Characterization of an IgG monoclonal antibody targeted to both tissue cyst and sporocyst walls of Toxoplasma gondii

Exp Parasitol. 2016 Jan 30. pii: S0014-4894(16)30014-5. doi: 10.1016/j.exppara.2016.01.014. [Epub ahead of print]

Gondim LF¹, Wolf A², Vrhovec MG³, Pantchev N³, Bauer C⁴, Langenmayer MC⁵, Bohne W⁶, Teifke JP⁷, Dubey JP⁸, Conraths FJ², Schares G².

Toxoplasma gondii infects animals habiting terrestrial and aquatic environments. Its oocysts and tissue cysts are important for the horizontal transmission of this parasite. The oocyst and tissue cyst walls are crucial for the ability of the parasite to persist in the environment or in animal tissues, respectively. However, the composition of these walls is not well understood. We report the generation of monoclonal antibodies directed against wall components using mice immunized with oocyst antigens of T. gondii. One monoclonal antibody (mAb) G1/19 reacted solely with T. gondii sporozoites. The respective antigen had a relative molecular weight (Mr) of 30 kDa. MAb G1/19 failed to react with sporozoites of any other coccidian parasite species tested (Hammondia hammondi, Hammondia heydorni, Cystoisospora felis, Eimeria bovis, Sarcocystis sp.). Another mAb, designated K8/15-15, recognized antigens in sporocyst walls of the parasite and in the walls of in vivo or in vitro produced tissue cysts, as demonstrated by immunofluorescence and immunoblot assays. Antigens of 80 to a high molecular weight protein of about 350 kDa Mr were recognized by this antibody using antigen extracts from sporocysts, and from in vitro or in vivo generated tissue cysts of the parasite. Tissue cyst and sporocyst walls of H. hammondi and H. heydorni, and tissue cysts of Neospora caninum were also recognized by mAb K8/15-15. Sporocyst walls of C. felis also reacted to this mAb. The cyst walls of Sarcocystis sp. and B. besnoiti were not recognized by mAbK8/15-15. Reactivity by a single mAb against T. gondii antigens in tissue cysts and sporocysts had not been reported previously. MAb K8/15-15 may be a practical tool for the identification of both cysts and sporocysts of the parasite, and may also be potentially employed in proteomic studies on the identification of new components of the cyst and sporocyst walls of T. gondii.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

Toxoplasma; immunoblot; monoclonal antibody; oocyst; sporocyst; tissue cyst

PMID:

26836446

[PubMed - as supplied by publisher]

The Apoptotic Role of Metacaspase in Toxoplasma gondii

Front Microbiol. 2016 Jan 19;6:1560. doi: 10.3389/fmicb.2015.01560. eCollection 2015.

Li M¹, Wang H¹, Liu J¹, Hao P¹, Ma L¹, Liu Q¹.

Toxoplasma gondii is a major opportunistic pathogen that spreads in a range of animal species and human beings. Quite a few characterizations of apoptosis have been identified in T. gondii treated with apoptosis inducers, but the molecular mechanisms of the pathway are not clearly understood. Metacaspases are caspase-like cysteine proteases that can be found in plants, fungi, and protozoa in which caspases are absent. Metacaspases are multifunctional proteases involved in apoptosis-like cell death, insoluble protein aggregate clearance, and cell proliferation. To investigate whether T. gondii metacaspase (TgMCA) is involved in the apoptosis of the parasites, we generated TgMCA mutant strains. Western blot analysis indicated that the autoproteolytic processing of TgMCA was the same as that for metacaspases of some other species. Indirect immunofluorescence assay (IFA) showed that TgMCA was dispersed throughout the cytoplasm and relocated to the nucleus when the parasites were exposed to the extracellular environment, which indicated the execution of its function in the nucleus. The number of apoptosis parasites was significantly diminished in the TgMCA knockout strain and increased in the TgMCA overexpression strain after treatment with extracellular buffer, as determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The lack of TgMCA did not affect the parasite propagation in vitro and virulence in vivo, suggesting that it is probably redundant in parasite propagation. But overexpression of TgMCA reduced the intracellular parasites growth in vitro. The TgMCA knockout strain showed more viability in extracellular buffer compared to the parental and overexpression lines. In this study, we demonstrated that TgMCA contributes to the apoptosis of T. gondii.

KEYWORDS:

T. gondii; apoptosis; knockout; metacaspase; programmed cell death

PMID:

26834715

[PubMed]

Identification of new palmitoylated proteins in Toxoplasma gondii

Biochim Biophys Acta. 2016 Jan 26. pii: S1570-9639(16)30002-4. doi: 10.1016/j.bbapap.2016.01.010. [Epub ahead of print]

Caballero MC1, Alonso AM1, Deng B2, Attias M3, de Souza W3, Corvi MM4.

Author information

Abstract

Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In recent years, protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes.

Copyright © 2016. Published by Elsevier B.V.

KEYWORDS: 

Acyl-biotin exchange; Host-cell invasion; Palmitoylome; Protein identification; Rhoptry; Toxoplasma gondii

PMID:

 

26825284

 

[PubMed - as supplied by publisher]

Toxoplasma gondii peptide ligands open the gate of the HLA class I binding groove

Elife. 2016 Jan 29;5. pii: e12556. doi: 10.7554/eLife.12556. [Epub ahead of print]

McMurtrey C1, Trolle T2, Sansom T3, Remesh SG4, Kaever T4, Bardet W1, Jackson K1, Nielsen M2, McLeod R5, Zajonc DM4, Blader IJ3, Peters B4, Sette A4, Hildebrand W6.

Author information

Abstract

HLA class I presentation of pathogen-derived peptide ligands is essential for CD8+ T-cell recognition of Toxoplasma gondiiinfected cells. Currently, little data exist pertaining to peptides that are presented after T. gondii infection. Herein we purify HLA-A*02:01 complexes from T. gondii infected cells and characterize the peptide ligands using LCMS. We identify 195 T. gondiiencoded ligands originating from both secreted and cytoplasmic proteins. Surprisingly, T. gondii ligands are significantly longer than uninfected host ligands, and these longer pathogen-derived peptides maintain a canonical N-terminal binding core yet exhibit a C-terminal extension of 1-30 amino acids. Structural analysis demonstrates that binding of extended peptides opens the HLA class I F' pocket, allowing the C-terminal extension to protrude through one end of the binding groove. In summary, we demonstrate that unrealized structural flexibility makes MHC class I receptive to parasite-derived ligands that exhibit unique C-terminal peptide extensions.

KEYWORDS: 

human; immunology; infectious disease; microbiology

PMID:

 

26824387

 

[PubMed - as supplied by publisher] 

Garcinol inhibits GCN5-mediated lysine acetyltransferase activity and prevents replication of the parasite Toxoplasma gondii

Antimicrob Agents Chemother. 2016 Jan 25. pii: AAC.03059-15. [Epub ahead of print]

Jeffers V¹, Gao H², Checkley LA³, Liu Y², Ferdig MT³, Sullivan WJ Jr⁴.

Lysine acetylation is a critical post-translational modification that influences protein activity, stability, and binding properties. The acetylation of histone proteins in particular is a well-characterized feature of gene expression regulation. In the protozoan parasite Toxoplasma gondii, a number of lysine acetyltransferases (KATs) contribute to gene expression and are essential for parasite viability. The natural product, garcinol, was recently reported to inhibit enzymatic activities of GCN5 and p300 family KATs in other species. Here we show that garcinol inhibits TgGCN5b, the only nuclear GCN5-family KAT known to be required for Toxoplasma tachyzoite replication. Treatment of tachyzoites with garcinol led to a reduction of global lysine acetylation, particularly on histone H3 and TgGCN5b itself. We also performed RNAseq analysis on intracellular tachyzoites treated with garcinol, which revealed increasing aberrant gene expression coincident with increasing concentrations of garcinol. The majority of the genes that were most significantly affected by garcinol were also associated with TgGCN5b in a previous ChIP-chip analysis. The dysregulated gene expression induced by garcinol significantly inhibits Toxoplasma tachyzoite replication, and the concentrations used exhibit no overt toxicity on human host cells. Garcinol also inhibits Plasmodium falciparum asexual replication with a similar IC₅₀ as Toxoplasma. Together, these data support that pharmacological inhibition of TgGCN5b leads to a catastrophic failure in gene expression control that prevents parasite replication.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PMID:

26810649

[PubMed - as supplied by publisher]

Infection of male rats with Toxoplasma gondii induces effort-aversion in a T-maze decision-making task

Brain Behav Immun. 2016 Jan 16. pii: S0889-1591(16)30015-0. doi: 10.1016/j.bbi.2016.01.015. [Epub ahead of print]

Tan D¹, Vyas A².

Rats chronically infected with protozoan Toxoplasma gondii exhibit greater delay aversion in an inter-temporal task. Moreover Toxoplasma gondii infection also results in dendritic atrophy of basolateral amygdala neurons. Basolateral amygdala is reported to bias decision making towards greater effortful alternatives. In this context, we report that Toxoplasma gondii increases effort aversion in infected male rats. This host-parasite association has been widely studied in the context of loss of innate fear in the infected males. It is suggested that reduced fear towards predators reflects a parasitic behavioral manipulation to enhance trophic transmission of Toxoplasma gondii. Observations reported here extend this paradigm away from a monolithic change in fear and towards a multi-dimensional change in decision making.
Copyright © 2016. Published by Elsevier Inc.

KEYWORDS:

Behavioral manipulation; Decision making; Effort discounting; Operant learning; Parasites

PMID:

26783701

[PubMed - as supplied by publisher]

Toxoplasma gondii infection and testosterone congruently increase tolerance of male rats for risk of reward forfeiture

Horm Behav. 2016 Jan 13. pii: S0018-506X(16)30020-4. doi: 10.1016/j.yhbeh.2016.01.003. [Epub ahead of print]

Tan D¹, Vyas A².

Decision making under risk involves balancing the potential of gaining rewards with the possibility of loss and/or punishment. Tolerance to risk varies between individuals. Understanding the biological basis of risk tolerance is pertinent because excessive tolerance contributes to adverse health and safety outcomes. Yet, not much is known about biological factors mediating inter-individual variability in this regard. We investigate if latent Toxoplasma gondii infection can cause risk tolerance. Using a rodent model of the balloon analogous risk task, we show that latent T. gondii infection leads to a greater tolerance of reward forfeiture. Furthermore, effects of the infection on risk can be recapitulated with testosterone supplementation alone, demonstrating that greater testosterone synthesis by the host post-infection is sufficient to change risk tolerance. T. gondii is a frequent parasite of humans and animals. Thus, the infection status can potentially explain some of the inter-individual variability in the risky decision making.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS:

BART; Behavioral manipulation; Decision making; Impulsivity; Parasitism; Steroids

PMID:

26774464

[PubMed - as supplied by publisher]

The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells

PLoS Pathog. 2016 Jan 13;12(1):e1005388. doi: 10.1371/journal.ppat.1005388. eCollection 2016.

Graindorge A1, Frénal K1, Jacot D1, Salamun J1, Marq JB1, Soldati-Favre D1.

Author information

Abstract

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.

PMID:

 

26760042

 

[PubMed - as supplied by publisher]

Analysis of non-canonical calcium dependent protein kinases in Toxoplasma gondii by targeted gene deletion using CRISPR/Cas9

Infect Immun. 2016 Jan 11. pii: IAI.01173-15. [Epub ahead of print]

Long S1, Wang Q1, Sibley LD2.

Author information

Abstract

Calcium dependent protein kinases are expanded in apicomplexan parasites, especially in T. gondii where 14 separate genes encoding these enzymes are found. Although previous studies have shown a role for several CDPKs in controlling invasion, egress, and cell division in T. gondii, the role of most of these genes is unexplored. Here we developed a more efficient method for gene disruption using CRISRP/Cas9 that was modified to completely delete large, multi-exonic genes from the genome and to allow serial replacement by recycling of the selectable marker using Cre-loxP. Using this system we generated a total of 24 mutants in type 1 and 2 genetic backgrounds to ascertain the function of non-canonical CPDKs. Remarkably, although we were able to confirm the essentiality of CDPK1 and CDPK7, the majority of CDPKs had no discernible phenotype for growth in vitro or infection in the mouse model. The exception to this was CDPK6, loss of which lead to reduced plaquing, fitness defect in a competition assay, and reduced tissue cysts formation in chronically infected mice. Our findings highlight the utility of CRISPR/cas9 for rapid serial gene deletion, and also suggest that additional models are needed to reveal the function of many genes in T. gondii.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PMID:

 

26755159

 

[PubMed - as supplied by publisher]

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

Nat Med. 2016 Jan 11. doi: 10.1038/nm.4023. [Epub ahead of print]

 

Dotiwala F^1,2, Mulik S^1,2, Polidoro RB^1,2,3, Ansara JA¹, Burleigh BA⁴, Walch M⁵, Gazzinelli RT^3,4,6, Lieberman J^1,2.

 

Protozoan infections are a serious global health problem. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents-granzyme (Gzm) proteases and the pore-forming perforin (PFN)-into the infected cell. However, these cytotoxic molecules do not kill intracellular parasites. CD8⁺ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes, and GNLY, PFN and Gzms rapidly kill intracellular bacteria. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell-mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism.

PMID:

26752517

[PubMed - as supplied by publisher]

Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes

Nat Commun. 2016 Jan 7;7:10147. doi: 10.1038/ncomms10147.

Lorenzi H1, Khan A2,3, Behnke MS2,4, Namasivayam S5,6, Swapna LS7,8, Hadjithomas M1, Karamycheva S1, Pinney D9, Brunk BP9, Ajioka JW10, Ajzenberg D11, Boothroyd JC12, Boyle JP13, Dardé ML11, Diaz-Miranda MA9, Dubey JP14, Fritz HM15, Gennari SM16, Gregory BD9, Kim K17, Saeij JP18, Su C19, White MW20, Zhu XQ21, Howe DK22, Rosenthal BM14, Grigg ME3, Parkinson J7,8, Liu L23,24, Kissinger JC5,6,24, Roos DS9, David Sibley L2.

Author information

Abstract

Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity. 

PMID:

 

26738725

 

[PubMed - in process]

Antigen Presentation of Vacuolated Apicomplexans - Two Gateways to a Vaccine Antigen

Trends Parasitol. 2015 Dec 27. pii: S1471-4922(15)00284-6. doi: 10.1016/j.pt.2015.12.011. [Epub ahead of print]

 

For parasites that sequester themselves within a vacuole, new rules governing antigen presentation are coming into focus. Components of the host's autophagy machinery and the parasite's membranous nanotubular network within the parasitophorous vacuole play a major role in determining antigenicity of Toxoplasma proteins. As such, both parasite and vaccinologist may exploit these pathways to regulate the ever important CD8 T cell response to apicomplexan parasites.
Copyright © 2015. Published by Elsevier Ltd.

PMID:

26733404

[PubMed - as supplied by publisher]

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites

Med Microbiol Immunol. 2016 Jan 5. [Epub ahead of print]

 

Zorgi NE^1,2, Galisteo AJ Jr², Sato MN³, do Nascimento N⁴, de Andrade HF Jr^5,6,7.

 

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19⁺), plasma cells (CD138⁺) and the CD4⁺ and CD8⁺ T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4⁺ T cells, B cells and likely CD8⁺ T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8⁺ T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4⁺ and CD8⁺ T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

KEYWORDS:

B lymphocytes; CD4+ T lymphocytes; CD8+ T lymphocytes; Ionizing radiation; Toxoplasma gondii; Vaccine

PMID:

26732075

[PubMed - as supplied by publisher]

Antisense technologies in the studying of Toxoplasma gondii

J Microbiol Methods. 2015 Dec 24. pii: S0167-7012(15)30136-6. doi: 10.1016/j.mimet.2015.12.013. [Epub ahead of print]

 

Crater AK¹, Roscoe S¹, Roberts M¹, Ananvoranich S².

 

This review covers a brief history of antisense RNAs and its applications, and summarizes the current stage of antisense technologies used in Toxoplasma gondii, a fascinating model organism with a unique characteristic blend of genetic regulatory systems normally found in plants or animals. Based on the current knowledge of regulatory RNAs and non-coding RNA (ncRNA), the antisense technologies are reviewed according to the classification of ncRNAs, which are roughly categorized into small, ranging from ~20-200 nucleotides in length, and long >200 nucleotides. Techniques utilizing small regulatory RNAs such as siRNA, miRNA, antagomirs, ribozymes and morpholino oligomers are discussed along with long non-coding RNA (lncRNA) including antisense and double stranded. These antisense technologies can be used in forward and reverse genetics studies. The future of technologies is limitless, particularly by combining these technologies with conventional methods, and should allow for ever greater understanding of gene regulation of the organism and related pathogenic microorganisms.
Copyright © 2015. Published by Elsevier B.V.

KEYWORDS:

Antisense technologies; Genetic tools; Toxoplasma gondii

PMID:

26724749

[PubMed - as supplied by publisher]

The E3-ubiquitin ligase adaptor protein Skp1 is glycosylated by an evolutionarily conserved pathway that regulates protist growth and development

J Biol Chem. 2015 Dec 30. pii: jbc.M115.703751. [Epub ahead of print]

Rahman K1, Zhao P1, Mandalasi M1, van der Wel H1, Wells L1, Blader IJ2, West CM3.

Author information

• 1University of Georgia, United States;
• 2University at Buffalo, United States.
• 3University of Georgia, United States; westcm@uga.edu.

Abstract

Toxoplasma gondii is a protist parasite of warm-blooded animals that causes disease by proliferating intracellularly in muscle and the central nervous system. Previous studies showed that a prolyl 4-hydroxylase related to animal HIFα prolyl hydroxylases is required for optimal parasite proliferation, especially at low O2. We also observed that Pro154 of Skp1, a subunit of the Skp1/Cullin-1/F-box protein (SCF)-class of E3-ubiquitin ligases, is a natural substrate of this enzyme. In an unrelated protist, Dictyostelium discoideum, Skp1 hydroxyproline is modified by 5 sugars via the action of three glycosyltransferases, Gnt1, PgtA and AgtA, which are required for optimal O2-dependent development. We show here that TgSkp1 hydroxyproline is modified by a similar pentasaccharide, based on mass spectrometry, and that assembly of the first three sugars is dependent on Toxoplasma homologs of Gnt1 and PgtA. Reconstitution of the glycosyltransferase reactions in extracts with radioactive sugar nucleotide substrates and appropriate Skp1 glycoforms, followed by chromatographic analysis of acid hydrolysates of the reaction products, confirmed the predicted sugar identities as GlcNAc, Gal and Fuc. Disruptions of gnt1 or pgtA resulted in decreased parasite growth. Off target effects were excluded based on restoration of the normal glycan chain and growth upon genetic complementation. By analogy to Dictyostelium Skp1, the mechanism may involve regulation of assembly of the SCF complex. Understanding the mechanism of Toxoplasma Skp1 glycosylation is expected to help develop it as a drug target for control of the pathogen, as the glycosyltransferases are absent from mammalian hosts.

Copyright © 2015, The American Society for Biochemistry and Molecular Biology.

KEYWORDS: 

E3 ubiquitin ligase; SCF-complex; Skp1; Toxoplasma gondii; cytoplasmic glycosylation; glycobiology; glycosyltransferase; parasitology

PMID:

 

26719340

 

[PubMed - as supplied by publisher] 

High Seroprevalence of Toxoplasma Gondii Infection in Female Sex Workers: A Case-Control Study

Eur J Microbiol Immunol (Bp). 2015 Nov 4;5(4):285-92. doi: 10.1556/1886.2015.00039. eCollection 2015.

Alvarado-Esquivel C1, Sánchez-Anguiano LF2, Hernández-Tinoco J2, Arreola-Cháidez E3, López J3, Salcido-Meraz KI3, Estrada-Martínez S2, Navarrete-Flores JA2, Pérez-Álamos AR2, Hernández-Ochoa M3, Rábago-Sánchez E4, Liesenfeld O5.

Author information

Abstract

Through an age- and sex-matched case-control study, we sought to determine whether female sex workers have an increased risk of Toxoplasma gondii exposure and to determine the sociodemographic, work, clinical, and behavioral characteristics of these workers associated with T. gondii exposure. Female workers (n = 136) and controls (n = 272) were examined with enzyme-linked immunoassays (EIA) for the presence of anti-Toxoplasma IgG and IgM antibodies. IgM positive sera were additionally tested with enzyme linked-fluorescence immunoassay (ELFA). Anti-T. gondii IgG antibodies were found in 21 (15.44%) of 136 cases and in 10 (3.67%) of 272 controls (OR = 4.05; 95% CI: 1.84-8.89; P = 0.0001). Anti-T. gondii IgG levels higher than 150 IU/ml were found in 13 (9.6%) of 136 cases and in 8 (2.9%) of 272 controls (P = 0.007). Anti-T. gondii IgM antibodies were found in two cases and in six controls by EIA, but all were negative by ELFA. T. gondii seropositivity was associated with being born out of Durango State (OR = 10.47; 95% CI: 2.9-36.8; P < 0.01), injuries during sex work (OR = 6.30; 95% CI: 1.1-33.7; P = 0.03), and soil contact (OR = 4.11; 95% CI: 1.2-14.0; P = 0.02). This is the first report of an association of T. gondii infection and female sex workers. 

KEYWORDS: 

Toxoplasma gondii; case-control study; female sex workers; risk factors; seroprevalence

PMID:

 

26716017

 

[PubMed]

Preventive Anti-CD2 Treatment does not Impair Parasite Control in a Murine Toxoplasmosis Model

Eur J Microbiol Immunol (Bp). 2015 Nov 12;5(4):306-15. doi: 10.1556/1886.2015.00040. eCollection 2015.

Erben U1, Pawlowski NN2, Heimesaat MM3, Loddenkemper C4, Doerfel K5, Spieckermann S6, Siegmund B1, Kühl AA1.

Author information

Abstract

Targeting human CD2 with the monoclonal antibody (mAb) CB.219 reduces intestinal inflammation in a colitis model where T cells carry human CD2. Here, we asked whether this mAb has adverse effects on infection control. Mice expressing human CD2 on T cells (huCD2tg) were orally infected with Toxoplasma (T.) gondii and treated with the human CD2-specific mAb CB.219 in a preventive setting. The intestinal T. gondii loads in CB.219 treated mice did not differ from the control group. Histologically, huCD2tg mice showed moderate ileal inflammation that did not change with CB.219 treatment. In the ileum, CB.219 treatment reduced the protein levels of interferon-γ, transforming growth factor β and interleukin-6, whereas interleukin-18 mRNA was slightly increased. The infiltration of neutrophils, macrophages, and T cells into the ileum was unaffected by CB.219 treatment. However, CB.219 treatment decreased the numbers of forkhead box P3(+) regulatory T cells (Treg) in ileum and liver of huCD2tg mice. This was confirmed in vitro using human peripheral blood mononuclear cells. Taken together, targeting CD2(+) T cells by the human CD2 mAb CB.219 does not prevent beneficial immune reactions necessary for pathogen control. Further experiments will address gut specificity, underlying mechanisms, and general applicability of CB.219 treatment. 

KEYWORDS: 

CD2; experimental ileitis; infection model; inflammatory bowel disease

PMID:

 

26716019

 

[PubMed]

Toxoplasma Gondii Infection of Chicken Embryos Causes Retinal Changes and Modulates HSP90B1 Gene Expression: A Promising Ocular Toxoplasmosis Model

Eur J Microbiol Immunol (Bp). 2015 Nov 12;5(4):316-20. doi: 10.1556/1886.2015.00024. eCollection 2015.

Nasaré AM1, Tedesco RC1, Cristovam PC2, Cenedese MA3, Galisteo AJ Jr4, Andrade HF Jr4, Gomes JÁ2, Guimarães ÉV5, Barbosa HS5, Alonso LG1.

Author information

Abstract

HSP90B1 is a gene that codifies heat shock protein 108 (HSP108) that belongs to a group of proteins induced under stress situation, and it has close relation with the nervous system, especially in the retina. Toxoplasma gondii causes ocular toxoplasmosis that has been associated with a late manifestation of the congenital toxoplasmosis although experimental models show that morphological alterations are already present during embryological development. Here, we used 18 eyes of Gallus domesticus embryos in 7th and 20th embryonic days to establish a model of congenital ocular toxoplasmosis, experimentally infected in its fifth day correlating with HSP90B1 gene expression. Embryos' eyes were histologically evaluated, and gene expression was performed by real-time polymerase chain reaction (PCR). Our data showed parasite present in the choroid, unusual migration of retinal pigment epithelium, and chorioretinal scars, and a tendency to a lower expression of the HSP90B1 gene upon experimental infection. This is a promising model to better understand T. gondii etiopathogeny. 

KEYWORDS: 

embryo development; experimental models; ocular toxoplasmosis; parasitic ocular infections; regulation of gene expression in development; retina

PMID:

 

26716020

 

[PubMed]