J Vis Exp. 2013 Jul 12;(77). doi: 10.3791/50598.

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Rommereim LM, Hortua Triana MA, Falla A, Sanders KL, Guevara RB, Bzik DJ, Fox BA.

Source

Department of Microbiology and Immunology, The Geisel School of Medicine at Dartmouth.

Abstract

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein(1,2). The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale(1-4). Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

PMID:

23892917

[PubMed - in process]

Int J Parasitol. 2013 Jul 23. pii: S0020-7519(13)00179-3. doi: 10.1016/j.ijpara.2013.06.007. [Epub ahead of print]

Parasite zoonoses and wildlife: One health, spillover and human activity

Thompson RC.

Source

School of Veterinary and Health Sciences, Murdoch University, Murdoch, WA 6150, Australia. Electronic address: a.thompson@murdoch.edu.au.

Abstract

This review examines parasite zoonoses and wildlife in the context of the One Health triad that encompasses humans, domestic animals, wildlife and the changing ecosystems in which they live. Human (anthropogenic) activities influence the flow of all parasite infections within the One Health triad and the nature and impact of resulting spillover events are examined. Examples of spillover from wildlife to humans and/or domestic animals, and vice versa, are discussed, as well as emerging issues, particularly the need for parasite surveillance of wildlife populations. Emphasis is given to Trypanosoma cruzi and related species in Australian wildlife, Trichinella, Echinococcus, Giardia, Baylisascaris, Toxoplasma and Leishmania.
Copyright © 2013. Published by Elsevier Ltd.

KEYWORDS:

Echinococcus spp., Giardia, One health, Spillover, Toxoplasma, Trichinella, Trypanosoma spp., Zoonoses

PMID:

23892130

[PubMed - as supplied by publisher]

Curr Opin Microbiol. 2013 Jul 26. pii: S1369-5274(13)00092-1. doi: 10.1016/j.mib.2013.07.004. [Epub ahead of print]

Key molecular events during host cell invasion by Apicomplexan pathogens

Sharma P, Chitnis CE.

Source

National Institute of Immunology (NII), New Delhi, India. Electronic address: pushkar@nii.ac.in.

Abstract

The ability of Apicomplexan parasites to invade host cells is key to their survival and pathogenesis. Plasmodium and Toxoplasma parasites share common mechanisms for invasion of host cells. Secretion of microneme and rhoptry proteins, tight junction formation and assembly of an acto-myosin motor are key steps for successful invasion by both parasites. Here, we review our understanding of the molecular basis for these steps.
Copyright © 2013 Elsevier Ltd. All rights reserved.

PMID:

23895827

[PubMed - as supplied by publisher]

J Biol Chem. 2013 Jul 22. [Epub ahead of print]

Elp3 lysine acetyltransferase is a tail-anchored mitochondrial protein in Toxoplasma gondii

Stilger KL, Sullivan WJ Jr.

Source

Indiana University School of Medicine, United States.

Abstract

Lysine acetylation has recently emerged as an important, widespread post-translational modification occurring on proteins that reside in multiple cellular compartments, including the mitochondria. However, no lysine acetyltransferase (KAT) has been definitively localized to this organelle to date. Here we describe the identification of an unusual homologue of Elp3 in early-branching protozoa in the phylum Apicomplexa. Elp3 is the catalytic subunit of the well-conserved transcription Elongator complex; however, Apicomplexa lack all other Elongator subunits, suggesting that the Elp3 in these organisms plays a role independent of transcription. Surprisingly, Elp3 in the parasites of this phylum, including Toxoplasma gondii (TgElp3), possesses a unique C-terminal transmembrane domain (TMD) that localizes the protein to the mitochondrion. As TgElp3 is devoid of known mitochondrial targeting signals, we used selective permeabilization studies to reveal that this KAT is oriented with its catalytic components facing the cytosol and its C-terminal TMD inserted into the outer mitochondrial membrane, consistent with a tail-anchored membrane protein. Elp3 trafficking to mitochondria is not exclusive to Toxoplasma as we also present evidence that a form of Elp3 localizes to these organelles in mammalian cells, supporting the idea that Elp3 performs novel functions across eukaryotes that are independent of transcriptional elongation. Importantly, we also present genetic studies that suggest TgElp3 is essential in Toxoplasma and must be positioned at the mitochondrial surface for parasite viability.

KEYWORDS:

Apicomplexa, Elongator, Iron-sulfur protein, Membrane enzymes, Mitochondria, Parasite, Post translational modification, Protein targeting, Protozoan, acetylation

PMID:

 

23878194

 

[PubMed - as supplied by publisher]

J Infect Dis. 2013 Jul 21. [Epub ahead of print]

Toxoplasma serotype is associated with development of ocular toxoplasmosis

Shobab L, Pleyer U, Johnsen J, Metzner S, James ER, Torun N, Fay MP, Liesenfeld O, Grigg ME.

Source

Laboratory of Parasitic Diseases.

Abstract

Background. Ocular toxoplasmosis (OT) is the principal cause of posterior uveitis, a severe, life-altering disease worldwide. A Toxoplasma gondii serotyping ELISA that detects strain-specific antibodies present in serum was used to correlate serotype with disease manifestations.Methods. Toxoplasma serotypes in consecutive sera from German uveitis patients with OT were compared with non-OT seropositive patients with noninfectious autoimmune posterior uveitis. OT patients were tested for association of parasite serotype with age, gender, location, clinical onset, size, visual acuity, or number of lesions, and followed (mean follow-up 3.8 years) to determine association with recurrences.Results. A novel, non-reactive (NR) serotype was detected more frequently in sera of OT patients (50/114, 44%) than non-OT patients (4/56, 7%), (odds ratio 10.0, 95% confidence interval [3.4, 40.8], p<0 .0001="" 70="" 8="" alleles.conclusions.="" among="" and="" aqueous="" associated="" but="" central="" consistent="" detected="" disease.="" dna="" europe.="" expected="" experienced="" failed="" for="" frequencies="" frequent="" german="" humor="" identify="" ii="" in="" infected="" is="" management="" more="" non-ot="" nr="" of="" ot="" p="0.037)." parasite="" patients.="" patients="" pcr="" predominantly="" predominate="" recurrences="" screening="" serotype="" serotypes="" specimens="" strain="" strains="" the="" those="" to="" toxoplasma="" type="" underscoring="" value="" were="" with="">

PMID: 23878321

 

[PubMed - as supplied by publisher]

PLoS One. 2013 Jul 17;8(7):e69012. doi: 10.1371/journal.pone.0069012. Print 2013.

The Dysfunction of CD4(+)CD25(+) Regulatory T Cells Contributes to the Abortion of Mice Caused by Toxoplasma gondii Excreted-Secreted Antigens in Early Pregnancy

Chen JL, Ge YY, Zhang J, Qiu XY, Qiu JF, Wu JP, Wang Y.

Source

Department of Pathogen Biology, Key Laboratory of Pathogen Biology of Jiangsu Province, Nanjing Medical University, Nanjing, Jiangsu, China ; Department of Parasitology and Microbiology, School of Medicine, Nantong University, Nantong, Jiangsu, China.

Abstract

Toxoplasma gondii is an opportunistic intracellular parasite that is highly prevalent in human and warm-blooded animals throughout the world, leading to potentially severe congenital infections. Although the abortion caused by T. gondii is believed to be dependent on the timing of maternal infection during pregnancy, the mechanism remains unclear. This study was focused on the effects of T. gondii excreted-secreted antigens on pregnant outcomes and CD4(+)CD25(+) Foxp3(+) regulatory T cells at different stages of pregnancy. The results showed that in mice the frequency and suppressive function of CD4(+)CD25(+) regulatory cells were diminished after injection of T. gondii excreted-secreted antigens at early and intermediate stages of pregnancy. The abortion caused by T. gondii excreted-secreted antigens at early pregnancy could be partly prevented by adoptively transferring of CD4(+)CD25(+) cells from the mice injected with T. gondii excreted-secreted antigens at late pregnancy, but not from the mice with the same treatment at early pregnancy. Furthermore, T. gondii excreted-secreted antigens induced apoptosis of CD4(+)CD25(+) regulatory cells of mice in early and intermediate stages of pregnancy by down-regulating their Bcl-2 expressions and Bcl-2/Bax ratio. This study provides new insights into the mechanism that T. gondii infection is the high risk factor for abortion in early pregnancy.

PMID:

23874852

[PubMed - in process]

Inflammation. 2013 Jul 20. [Epub ahead of print]

Changes of Inhibitory Receptors on NK-92 Cells and HLA-G on BeWo Cells with Toxoplasma gondii Infection

Liu Y, Zhang L, Gao M, Zhang F, Xu X, Liu X, Hu X.

Source

Department of Immunology, Binzhou Medical University, No.346 Guan-Hai Road, Lai-shan, Yantai, Shandong, 264003, People's Republic of China.

Abstract

The aim of this study were to demonstrate the effects of Yellow fluorescent protein-Toxoplasma gondii infection on the NK-92 cells co-cultured with BeWo cells in vitro and to verify the implications upon adverse pregnancy outcome. There are four groups including NK-92 cells infected or uninfected with T. gondii, and NK-92 cells co-cultured with BeWo cells in the presence or absent of T. gondii infection. Cells were observed by fluorescence microscope at 12, 24, 36, 48, and 60 h after infection. Levels of inhibitory receptors killer immunoglobulin-like receptor (KIR)2DL4 and ILT-2 expressed on NK-92 cells, and their common ligand HLA-G expressed on BeWo cells were measured by real-time PCR and flow cytometry in all groups. Levels of KIR2DL4, ILT-2 and HLA-G increased at 12, 24, 36, and 48 h, while decreased at 60 h in all T. gondii-infected groups compared with their parallel control groups, respectively. KIR2DL4 and ILT-2 expression in co-culture groups with infection were higher than those in infected NK-92 groups at 12, 24, 36, and 48 h, while there were no significant differences at 60 h. The expression of the two inhibitory receptors was correlated with HLA-G expression in infected co-culture groups. The changes of inhibitory receptors on NK-92 cells and their common ligand HLA-G on BeWo cells with T. gondii infection might contribute to the occurrence of adverse pregnancy outcome.

PMID:

23872718

[PubMed - as supplied by publisher]

Eukaryot Cell. 2013 Jul 19. [Epub ahead of print]

Novel thioredoxin-like proteins are components of a protein complex coating the cortical microtubules of Toxoplasma gondii

Liu J, Wetzel L, Zhang Y, Nagayasu E, Ems-McClung S, Florens L, Hu K.

Source

Department of Biology, Indiana University, Bloomington, IN, 47405, USA.

Abstract

Microtubules are versatile biopolymers that support numerous vital cellular functions in eukaryotes. The specific properties of microtubules are dependent on distinct microtubule-associated proteins, as the tubulin subunits and microtubule structure are exceptionally conserved. Highly specialized microtubule-containing assemblies are often found in protists, which are rich sources for novel microtubule-associated proteins. A protozoan parasite, Toxoplasma gondii, possesses several distinct tubulin-containing structures, including 22 microtubules closely associated with the cortical membrane. Early ultrastructural studies have shown that the cortical microtubules are heavily decorated with associating proteins. However, little is known about the identities of these proteins. Here we report the discovery of a novel protein, TrxL1 (for Thioredoxin-Like protein 1), and an associating complex that coats the cortical microtubules. TrxL1 contains a thioredoxin-like fold. To visualize its localization in live parasites by fluorescence, we replaced the endogenous TrxL1 gene with a mEmeraldFP-TrxL1 fusion gene. Structured-illumination based super-resolution imaging of this parasite line produced a detailed view of the microtubule cytoskeleton. Despite its stable association with the cortical microtubules in the parasite, TrxL1 does not seem to bind to microtubules directly. Co-immunoprecipitation experiments showed that TrxL1 associates with a protein complex containing SPM1, a previously reported microtubule-associated protein in T. gondii. We also found that SPM1 recruits TrxL1 to the cortical microtubules. Besides SPM1, several other novel proteins are found in the TrxL1-containing complex, including TrxL2, a close homolog of TrxL1. Our results thus for the first time reveal a microtubule-associated complex in T. gondii.

PMID:

23873863

[PubMed - as supplied by publisher]

Korean J Parasitol. 2013 Jun;51(3):279-87. doi: 10.3347/kjp.2013.51.3.279. Epub 2013 Jun 30.

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Lee YJ, Song HO, Lee YH, Ryu JS, Ahn MH.

Source

Department of Environmental Biology and Medical Parasitology, Hanyang University College of Medicine, Seoul 133-791, Korea.

Abstract

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.

KEYWORDS:

Beclin 1, LC3 II, Toxoplasma gondii, autophagy, rapamycin

PMID:

 

23864738

 

[PubMed - in process]

Infect Immun. 2013 Jul 15. [Epub ahead of print]

Transcriptome Analysis of Mouse Brain Infected with Toxoplasma gondii

Tanaka S, Nishimura M, Ihara F, Yamagishi J, Suzuki Y, Nishikawa Y.

Source

National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.

Abstract

Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were up-regulated, whereas 12 genes were down-regulated. GOstat analysis predicted that the up-regulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the number of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological function such as small GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.

PMID:

23856619

[PubMed - as supplied by publisher]

Trends Parasitol. 2013 Jul 3. pii: S1471-4922(13)00090-1. doi: 10.1016/j.pt.2013.06.001. [Epub ahead of print]

Toxoplasma oocysts as a public health problem

Torrey EF, Yolken RH.

Source

The Stanley Medical Research Institute, 8401 Connecticut Avenue, Suite 200, Chevy Chase, MD 20815, USA. Electronic address: torreyf@stanleyresearch.org.

Abstract

Waterborne outbreaks of Toxoplasma gondii have focused attention on the importance of oocysts shed in the feces of infected cats. Cat feces deposited annually into the environment in the United States total approximately 1.2 million metric tons. The annual oocyst burden measured in community surveys is 3 to 434 oocysts per square foot and is greater in areas where cats selectively defecate. Because a single oocyst can possibly cause infection, this oocyst burden represents a major potential public health problem. The proper disposal of cat litter, keeping cats indoors, reducing the feral cat population, and protecting the play areas of children might potentially reduce the oocyst burden.

Copyright © 2013 Elsevier Ltd. All rights reserved.

KEYWORDS:

Toxoplasma gondii, oocysts, prevention, toxoplasmosis

PMID:

 

23849140

 

[PubMed - as supplied by publisher]

Front Microbiol. 2013 Jul 4;4:179. Print 2013.

Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca2+ mobilization

Smith NL, Abi Abdallah DS, Butcher BA, Denkers EY, Baird B, Holowka D.

Source

Baker Laboratory, Department of Chemistry and Chemical Biology, Cornell University Ithaca, NY, USA.

Abstract

Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca²⁺-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca²⁺ responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca²⁺ mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca²⁺ mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca²⁺mobilization, particularly antigen-mediated Ca²⁺ release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion.

KEYWORDS:

FcεRI, IgE, Syk kinase

PMID:

 

23847603

 

[PubMed - as supplied by publisher]

PLoS One. 2013 Jun 28;8(6):e68129. doi: 10.1371/journal.pone.0068129. Print 2013.

A Genome-Wide siRNA Screen to Identify Host Factors Necessary for Growth of the Parasite Toxoplasma gondii

Moser LA, Pollard AM, Knoll LJ.

Source

Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Abstract

Toxoplasma gondii is an obligate intracellular parasite that is able to infect virtually any nucleated cell of all warm-blooded animals. The host cell factors important for parasite attachment, invasion, and replication are poorly understood. We screened a siRNA library targeting 18,200 individual human genes in order to identify host proteins with a role in T. gondii growth. Our screen identified 19 genes whose inhibition by siRNA consistently and significantly lowered parasite replication. The gene ontology categories for those 19 genes represented a wide variety of functions with several genes implicated in regulation of the cell cycle, ion channels and receptors, G-protein coupled receptors, and cytoskeletal structure as well as genes involved in transcription, translation and protein degradation. Further investigation of 5 of the 19 genes demonstrated that the primary reason for the reduction in parasite growth was death of the host cell. Our results suggest that once T. gondii has invaded and established an infection, global changes in the host cell may be necessary to reduce parasite replication. While siRNA screens have been used, albeit rarely, in other parasite systems, this is the first report to describe a high-throughput siRNA screen for host proteins that affect T. gondii replication.

PMID:

 

23840822

 

[PubMed - in process] 

MBio. 2013 Jul 9;4(4). pii: e00255-13. doi: 10.1128/mBio.00255-13.

Human Innate Immunity to Toxoplasma gondii Is Mediated by Host Caspase-1 and ASC and Parasite GRA15

Gov L, Karimzadeh A, Ueno N, Lodoen MB.

Source

Department of Molecular Biology and Biochemistry and the Institute for Immunology, University of California, Irvine, California, USA.

Abstract

ABSTRACT   Interleukin-1β (IL-1β) functions as a key regulator of inflammation and innate immunity. The protozoan parasite Toxoplasma gondii actively infects human blood monocytes and induces the production of IL-1β; however, the host and parasite factors that mediate IL-1β production during T. gondii infection are poorly understood. We report that T. gondii induces IL-1β transcript, processing/cleavage, and release from infected primary human monocytes and THP-1 cells. Treating monocytes with the caspase-1 inhibitor Ac-YVAD-CMK reduced IL-1β release, suggesting a role for the inflammasome in T. gondii-induced IL-1β production. This was confirmed by performing short hairpin RNA (shRNA) knockdown of caspase-1 and of the inflammasome adaptor protein ASC. IL-1β induction required active parasite invasion of monocytes, since heat-killed or mycalolide B-treated parasites did not induce IL-1β. Among the type I, II, and III strains of T. gondii, the type II strain induced substantially more IL-1β mRNA and protein release than did the type I and III strains. Since IL-1β transcript is known to be induced downstream of NF-κB signaling, we investigated a role for the GRA15 protein, which induces sustained NF-κB signaling in a parasite strain-specific manner. By infecting human monocytes with a GRA15-knockout type II strain and a type I strain stably expressing type II GRA15, we determined that GRA15 is responsible for IL-1β induction during T. gondii infection of human monocytes. This research defines a pathway driving human innate immunity by describing a role for the classical inflammasome components caspase-1 and ASC and the parasite GRA15 protein in T. gondii-induced IL-1β production. IMPORTANCE Monocytes are immune cells that protect against infection by increasing inflammation and antimicrobial activities in the body. Upon infection with the parasitic pathogen Toxoplasma gondii, human monocytes release interleukin-1β (IL-1β), a "master regulator" of inflammation, which amplifies immune responses. Although inflammatory responses are critical for host defense against infection, excessive inflammation can result in tissue damage and pathology. This delicate balance underscores the importance of understanding the mechanisms that regulate IL-1β during infection. We have investigated the molecular pathway by which T. gondii induces the synthesis and release of IL-1β in human monocytes. We found that specific proteins in the parasite and the host cell coordinate to induce IL-1β production. This research is significant because it contributes to a greater understanding of human innate immunity to infection and IL-1β regulation, thereby enhancing our potential to modulate inflammation in the body.

PMID:

 

23839215

 

[PubMed - in process] 

BMC Genomics. 2013 Jul 10;14(1):467. [Epub ahead of print]

Genetic basis for phenotypic differences between different Toxoplasma gondii type I strains.

Yang N, Farrell A, Niedelman W, Melo M, Lu D, Julien L, Marth GT, Gubbels MJ, Saeij JP.

Abstract

BACKGROUND:

Toxoplasma gondii has a largely clonal population in North America and Europe, with types I, II and III clonal lineages accounting for the majority of strains isolated from patients. RH, a particular type I strain, is most frequently used to characterize Toxoplasma biology. However, compared to other type I strains, RH has unique characteristics such as faster growth, increased extracellular survival rate and inability to form orally infectious cysts. Thus, to identify candidate genes that could account for these parasite phenotypic differences, we determined genetic differences and differential parasite gene expression between RH and another type I strain, GT1. Moreover, as differences in host cell modulation could affect Toxoplasma replication in the host, we determined differentially modulated host processes among the type I strains through host transcriptional profiling.

RESULTS:

Through whole genome sequencing, we identified 1,394 single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) between RH and GT1. These SNPs/indels together with parasite gene expression differences between RH and GT1 were used to identify candidate genes that could account for type I phenotypic differences. A polymorphism in dense granule protein, GRA2, determined RH and GT1 differences in the evasion of the interferon gamma response. In addition, host transcriptional profiling identified that genes regulated by NF-[latin small letter kra]B, such as interleukin (IL)-12p40, were differentially modulated by the different type I strains. We subsequently showed that this difference in NF-[latin small letter kra]B activation was due to polymorphisms in GRA15. Furthermore, we observed that RH, but not other type I strains, recruited phosphorylated I[latin small letter kra]Balpha (a component of the NF-[latin small letter kra]B complex) to the parasitophorous vacuole membrane and this recruitment of p- I[latin small letter kra]Balpha was partially dependent on GRA2.

CONCLUSIONS:

We identified candidate parasite genes that could be responsible for phenotypic variation among the type I strains through comparative genomics and transcriptomics. We also identified differentially modulated host pathways among the type I strains, and these can serve as a guideline for future studies in examining the phenotypic differences among type I strains.

PMID:

 

23837824

 

[PubMed - as supplied by publisher] 

Toxoplasmosis

Handb Clin Neurol. 2013;114:125-45. doi: 10.1016/B978-0-444-53490-3.00008-X.

Toxoplasmosis

Halonen SK, Weiss LM.

Department of Microbiology, Montana State University, Bozeman, MT, USA.

Toxoplasma gondii, an Apicomplexan, is a pathogic protozoan that can infect the central nervous system. Infection during pregnancy can result in a congenial infection with severe neurological sequelae. In immunocompromisedindividuals reactivation of latent neurological foci can result in encephalitis. Immunocompetent individuals infected with T. gondii are typically asymptomatic and maintain this infection for life. However, recent studies suggest that these asymptomatic infections may have effects on behavior and other physiological processes. Toxoplasma gondii infects approximately one-third of the world population, making it one of the most successful parasitic organisms. Cats and other felidae serve as the definite host producing oocysts, an environmentally resistant life cycle stage found in cat feces, which can transmit the infection when ingested orally. A wide variety of warm-blooded animals, including humans, can serve as the intermediate host in which tissue cysts (containing bradyzoites) develop. Transmission also occurs due to ingestion of the tissue cysts. There are three predominant clonal lineages, termed Types I, II and III, and an association with higher pathogenicity with the Type I strains in humans has emerged. This chapter presents a review of the biology of this infection including the life cycle, transmission, epidemiology, parasite strains, and the host immune response. The major clinical outcomes of congenital infection, chorioretinitis and encephalitis, and the possible association of infection of toxoplasmosis with neuropsychiatric disorders such as schizophrenia, are reviewed.

PMID: 23829904 [PubMed - in process]

Lipid Synthesis in Protozoan Parasites: a Comparison Between Kinetoplastids and Apicomplexans

Prog Lipid Res. 2013 Jul 1. pii: S0163-7827(13)00043-X. doi: 10.1016/j.plipres.2013.06.003. [Epub ahead of print]

Lipid Synthesis in Protozoan Parasites: a Comparison Between Kinetoplastids and Apicomplexans

Ramakrishnan S, Serricchio M, Striepen B, Bütikofer P.

Department of Cellular Biology, University of Georgia, Athens, GA 30602, U.S.A.

Lipid metabolism is of crucial importance for pathogens. Lipids serve as cellular building blocks, signalling molecules, energy stores, posttranslational modifiers, and pathogenesis factors. Parasites rely on a complex system of uptake and synthesis mechanisms to satisfy their lipid needs. The parameters of this system change dramatically as the parasite transits through the various stages of its life cycle. Here we discuss the tremendous recent advances that have been made in the understanding of the synthesis and uptake pathways for fatty acids and phospholipids in apicomplexan and kinetoplastid parasites, including Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania. Lipid synthesis differs in significant ways between parasites from both phyla and the human host. Parasites have acquired novel pathways through endosymbiosis, as in the case of the apicoplast, have dramatically reshaped substrate and product profiles, and have evolved specialized lipids to interact with or manipulate the host. These differences potentially provide opportunities for drug development. We outline the lipid pathways for key species in detail as they progress through the developmental cycle and highlight those that are of particular importance to the biology of the
pathogens and/or are the most promising targets for parasite-specific treatment.

PMID: 23827884 [PubMed - as supplied by publisher]

Host Organelle Hijackers: A similar modus operandi for Toxoplasma gondii and Chlamydia trachomatis-Co-infection model as a tool to investigate pathogenesis

Pathog Dis. 2013 Jul 2. doi: 10.1111/2049-632X.12057. [Epub ahead of print]

Host Organelle Hijackers: A similar modus operandi for Toxoplasma gondii and Chlamydia trachomatis-Co-infection model as a tool to investigate pathogenesis
Romano JD, Coppens I.

Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, 21205, USA.

The bacterium Chlamydia trachomatis and the protozoan parasite Toxoplasma gondii are the causative agents of chlamydiosis and toxoplasmosis in humans, respectively. Both micro-organisms are obligate intracellular pathogens and notorious for extensively modifying the cytoskeletal architecture and the endomembrane system of their host cells to establish productive infections. This review highlights the similar tactics developed by these two pathogens to manipulate their host cell despite their genetic unrelatedness. By using an in vitro cell culture model whereby single fibroblasts are infected by C. trachomatis and T. gondii simultaneously, thus setting up an intracellular competition, we demonstrate that the solutions to the problem of intracellular survival deployed by the parasite and the bacterium may represent an example of convergent evolution, driven by the necessity to acquire nutrients in a hostile environment. This article is protected by copyright. All rights reserved.

PMID: 23821471 [PubMed - as supplied by publisher]

Abnormalities of neurotransmitter and neuropeptide systems in human neuroepithelioma cells infected by three Toxoplasma strains

J Neural Transm. 2013 Jul 3. [Epub ahead of print]

Abnormalities of neurotransmitter and neuropeptide systems in human neuroepithelioma cells infected by three Toxoplasma strains

Xiao J, Li Y, Jones-Brando L, Yolken RH.

Stanley Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins School of Medicine, Baltimore, MD, 21287, USA, jxiao4@jhmi.edu.

Since Toxoplasma gondii can establish a persistent infection in the central nervous system in humans, we studied its effects on a host's neurotransmitter and neuropeptide systems (NNS). Using microarray technology, we have screened the expression of genes coding for NNS in human neuroepithelioma cells in response to representative strains of Toxoplasma to identify potential target genes. Transcripts that displayed expression levels distinct from uninfected controls were examined by RT-PCR and Western blot. Our results indicate the presence of disturbed NNS upon Toxoplasma infection and the extent of this disturbance varies considerably among the three strains. In cells infected by type I strain, three neurotransmitter systems (dopamine, glutamate and serotonin) and two neuropeptides (PROK2 and TAC1) displayed abnormalities relative to controls. Type III infection led to the change of a critical enzyme, TDO2, in the kynurenine pathway. No significant effects of type II infection were found in the NNS. These data may have implications for understanding the pathogenesis and heterogeneity of neurologic disturbances in toxoplasmosis.

PMID: 23821371 [PubMed - as supplied by publisher]

Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite

PLoS Pathog. 2013 Jun;9(6):e1003449. Epub 2013 Jun 20.

Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite

Feliu V, Vasseur V, Grover HS, Chu HH, Brown MJ, Wang J, Boyle JP, Robey EA, Shastri N, Blanchard N.

INSERM, U1043, Toulouse, France ; CNRS, U5282, Toulouse, France ; Université de Toulouse, UPS, Centre de Physiopathologie de Toulouse Purpan (CPTP), Toulouse, France.

CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood. Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells.

PMID: 23818852 [PubMed - as supplied by publisher]

Donor CD8+ T cells prevent Toxoplasma de-encystation but fail to rescue exhausted endogenous CD8 population

Infect Immun. 2013 Jul 1. [Epub ahead of print]

Donor CD8+ T cells prevent Toxoplasma de-encystation but fail to rescue exhausted endogenous CD8 population

Bhadra R, Cobb DA, Khan IA.

Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, DC 20037, USA.

Functional exhaustion of CD8+ T cells due to increased expression of inhibitory molecule PD-1 causes reactivation of latent disease during later phases of chronic toxoplasmosis. Onset of disease recrudescence, results in decreased parasite cyst burden concomitant with parasites undergoing stage conversion from primarily encysted, quiescent bradyzoite to fast replicating, highly motile tachyzoite. Thus reduced cyst burden is one of the early hallmarks of disease recrudescence. This was further validated by depleting IFNγ, a cytokine known to control latent toxoplasmosis, in chronically infected pre-recrudescent mice. Since CD8+ T cells (an important source of IFNγ) lose their functionality during the later phases of chronic toxoplasmosis, we next examined if adoptive transfer of functional CD8+ T cells from acutely infected donors to the chronically infected pre-recrudescent hosts could impede parasite de-encystation and rescue exhausted CD8+ T cells. While the transfer of immune CD8+ T cells temporarily restricted the breakdown of cysts, exhausted endogenous CD8+ T cell population was not rescued. Over time, the donor population got deleted resulting in parasite de-encystation and host mortality. Considering that donor CD8+ T cells fail to become long-lived, one of the cardinal features of memory CD8+ T cells, it bears the implication that memory CD8 differentiation is impaired during chronic toxoplasmosis. Moreover our data strongly suggests that while adoptive immunotherapy can prevent parasite de-encystation transiently, reduced antigen burden in chronic phase by itself is insufficient for rescue of exhausted CD8+ T cells. The conclusions of this study have profound ramifications in designing immunotherapeutics against chronic toxoplasmosis.

PMID: 23817617 [PubMed - as supplied by publisher]

The Algal Past and Parasite Present of the Apicoplast

Annu Rev Microbiol. 2013 Jun 26. [Epub ahead of print]

The Algal Past and Parasite Present of the Apicoplast

van Dooren GG, Striepen B.

Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia; email: giel.vandooren@anu.edu.au.

Plasmodium and Toxoplasma are genera of apicomplexan parasites that infect millions of people each year. The former causes malaria, and the latter causes neurotropic infections associated with a weakened or developing immune system. These parasites harbor a peculiar organelle, the apicoplast. The apicoplast is the product of an ancient endosymbiosis between a heterotrophic and a photosynthetic protist. We explore the cellular and molecular mechanisms that enabled a stable union of two previously independent organisms. These include the exchange of metabolites, transfer of genes, transport of proteins, and overall coordination of biogenesis and proliferation. These mechanisms are still active today and can be exploited to treat parasite infection. They were shaped by the dramatic changes that occurred in the evolution of the phylum Apicomplexa-including the gain and loss of photosynthesis, adaptation to symbiosis and parasitism, and the explosion of animal diversity-that ultimately provided an aquatic alga access to every biotope on this planet. Expected final online publication date for the Annual Review of Microbiology Volume 67 is September 08, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

PMID: 23808340 [PubMed - as supplied by publisher]

Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii

Protein Sci. 2013 Jun 27. doi: 10.1002/pro.2300. [Epub ahead of print]

Selective peptide inhibitors of bifunctional thymidylate synthase-dihydrofolate reductase from Toxoplasma gondii provide insights into domain-domain communication and allosteric regulation

Landau MJ, Sharma H, Anderson KS.

The Departments of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510; Molecular Biophysics and Biochemistry, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.

The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking β-strands at the interface, revealing a previously unknown allosteric target. The current study shows that these β-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

PMID: 23813474 [PubMed - as supplied by publisher]