Wednesday, December 31, 2014

Reassessment of the role of aromatic amino acid hydroxylases and the effect of infection by Toxoplasma gondii on host dopamine levels

 2014 Dec 29. pii: IAI.02465-14. [Epub ahead of print]

Abstract

Toxoplasma gondii infection has previously been described to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was over-expressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may locally affect this pathway. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii since AAH1 is essential for growth in non-dopaminergic cells.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
 
25547791
 
[PubMed - as supplied by publisher]

3-D Imaging and Analysis of Neurons Infected In Vivo with Toxoplasma gondii

 2014 Dec 9;(94). doi: 10.3791/52237.

Abstract

Toxoplasma gondii is an obligate, intracellular parasite with a broad host range, including humans and rodents. In both humans and rodents, Toxoplasma establishes a lifelong persistent infection in the brain. While this brain infection is asymptomatic in most immunocompetent people, in the developing fetus or immunocompromised individuals such as acquired immune deficiency syndrome (AIDS) patients, this predilection for and persistence in the brain can lead to devastating neurologic disease. Thus, it is clear that the brain-Toxoplasma interaction is critical to the symptomatic disease produced by Toxoplasma, yet we have little understanding of the cellular or molecular interaction between cells of the central nervous system (CNS) and the parasite. In the mouse model of CNS toxoplasmosis it has been known for over 30 years that neurons are the cells in which the parasite persists, but little information is available about which part of the neuron is generally infected (soma, dendrite, axon) and if this cellular relationship changes between strains. In part, this lack is secondary to the difficulty of imaging and visualizing whole infected neurons from an animal. Such images would typically require serial sectioning and stitching of tissue imaged by electron microscopy or confocal microscopy after immunostaining. By combining several techniques, the method described here enables the use of thick sections (160 µm) to identify and image whole cells that contain cysts, allowing three-dimensional visualization and analysis of individual, chronically infected neurons without the need for immunostaining, electron microscopy, or serial sectioning and stitching. Using this technique, we can begin to understand the cellular relationship between the parasite and the infected neuron.
PMID:
 
25549001
 
[PubMed - as supplied by publisher]

Friday, December 26, 2014

NextGen sequencing reveals short double crossovers contribute disproportionately to genetic diversity in Toxoplasma gondii

 2014 Dec 23;15(1):1168. [Epub ahead of print]

Abstract

BACKGROUND: 

Toxoplasma gondii is a widespread protozoan parasite of animals that causes zoonotic disease in humans. Three clonal variants predominate in North America and Europe, while South American strains are genetically diverse, and undergo more frequent recombination. All three northern clonal variants share a monomorphic version of chromosome Ia (ChrIa), which is also found in unrelated, but successful southern lineages. Although this pattern could reflect a selective advantage, it might also arise from non-Mendelian segregation during meiosis. To understand the inheritance of ChrIa, we performed a genetic cross between the northern clonal type 2 ME49 strain and a divergent southern type 10 strain called VAND, which harbors a divergent ChrIa.

RESULTS: 

NextGen sequencing of haploid F1 progeny was used to generate a genetic map revealing a low level of conventional recombination, with an unexpectedly high frequency of short, double crossovers. Notably, both the monomorphic and divergent versions of ChrIa were isolated with equal frequency. As well, ChrIa showed no evidence of being a sex chromosome, of harboring an inversion, or distorting patterns of segregation. Although VAND was unable to self fertilize in the cat, it underwent successful out-crossing with ME49 and hybrid survival was strongly associated with inheritance of ChrIII from ME49 and ChrIb from VAND..

CONCLUSIONS: 

Our findings suggest that the successful spread of the monomorphic ChrIa in the wild has not been driven by meiotic drive or related processes, but rather is due to a fitness advantage. As well, the high frequency of short double crossovers is expected to greatly increase genetic diversity among progeny from genetic crosses, thereby providing an unexpected and likely important source of diversity.
PMID:
 
25532601
 
[PubMed - as supplied by publisher]

Regulatory cells induced by acute toxoplasmosis prevent the development of allergic lung inflammation

 2014 Dec 13. pii: S0171-2985(14)00262-9. doi: 10.1016/j.imbio.2014.11.016. [Epub ahead of print]

Abstract

The increased prevalence of allergies in developed countries has been attributed to a reduction of some infections. Supporting epidemiological studies, we previously showed that both acute and chronic Toxoplasma gondii infection can diminish allergic airway inflammation in BALB/c mice. The mechanisms involved when sensitization occurs during acute phase would be related to the strong Th1 response induced by the parasite. Here, we further investigated the mechanisms involved in T. gondii allergy protection in mice sensitized during acute T. gondii infection. Adoptive transference assays and ex vivo co-cultures experiments showed that not only thoracic lymph node cells from infected and sensitized mice but also from non-sensitized infected animals diminished both allergic lung inflammation and the proliferation of effector T cells from allergic mice. This ability was found to be contact-independent and correlated with high levels of CD4+FoxP3+ cells. IL-10 would not be involved in allergy suppression since IL-10-deficient mice behaved similar to wild type mice. Our results extend earlier work and show that, in addition to immune deviation, acute T. gondii infection can suppress allergic airway inflammation through immune suppression.
Copyright © 2014 Elsevier GmbH. All rights reserved.

KEYWORDS: 

Allergy; Immune suppression; Inflammation; Lung; Regulatory cells; Toxoplasma gondii
PMID:
 
25532793
 
[PubMed - as supplied by publisher]

Toxoplasma gondii-infected natural killer cells display a hypermotility phenotype in vivo

 2014 Dec 23. doi: 10.1038/icb.2014.106. [Epub ahead of print]

Abstract

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two-photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected 'bystander' NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA-1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii-infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host.Immunology and Cell Biology advance online publication, 23 December 2014; doi:10.1038/icb.2014.106.
PMID:
 
25533287
 
[PubMed - as supplied by publisher]

Saturday, December 20, 2014

Unique features of apicoplast DNA gyrases from Toxoplasma gondii and Plasmodium falciparum

 2014 Dec 19;15(1):416. [Epub ahead of print]

Abstract

BackgroundDNA gyrase, an enzyme once thought to be unique to bacteria, is also found in some eukaryotic plastids including the apicoplast of Apicomplexa such as Plasmodium falciparum and Toxoplasma gondii which are important disease-causing organisms. DNA gyrase is an excellent target for antibacterial drugs, yet such antibacterials seem ineffective against Apicomplexa. Characterisation of the apicoplast gyrases would be a useful step towards understanding why this should be so. While purification of active apicoplast gyrase has proved impossible to date, in silico analyses have allowed us to discover differences in the apicoplast proteins. The resulting predicted structural and functional differences will be a first step towards development of apicoplast-gyrase specific inhibitors.ResultsWe have carried out sequence analysis and structural predictions of the enzymes from the two species and find that P. falciparum gyrase lacks a GyrA box, but T. gondii may retain one. All proteins contained signal/transport peptides for localization to the apicoplast but T. gondii Gyrase B protein lacks the expected hydrophobic region. The most significant difference is in the GyrA C-terminal domain: While the cores of the proteins, including DNA binding and cleavage regions are essentially unchanged, both apicoplast gyrase A proteins have C-terminal domains that are significantly larger than bacterial counterparts and are predicted to have different structures.ConclusionThe apicoplast gyrases differ significantly from bacterial gyrases while retaining similar core domains. T. gondii Gyrase B may have an unusual or inefficient mechanism of localisation to the apicoplast. P.falciparum gyrase, lacks a GyrA box and is therefore likely to be inefficient in DNA supercoiling. The C-terminal domains of both apicoplast Gyrase A proteins diverge significantly from the bacterial proteins. We predict that an additional structural element is present in the C-terminal domain of both apicoplast Gyrase A proteins, including the possibility of a ß-pinwheel with a non-canonical number of blades. These differences undoubtedly will affect the DNA supercoiling mechanism and have perhaps evolved to compensate for the lack of Topoisomerase IV in the apicoplast. These data will be useful first step towards further characterisation of and development of inhibitors for apicoplast gyrases.
PMID:
 
25523502
 
[PubMed - as supplied by publisher]

Identification and bioinformatic analysis of a putative calcium-dependent protein kinase (CDPK6) from Toxoplasma gondii

 2014 Dec 18;13(4):10669-10677.

Abstract

Toxoplasma gondii is recognized as an opportunistic human pathogen with a worldwide distribution. Development of effective vaccines is considered the only ideal way to control T. gondii infection. However, only one live vaccine is commercially available for use in sheep and goats. Therefore, the identification of more effective antigenic proteins is very important. In this study, we identified a novel putative calcium-dependent protein kinase of T. gondii, TgCDPK6, and further analyzed its potential antigenicity using a bioinformatic approach. The physical and chemical characteristics, transmembrane domain, epitopes, advanced structure, and functional sites of TgCDPK6 were predicted by multiple bioinformatic approaches. Twenty-six post-translational modification sites were identified in the protein. The secondary structure showed that 58.35% amino acids of TgCDPK6 are exposed to the solvent interface, and the high hydrophilic domains were distributed in amino acid positions 21-59, 68-81, 156-205, 245-271, 280-294, 297-324, 334-356, 367-393, 474-498, and 543-553. The advanced structure of TgCDPK6 was developed by a homology modeling method and was validated by PROCHECK, which showed that most amino acid residues were in the most favored regions. Using these analyses, 10 potential epitopes were predicted. The results indicated that TgCDPK6 could be a vaccine candidate antigen against T. gondii.
PMID:
 
25526188
 
[PubMed - as supplied by publisher]

Tuesday, December 09, 2014

Genetic mapping reveals that sinefungin resistance in Toxoplasma gondii is controlled by a putative amino acid transporter locus that can be used as a negative selectable marker

 2014 Dec 5. pii: EC.00229-14. [Epub ahead of print]

Abstract

Quantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasite Toxoplasma gondii. Here, we interrogate a different phenotype by mapping sinefungin (SNF) drug resistance in the genetic cross between type 2 ME49-FUDRR and type 10 VAND-SNFR. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of SNPs inherited from the parents. Based on this high density genetic map, we were able to pin point the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single non-synonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified occurring only in the sinefungin resistant progeny. Using CRISPR/CAS9 we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of the SNR1 gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole genome sequencing, and CRISPR mediated gene disruption for combined forward and reverse genetic strategies in T. gondii.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
 
25480939
 
[PubMed - as supplied by publisher]

Utilization of inherent miRNAs in functional analyses of Toxoplasma gondii genes

 2014 Dec 3. pii: S0167-7012(14)00329-7. doi: 10.1016/j.mimet.2014.11.014. [Epub ahead of print]

Abstract

MicroRNAs (miRNAs) are crucial genetic effectors partaking in numerous mechanisms of gene regulation in eukaryotic organisms. Recent discoveries of miRNA in Toxoplasma gondii, an intracellular obligate parasite of the phylum Apicomplexa, suggested possible roles of T. gondii miRNAs (Tg-miRNAs) in the post-transcriptional gene regulation and in the cell biology of the parasite. To gain a better understanding of the involvement of Tg-miRNAs in regulating the parasite gene expression, a dual luciferase reporter system was used in the examination and evaluation of the effects of endogenous Tg-miRNAs, their mimics and inhibitors. A Renilla luciferase (Rnluc) transcript was engineered to carry independent binding sites of two abundant species, namely Tg-miR-60a and Tg-miR-4a, so that the expression of Rnluc was silenced in a sequence specific manner by Tg-miR-60a and Tg-miR-4a. Notably, Tg-miR-60a, but not Tg-miR-4a, caused the levels of Rnluc transcripts to decrease. These findings strongly suggested that T. gondii employs the Tg-miRNA species-specific mode of silencing actions: transcript degradation by Tg-miR-60a, and translational suppression by Tg-miR-4a. Herein we developed a genetic system that exploits and directs the most abundant Tg-miR-60a for loss-of-function analyses in T. gondii. As a proof of principle, we showed that when the binding sites for Tg-miR-60a were introduced into the parasite transcripts via homologous recombination at the locus of (i) DEAD-box RNA helicase (TgHoDI), or (ii) lactate dehydrogenase isoform 1 (TgLDH1), the expression levels of the selected genes can be altered. It was thus proven that inherit Tg-miR-60a could be directed and used to assist in the loss-of-function analyses.
Copyright © 2014. Published by Elsevier B.V.

KEYWORDS: 

Gene modulation; Genetic tool; Toxoplasma; miRNA inhibitor; miRNA mimic
PMID:
 
25479428
 
[PubMed - as supplied by publisher]

Quantification and viability assays of Toxoplasma gondii in commercial "Serrano" ham

 2015 Apr;46:107-13. doi: 10.1016/j.fm.2014.07.003. Epub 2014 Jul 15.

Abstract

"Serrano" ham is a typical pork product from the Mediterranean area, highly valued for its flavour. To make Serrano ham, pork undergoes a salting and a subsequent fermentation process known as curing. Certain pigs used for meat production are an important source of Toxoplasma gondii infection in humans. We have developed a method for quantifying and assaying the viability of the T. gondii present in commercial Serrano ham samples. A magnetic capture method for the isolation of T. gondii DNA and a qRT-PCR were used to estimate the T. gondii burden in 475 commercial samples of "Serrano" ham in two presentation formats: ham pieces and sliced ham. The infectivity capacity of T. gondii in positive samples was assayed in mice. The global prevalence of T. gondii was 8.84%, ranging from 32.35% in one of the companies to 0% prevalence in three other companies. The infectivity assays revealed that only 4.84% of the positive samples were infective. To the best of our knowledge this is the first report focussing on the prevalence of T. gondii in commercial "Serrano" ham. The method described here could be useful for producers to guarantee the safety of their products. 
Copyright © 2014 Elsevier Ltd. All rights reserved.

KEYWORDS: 

Bioassay; Commercial cured “Serrano Ham”; DNA magnetic capture; Infectivity; Quantitative real-time PCR; Toxoplasma gondii
PMID:
 
25475273
 
[PubMed - in process]

Friday, December 05, 2014

Genetic Manipulation of the Toxoplasma gondii Genome by Fosmid Recombineering

2014 Dec 2;5(6). pii: e02021-14. doi: 10.1128/mBio.02021-14.
 
 
Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.

IMPORTANCE:

Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.
Copyright © 2014 Vinayak et al.
PMID:
25467441
[PubMed - in process]

Wednesday, December 03, 2014

A small-molecule cell-based screen led to the identification of biphenylimidazoazines with highly potent and broad-spectrum anti-apicomplexan activity

2014 Oct 22;89C:386-400. doi: 10.1016/j.ejmech.2014.10.057. [Epub ahead of print]
 
 
An in vitro screening of the anti-apicomplexan activity of 51 compounds, stemming from our chemical library and from chemical synthesis, was performed. As a study model, we used Toxoplasma gondii (T. gondii), expressing β-galactosidase for the colorimetric assessment of drug activity on parasites cultivated in vitro. This approach allowed the validation of a new series of molecules with a biphenylimidazoazine scaffold as inhibitors of T. gondii growth in vitro. Hence, 8 molecules significantly inhibited intracellular replication of T. gondii in vitro, with EC50 < 1 μM, while being non-toxic for human fibroblasts at these concentrations. Most attractive candidates were then selected for further biological investigations on other apicomplexan parasites (Neospora caninum, Besnoitia besnoiti, Eimeria tenella and Plasmodium falciparum). Finally, two compounds were able to inhibit growth of four different apicomplexans with EC50 in the submicromolar to nanomolar range, for each parasite. These data, including the broad anti-parasite spectrum of these inhibitors, define a new generation of potential anti-parasite compounds of wide interest, including for veterinary application. Studies realized on E. tenella suggest that these molecules act during the intracellular development steps of the parasite. Further experiments should be done to identify the molecular target(s) of these compounds.
Copyright © 2014 Elsevier Masson SAS. All rights reserved.

KEYWORDS:

Anti-apicomplexan; Besnoitia besnoiti; Eimeria tenella; Imidazo[1,2-a]pyridine; Imidazo[1,2-b]pyridazine; Neospora caninum; Plasmodium falciparum; Toxoplasma gondii
PMID:
25462254
[PubMed - as supplied by publisher]

Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9

2014 Nov 13. pii: S0020-7519(14)00267-7. doi: 10.1016/j.ijpara.2014.09.003. [Epub ahead of print]
 
 
Leucine aminopeptidases of the M17 peptidase family represent ideal drug targets for therapies directed against the pathogens Plasmodium, Babesia and Trypanosoma. Previously, we characterised Toxoplasma gondii leucine aminopeptidase and demonstrated its role in regulating the levels of free amino acids. In this study, we evaluated the potential of T. gondii leucine aminopeptidase as a drug target in T. gondii by a knockout method. Existing knockout methods for T. gondii have many drawbacks; therefore, we developed a new technique that takes advantage of the CRISPR/Cas9 system. We first chose a Cas9 target site in the gene encoding T. gondii leucine aminopeptidase and then constructed a knockout vector containing Cas9 and the single guide RNA. After transfection, single tachyzoites were cloned in 96-well plates by limiting dilution. Two transfected strains derived from a single clone were cultured in Vero cells, and then subjected to expression analysis by western blotting. The phenotypic analysis revealed that knockout of T. gondii leucine aminopeptidase resulted in inhibition of attachment/invasion and replication; both the growth and attachment/invasion capacity of knockout parasites were restored by complementation with a synonymously substituted allele of T. gondii leucine aminopeptidase. Mouse experiments demonstrated that T. gondii leucine aminopeptidase knockout somewhat reduced the pathogenicity of T. gondii. An enzymatic activity assay showed that T. gondii leucine aminopeptidase knockout reduced the processing of a leucine aminopeptidase-specific substrate in T. gondii. The absence of leucine aminopeptidase activity could be slightly compensated for in T. gondii. Overall, T. gondii leucine aminopeptidase knockout influenced the growth of T. gondii, but did not completely block parasite development, virulence or enzymatic activity. Therefore, we conclude that leucine aminopeptidase would be useful only as an adjunctive drug target in T. gondii.
Copyright © 2014. Published by Elsevier Ltd.

KEYWORDS:

CRISP/Cas9; Growth; Invasion; Leucine aminopeptidase; Toxoplasma gondii
PMID:
25444863
[PubMed - as supplied by publisher]

Mini-FLOTAC for counting Toxoplasma gondii oocysts from cat feces - Comparison with cell counting plates

2014 Oct 18;147C:67-71. doi: 10.1016/j.exppara.2014.10.009. [Epub ahead of print]
 
 
Oocysts of Toxoplasma gondii represent one of the most common environmental contaminants causing the zoonotic infection toxoplasmosis. The aim of the present study was to compare the Mini-FLOTAC device with traditional cell counting plates (Kova Slide) for the detection of T. gondii oocysts from feline feces. Two types of experiments were performed: (i) purified oocysts were counted in different dilutions and (ii) specific pathogen free T. gondii-negative cat feces was inoculated with numbers of purified oocysts and counting was performed directly from feces. Our analysis showed a thousand times higher sensitivity of Mini-FLOTAC (5 × 102 oocysts) compared to Kova Slide (5 × 105 oocysts). Also, when compared by McNemar's test, counting of the purified oocysts showed a higher sensitivity of Mini-FLOTAC compared to Kova Slide, for a dilution of 103 oocysts/ml
Our results show that Mini-FLOTAC is more sensitive than traditional methods of T. gondii oocysts detection and quantification is more accurate. Furthermore, Mini-FLOTAC simplicity and cost effectiveness allow it to be used with light microscopes in any laboratory or field conditions. We therefore recommend its use for regular screening. Further studies are needed to validate Mini-FLOTAC for the detection of oocysts in soil and water samples in field conditions.

KEYWORDS:

Detection; Kova slide; Mini-FLOTAC; Oocysts; Toxoplasma gondii
PMID:
25448359
[PubMed - as supplied by publisher]

Phylogeny and evolution of apicoplasts and apicomplexan parasites

2014 Oct 14. pii: S1383-5769(14)00141-X. doi: 10.1016/j.parint.2014.10.005. [Epub ahead of print]
 
 
The phylum Apicomplexa includes many parasitic genera of medical and veterinary importance including Plasmodium (causative agent of malaria), Toxoplasma (toxoplasmosis), and Babesia (babesiosis). Most of the apicomplexan parasites possess a unique, essential organelle, the apicoplast, which is a plastid without photosynthetic ability. Although the apicoplast is considered to have evolved through secondary endosymbiosis of a red alga into the common ancestral cell of apicomplexans, its evolutionary history has been under debate until recently. The apicoplast has a genome around 30-40kb in length. Repertoire and arrangement of the apicoplast genome-encoded genes differ among apicomplexan genera, although within the genus Plasmodium these are almost conserved. Genes in the apicoplast genome may be useful markers for Plasmodium phylogeny, because these are single copy (except for the inverted repeat region) and may have more phylogenetic signal than the mitochondrial genome that have been most commonly used for Plasmodium phylogeny. This review describes recent studies concerning the evolutionary origin of the apicoplast, presents evolutionary comparison of the primary structures of apicoplast genomes from apicomplexan parasites, and summarizes recent findings of malaria phylogeny based on apicoplast genome-encoded genes.
Copyright © 2014. Published by Elsevier Ireland Ltd.

KEYWORDS:

Apicoplast; Evolution; Phylogeny; Plasmodium
PMID:
25451217
[PubMed - as supplied by publisher]

CD73-Generated Adenosine is Critical for Immune Regulation During Toxoplasma gondii Infection

2014 Dec 1. pii: IAI.02536-14. [Epub ahead of print]
 
 
As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii evades immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. Previously, we found that mice deficient in the ecto-enzyme CD73, which generates adenosine in the extracellular matrix, were resistant to chronic toxoplasmosis after oral infection with T. gondii. Resistance in CD73-knockout mice was due to a delay in parasite differentiation in the CNS. To further clarify the role of CD73 and extracellular adenosine in T. gondii pathogenesis, we infected WT and CD73-/- mice with T. gondii cysts systemically by the intraperitoneal (i.p.) route. In contrast to oral infection, i.p.-infected CD73-/- mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. Administration of the broad spectrum adenosine receptor agonist NECA protected CD73-/- mice against T. gondii induced immunopathology, suggesting absence of CD73-generated adenosine led to the increased susceptibility in these mice. Peritoneal exudate cells from infected CD73-/- mice produced higher levels of the inflammatory mediators, nitric oxide, TNFα, and IL1β, without enhanced parasite killing or clearance. Bone marrow chimeras established that CD73 expression on both hematopoietic and nonhematopoietic compartments contributes to limiting T. gondii-induced immunopathology. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii, compared to WT mice. Thus, extracellular adenosine is a key immune regulator that limits collateral tissue damage to an intracellular pathogen and promotes host survival.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
25452548
[PubMed - as supplied by publisher]

Monday, December 01, 2014

Protein-protein interaction studies provide evidence for electron transfer from ferredoxin to lipoic acid synthase in Toxoplasma gondii

 2014 Nov 26. pii: S0014-5793(14)00825-4. doi: 10.1016/j.febslet.2014.11.020. [Epub ahead of print]

Abstract

The only known redox system in the apicoplast, a plastid-like organelle of apicomplexan parasites, is ferredoxin and ferredoxin-associated reductase. Ferredoxin donates electrons to different enzymes, presumably including lipoate synthase (LipA), which is essential for fatty acid biosynthesis. We recombinantly expressed and characterized LipA from the protozoan parasite Toxoplasma gondii, generated LipA-specific antibodies and confirmed the apicoplast localization of LipA. Electron transfer from ferredoxin to LipA would require direct protein-protein interaction. Such a robust interaction between the two proteins was demonstrated in both yeast and bacterial two-hybrid systems. Taken together, our results provide strong evidence for a role of ferredoxin as an electron donor to LipA.
Copyright © 2014. Published by Elsevier B.V.

KEYWORDS: 

Apicomplexa; Ferredoxin; Lipoic acid synthesis; Plastid; Protein interactions; Two-hybrid system
PMID:
 
25433292
 
[PubMed - as supplied by publisher]