role of duplications in the continuum of forces that have shaped these genomes.
PMID:
24919110
[PubMed - as supplied by publisher]

Monday, June 09, 2014

Toxoplasma gondii influences aversive behaviors of female rats in an estrus cycle dependent manner

2014 Jun 4. pii: S0031-9384(14)00326-6. doi: 10.1016/j.physbeh.2014.05.036. [Epub ahead of print]

Toxoplasma gondii influences aversive behaviors of female rats in an estrus cycle dependent manner

Abstract

The protozoan Toxoplasma gondii (T.gondii) manipulates the behavior of its rodent intermediate host to facilitate its passage to its feline definitive host. This is accomplished by a reduction of the aversive response that rodents show towards cat odors, which likely increases the predation risk. Females on average show similar changes as males. However, behaviors that relate to aversion and attraction are usually strongly influenced by the estrus cycle. In this study, we replicated behavioral effects of T.gondii in female rats, as well as expanded it to two novel behavioral paradigms. We also characterized the role of the estrus cycle in the behavioral effects of T.gondii on female rats. Uninfected females preferred to spend more time in proximity to rabbit rather than bobcat urine, and in a dark chamber rather than lit chamber. Infected females lost both of these preferences, and also spent more time investigating social novelty (foreign bedding in their environment). Taken together, these data suggest that infection makes females less risk averse and more exploratory. Furthermore, this effect was influenced by the estrus cycle. Uninfected rats preferred rabbit urine to bobcat urine throughout the cycle except at estrus and metestrus. In contrast, infected rats lost this preference at every stage of the cycle except estrus. Commensurate with the possibility that this was a hormone-dependent effect, infected rats had elevated levels of circulating progesterone, a known anxiolytic.
Copyright © 2014. Published by Elsevier Inc.

KEYWORDS:

Aversion; Behavior; Estrus; Female; Light aversion; Predator aversion; Progesterone; Rat; Toxoplasma gondii
PMID:
24907696
[PubMed - as supplied by publisher]

What Makes a Feline Fatal in Toxoplasma gondii's Fatal Feline Attraction? Infected Rats Choose Wild Cats

2014 Jun 6. pii: icu060. [Epub ahead of print]

What Makes a Feline Fatal in Toxoplasma gondii's Fatal Feline Attraction? Infected Rats Choose Wild Cats

Abstract

Toxoplasma gondii is an indirectly transmitted protozoan parasite, of which members of the cat family (Felidae) are the only definitive hosts and small mammals such as rats serve as intermediate hosts. The innate aversion of rodents to cat odor provides an obstacle for the parasite against successful predation by the feline definitive host. Previous research has demonstrated that T. gondii appears to alter a rat's perception of the risk of being preyed upon by cats. Although uninfected rats display normal aversion to cat odor, infected rats show no avoidance and in some cases even show attraction to cat odor, which we originally termed the "Fatal Feline Attraction." In this study, we tested for the first time whether the "Fatal Feline Attraction" of T. gondii-infected rats differed according to the type of feline odor used, specifically whether it came from domestic cats (Felis silvestris catus) or wild cats-cheetahs (Acinonyx jubatus) or pumas (Felis concolor). In two-choice odor trials, where wild and domestic cat odors were competed against one another, consistent with previous findings we demonstrated that infected rats spent more time in feline odor zones compared with uninfected rats. However, we further demonstrated that all cat odors are not equal: infected rats had a stronger preference for wild cat odor over that of domestic cats, an effect that did not differ significantly according to the type of wild cat odor used (cheetah or puma). We discuss these results in terms of the potential mechanism of action and their implications for the current and evolutionary role of wild, in addition to domestic, cats in transmission of T. gondii.
© The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.
PMID:
24907200
[PubMed - as supplied by publisher]