Saturday, May 28, 2016

Host cell membrane microdomain is a critical factor for organelle discharge by Toxoplasma gondii

 2016 May 20. pii: S1383-5769(16)30143-X. doi: 10.1016/j.parint.2016.05.012. [Epub ahead of print]

Abstract

Host cell microdomains are involved in the attachment, entry, and replication of intracellular microbial pathogens. Entry into the host cell of Toxoplasma gondii and the subsequent survival of this protozoan parasite are tightly coupled with the proteins secreted from organelle called rhoptry. The rhoptry proteins are rapidly discharged into clusters of vesicles, called evacuoles, which are then delivered to parasitophorous vacuoles (PVs) or nucleus. In this study, we examined the roles of two host cell microdomain components, cholesterol and glycosylphosphatidylinositol (GPI), in evacuole formation. The acute depletion of cholesterol from the host cell plasma membrane blocked evacuole formation but not invasion. Whereas the lack of host cell GPI also altered evacuole formation but not invasion, instead inducing excess evacuole formation. The latter effect was not influenced by the evacuole-inhibiting effects of host cell cholesterol depletion, indicating the independent roles of host GPI and cholesterol in evacuole formation. In addition, the excess formation of evacuoles resulted in the enhanced recruitment of host mitochondria and endoplasmic reticulum to PVs, which in turn stimulated the growth of the parasite.
Copyright © 2015. Published by Elsevier Ireland Ltd.

KEYWORDS: 

Toxoplasma gondii; cholesterol; evacuole; glycosylphosphatidylinositol-anchor; rhoptry protein
PMID:
 
27217289
 
[PubMed - as supplied by publisher] 

Tuesday, May 24, 2016

Anti-parasitic effect on Toxoplasma gondii induced by BnSP-7, a Lys49-phospholipase A2 homologue from Bothrops pauloensis venom

 2016 May 19. pii: S0041-0101(16)30139-8. doi: 10.1016/j.toxicon.2016.05.010. [Epub ahead of print]

Abstract

Toxoplasmosis affects a third of the global population and presents high incidence in tropical areas. Its great relevance in public health has led to a search for new therapeutic approaches. Herein, we report the antiparasitic effects of BnSP-7 toxin, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, on Toxoplasma gondii. In an MTT assay, BnSP-7 presented significant cytotoxicity against host HeLa cells at higher doses (200 μg/mL to 50 μg/mL), whereas lower doses (25 μg/mL to 1.56 μg/mL) produced low cytotoxicity. Furthermore, the toxin showed no effect on T. gondii tachyzoite viability when evaluated by trypan blue exclusion, but decreased both adhesion and parasite proliferation when tachyzoites were treated before infection. We also measured cytokines in supernatants collected from HeLa cells infected with T. gondii tachyzoites previously treated with RPMI or BnSP-7, which revealed enhancement of only MIF and IL-6 cytokines levels in supernatants of HeLa cells after BnSP-7 treatment. Our results showed that the BnSP-7 PLA2 exerts an anti-Toxoplasma effect at a lower dose than that required to induce cytotoxicity in HeLa cells, and also modulates the immune response of host cells. In this sense, the anti-parasitic effect of BnSP-7 PLA2 demonstrated in the present study opens perspectives for use of this toxin as a tool for future studies on toxoplasmosis.
Copyright © 2016. Published by Elsevier Ltd.

KEYWORDS: 

Bothrops pauloensis; Phospholipase A(2); Snake venom; Toxoplasma gondii
PMID:
 
27212627
 
[PubMed - as supplied by publisher]

Effects of Extracts from Thai Piperaceae Plants against Infection with Toxoplasma gondii

 2016 May 23;11(5):e0156116. doi: 10.1371/journal.pone.0156116.

Abstract

Herbal medicines and natural herb extracts are widely used as alternative treatments for various parasitic diseases, and such extracts may also have potential to decrease the side effects of the standard regimen drugs used to treat toxoplasmosis (sulfadiazine-pyrimethamine combination). We evaluated how effective the Thai piperaceae plants Piper betle, P. nigrum and P. sarmentosum are against Toxoplasma gondii infection in vitro and in vivo. Individually, we extracted the piperaceae plants with ethanol, passed them through a rotary evaporator and then lyophilized them to obtain crude extracts for each one. The in vitro study indicated that the P. betle extract was the most effective extract at inhibiting parasite growth in HFF cells (IC50 on RH-GFP: 23.2 μg/mL, IC50 on PLK-GFP: 21.4 μg/mL). Furthermore, treatment of experimental mice with the P. betle extract for 7 days after infection with 1,000 tachyzoites of the T. gondii PLK strain increased their survival (survival rates: 100% in 400 mg/kg-treated, 83.3% in 100 mg/kg-treated, 33.3% in 25 mg/kg-treated, 33.3% in untreated mice). Furthermore, treatment with 400 mg/kg of the P. betle extract resulted in 100% mouse survival following infection with 100,000 tachyzoites. The present study shows that P. betle extract has the potential to act as a medical plant for the treatment of toxoplasmosis.
PMID:
 
27213575
 
[PubMed - as supplied by publisher]

Id2 reinforces TH1 differentiation and inhibits E2A to repress TFH differentiation

 2016 May 23. doi: 10.1038/ni.3461. [Epub ahead of print]

Abstract

The differentiation of helper T cells into effector subsets is critical to host protection. Transcription factors of the E-protein and Id families are important arbiters of T cell development, but their role in the differentiation of the TH1 and TFH subsets of helper T cells is not well understood. Here, TH1 cells showed more robust Id2 expression than that of TFH cells, and depletion of Id2 via RNA-mediated interference increased the frequency of TFH cells. Furthermore, TH1 differentiation was blocked by Id2 deficiency, which led to E-protein-dependent accumulation of effector cells with mixed characteristics during viral infection and severely impaired the generation of TH1 cells following infection with Toxoplasma gondii. The TFH cell-defining transcriptional repressor Bcl6 bound the Id2 locus, which provides a mechanism for the bimodal Id2 expression and reciprocal development of TH1 cells and TFH cells.
PMID:
 
27213691
 
[PubMed - as supplied by publisher]

Saturday, May 21, 2016

The Knowns Unknowns: Exploring the Homologous Recombination Repair Pathway in Toxoplasma gondii

Front Microbiol. 2016 May 3;7:627. doi: 10.3389/fmicb.2016.00627. eCollection 2016.

Author information

  • 1Laboratorio de Parasitología Molecular, IIB-INTECH, CONICET-UNSAM Chascomús, Argentina.
  • 2Cell Cycle Genomic Instability Laboratory, Fundación Instituto Leloir, IIBBA-CONICET Chascomús, Argentina.

Abstract

Toxoplasma gondii is an apicomplexan parasite of medical and veterinary importance which causes toxoplasmosis in humans. Great effort is currently being devoted toward the identification of novel drugs capable of targeting such illness. In this context, we believe that the thorough understanding of the life cycle of this model parasite will facilitate the identification of new druggable targets in T. gondii. It is important to exploit the available knowledge of pathways which could modulate the sensitivity of the parasite to DNA damaging agents. The homologous recombination repair (HRR) pathway may be of particular interest in this regard as its inactivation sensitizes other cellular models such as human cancer to targeted therapy. Herein we discuss the information available on T. gondii's HRR pathway from the perspective of its conservation with respect to yeast and humans. Special attention was devoted to BRCT domain-containing and end-resection associated proteins in T. gondii as in other experimental models such proteins have crucial roles in early/late steps or HRR and in the pathway choice for double strand break resolution. We conclude that T. gondii HRR pathway is a source of several lines of investigation that allow to to comprehend the extent of diversification of HRR in T. gondii. Such an effort will serve to determine if HRR could represent a potential targer for the treatment of toxoplasmosis. 

KEYWORDS: 

DNA damage; Toxoplasma; chromatin; double strand break; fork collapse; homologous recombination repair
PMID:
 
27199954
 
[PubMed]

Thursday, May 19, 2016

The Past, Present, and Future of Genetic Manipulation in Toxoplasma gondii

 2016 May 12. pii: S1471-4922(16)30030-7. doi: 10.1016/j.pt.2016.04.013. [Epub ahead of print]

Abstract

Toxoplasma gondii is a classic model for studying obligate intracellular microorganisms as various genetic manipulation tools have been developed in T. gondii over the past 20 years. Here we summarize the major strategies for T. gondii genetic manipulation including genetic crosses, insertional mutagenesis, chemical mutagenesis, homologous gene replacement, conditional knockdown techniques, and the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. We evaluate the advantages and limitations of each of these tools in a historical perspective. We also discuss additional applications of modified CRISPR-Cas9 systems for use in T. gondii, such as regulation of gene expression, labeling of specific genomic loci, and epigenetic modifications. These approaches have the potential to revolutionize the analysis of T. gondii biology and help us to better develop new drugs and vaccines.
Copyright © 2016 Elsevier Ltd. All rights reserved.
PMID:
 
27184069
 
[PubMed - as supplied by publisher]

Saturday, May 14, 2016

Salivary IgA against sporozoite-specific embryogenesis-related protein (TgERP) in the study of horizontally transmitted toxoplasmosis via T. gondii oocysts in endemic settings

 2016 May 12:1-10. [Epub ahead of print]

Abstract

The aim of this study was to contribute to the better understanding of the relative epidemiological importance of different modes of infection with respect to horizontal transmission of Toxoplasma gondii in endemic settings. We investigated the prevalence of salivary IgA against a sporozoite-specific embryogenesis-related protein (TgERP) in a highly endemic area for toxoplasmosis in Brazil in order to pinpoint parasite transmission via oocysts. Prevalence calculated by salivary IgA specific to TgERP was compared to the prevalence calculated by serum IgG against both TgERP and tachyzoites (in conventional serological tests). Prevalence calculated by different serological and salivary parameters varied in the studied age groups. However, for the 15-21 years age group, values for T. gondii prevalence estimated by conventional serological tests and by anti-TgERP salivary IgA were similar; i.e. 68·7% and 66·6% or 66·7%, respectively, using two different cut-off parameters for salivary IgA anti-TgERP. Furthermore, salivary IgA anti-TgERP for this age group presented the highest specificity (93·33%), sensitivity (93·94%), and likelihood (14·09) compared to all the other age groups. These data demonstrate the importance of age for salivary IgA investigation against TgERP to estimate the mode of T. gondii transmission in endemic settings.

KEYWORDS: 

Toxoplasma gondii ; Diagnosis; oocysts; salivary IgA; waterborne infections
PMID:
 
27169485
 
[PubMed - as supplied by publisher]

Towards Universal Screening for Toxoplasmosis

 2016 May 11. pii: JCM.00913-16. [Epub ahead of print]

Towards Universal Screening for Toxoplasmosis: Rapid, Cost-effective and Simultaneous Detection of Toxoplasma Anti-IgG, IgM and IgA Antibodies Using Very Small Serum Volumes.

Abstract

Rapid, cost-effective and early determination of the serological status of potentially infected individuals, particularly pregnant women, can be critical in preventing life-threatening infections and subsequent fetal congenital abnormalities. An article in this issue of the Journal of Clinical Microbiology (X. Li, C. Pomares, G. Gonfrier, B. Koh, S. Zhu, M. Gong, J. G. Montoya, and H. Dai, J Clin Microbiol, 2016, doi: 10.1128/JCM.03371-15) describes an innovative multiplexed immunoassay that offers a path towards universal screening.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
 
27170019
 
[PubMed - as supplied by publisher]

Targeting by AutophaGy proteins (TAG): Targeting of IFNG-inducible GTPases to membranes by the LC3 conjugation system of autophagy

 2016 May 12:0. [Epub ahead of print]

Abstract

LC3 has been used as a marker to locate autophagosomes. However, it is also well established that LC3 can localize on various membranous structures other than autophagosomes. We recently demonstrated that the LC3 conjugation system (ATG7, ATG3, and ATG12-;ATG5-ATG16L1) is required to target LC3 and IFNG (interferon, gamma)-inducible GTPases to the parasitophorus vacuole membrane (PVM) of a protist parasite Toxoplasma gondii and consequently for IFNG to control T. gondii infection. Here we show that not only LC3, but also its homologs (GABARAP, GABARAPL1, and GABARAPL2) localize on the PVM of T. gondii in a conjugation-dependent manner. Knockout/knockdown of all LC3 homologs led to a significant reduction in targeting of the IFNG-inducible GTPases to the PVM of T. gondii and the IFNG-mediated control of T. gondii infection. Furthermore, when we relocated the ATG12-ATG5-ATG16L1 complex, which specifies the conjugation site of LC3 homologs, to alternative target membranes, the IFNG-inducible GTPases were targeted to the new target membranes rather than the PVM of T. gondii. These data suggest that the localization of LC3 homologs onto a membrane by the LC3 conjugation system is necessary and sufficient for targeting of the IFNG-inducible GTPases to the membrane, implying Targeting by AutophaGy proteins (TAG). Our data further suggest that the conjugation of ubiquitin-like LC3 homologs to the phospholipids of membranes may change the destiny of the membranes beyond degradation through lysosomal fusion, as the conjugation of ubiquitin to proteins changes the destiny of the proteins beyond proteasomal degradation.

KEYWORDS: 

GBP; IFN; IRG; LC3; TAG; Toxoplasma gondii; autophagy; targeting; ubiquitin
PMID:
 
27172324
 
[PubMed - as supplied by publisher]

Nucleotide variation in the Toxoplasma gondii micronemal protein 8 gene

 2016 May 9;15(2). doi: 10.4238/gmr.15028578.

Li ZY1,2Song HQ2Wang CR1Zhu XQ1,2.

Abstract

Toxoplasma gondii is a successful opportunistic protozoan distributed worldwide, which can infect all vertebrates, leading to serious infection, blindness, and abortion. Micronemal (MIC) proteins are critically important for T. gondii infection, as they participate in various stages of the Toxoplasma life cycle, including invasion and attachment to host cells. MIC8 secretion relies on the concentration of intracellular calcium, and can mediate the invasion of T. gondii by interacting with soluble MIC3. To investigate genetic diversity of the MIC8 gene, 16 T. gondii strains from different hosts and geographical locations, and two reference isolates (ToxoDB: TGME49_245490 and TGVEG_245490) were examined in this study. The results showed that all the examined MIC8 genes are 2055 bp, with an A+T content ranging from 50.2 to 50.6%. Conversely, lower levels of variation were detected within their nucleotide and amino acid sequences. Phylogenetic analyses indicated that three classical genotypes of T. gondii and the ToxoDB#9 genotype did not group exclusively via Bayesian inference, maximum parsimony, neighbor joining, and/or maximum likelihood assays based on the nucleotide and amino acid sequences of the MIC8 gene. In summary, the T. gondii MIC8 gene is not a suitable marker for population genetic studies of this parasite. 
PMID:
 
27173337
 
[PubMed - in process]

Thursday, May 12, 2016

An improved method for introducing site-directed point mutation into the Toxoplasma gondii genome using CRISPR/Cas9

 2016 May 7. pii: S1383-5769(16)30109-X. doi: 10.1016/j.parint.2016.05.002. [Epub ahead of print]

Abstract

Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa. Due to the ease of genetic manipulations in T. gondii it serves as a model organism for intracellular parasites. We utilized CRISPR/Cas9, which can be designed to target a specific genomic locus, to introduce site-directed point mutations directly into the T. gondii genomes. This paper contains step-by-step protocols for: (1) designing the guide RNA sequence; (2) constructing the CRISPR/Cas9 construct for a target gene and preparing a donor sequence; and (3) transfecting the CRISPR/Cas9 modules into the parasite and selecting the parasite with the desired point mutation. In brief: T. gondii strains PRU∆ku80∆hxgprt or RH∆ku80∆hxgprt were nucleofected with pDHFR-SAG1::Cas9-U6::sgGeneA and a mutation donor sequence at a molar ratio of ~1:3. After 10days of 1μM pyrimethamine selection the parasite population was enriched for parasites with the desired point mutation. This technique was also applied to evaluate the importance of genes of interest by introducing either knock out or silence mutations at the same time and then tracking the population kinetics of the resultant T. gondii strains. In addition to previously established high efficient knock out and knock in strategies in Toxoplasma, the site directed point mutation technique presented in this manuscript provides another powerful tool set for T. gondii research.
Copyright © 2015. Published by Elsevier Ireland Ltd.

KEYWORDS: 

CRISPR/Cas9; Genetic manipulation; Site-directed mutation; Toxoplasma gondii
PMID:
 
27167504
 
[PubMed - as supplied by publisher]

The Toxoplasma gondii Rhoptry Kinome Is Essential for Chronic Infection

 2016 May 10;7(3). pii: e00193-16. doi: 10.1128/mBio.00193-16.

Abstract

Ingestion of the obligate intracellular protozoan parasite Toxoplasma gondii causes an acute infection that leads to chronic infection of the host. To facilitate the acute phase of the infection, T. gondii manipulates the host response by secreting rhoptry organelle proteins (ROPs) into host cells during its invasion. A few key ROP proteins with signatures of kinases or pseudokinases (ROPKs) act as virulence factors that enhance parasite survival against host gamma interferon-stimulated innate immunity. However, the roles of these and other ROPK proteins in establishing chronic infection have not been tested. Here, we deleted 26 ROPK gene loci encoding 31 unique ROPK proteins of type II T. gondii and show that numerous ROPK proteins influence the development of chronic infection. Cyst burdens were increased in the Δrop16 knockout strain or moderately reduced in 11 ROPK knockout strains. In contrast, deletion of ROP5, ROP17, ROP18, ROP35, or ROP38/29/19 (ROP38, ROP29, and ROP19) severely reduced cyst burdens. Δrop5 and Δrop18 knockout strains were less resistant to host immunity-related GTPases (IRGs) and exhibited >100-fold-reduced virulence. ROP18 kinase activity and association with the parasitophorous vacuole membrane were necessary for resistance to host IRGs. The Δrop17 strain exhibited a >12-fold defect in virulence; however, virulence was not affected in the Δrop35 or Δrop38/29/19 strain. Resistance to host IRGs was not affected in the Δrop17, Δrop35, or Δrop38/29/19 strain. Collectively, these findings provide the first definitive evidence that the type II T. gondii ROPK proteome functions as virulence factors and facilitates additional mechanisms of host manipulation that are essential for chronic infection and transmission of T. gondii

IMPORTANCE: 

Reactivation of chronic Toxoplasma gondii infection in individuals with weakened immune systems causes severe toxoplasmosis. Existing treatments for toxoplasmosis are complicated by adverse reactions to chemotherapy. Understanding key parasite molecules required for chronic infection provides new insights into potential mechanisms that can interrupt parasite survival or persistence in the host. This study reveals that key secreted rhoptry molecules are used by the parasite to establish chronic infection of the host. Certain rhoptry proteins were found to be critical virulence factors that resist innate immunity, while other rhoptry proteins were found to influence chronic infection without affecting virulence. This study reveals that rhoptry proteins utilize multiple mechanisms of host manipulation to establish chronic infection of the host. Targeted disruption of parasite rhoptry proteins involved in these biological processes opens new avenues to interfere with chronic infection with the goal to either eliminate chronic infection or to prevent recrudescent infections.
Copyright © 2016 Fox et al.
PMID:
 
27165797
 
[PubMed - in process]

Wednesday, May 11, 2016

Innate recognition of Toxoplasma gondii in humans involves a mechanism distinct from that utilized by rodents

2016 May 9. doi: 10.1038/cmi.2016.12. [Epub ahead of print]


Toxoplasma gondii is an intracellular protozoan parasite that infects rodents as part of its natural transmission cycle and induces disease in humans, an end-stage host. As one of the natural hosts of T. gondii, the mouse has been used extensively for elucidating the cellular and molecular basis of immunity to this pathogen while relatively few studies have focused on the response of humans. In our recent work, we identified CD16+ monocytes and DC1 dendritic cells as the major myeloid cell populations that respond to T. gondii in human peripheral blood. Interestingly, these myeloid subsets represent the opposite counterparts from those triggered by the parasite in mice. Moreover, whereas the innate cytokine response to T. gondii in the mouse involves stimulation of Toll-like receptors by a soluble parasite ligand, the response of human cells instead requires phagocytosis of the live pathogen. We speculate that these marked distinctions in the pathways utilized for innate recognition of toxoplasma in mouse and man reflect the differing roles of the two hosts in the biology of this parasite.Cellular & Molecular Immunology advance online publication, 9 May 2016; doi:10.1038/cmi.2016.12.
PMID:
27157497
[PubMed - as supplied by publisher]

Toxoplasma gondii GRA15II effector-induced M1 cells ameliorate liver fibrosis in mice infected with Schistosomiasis japonica

2016 May 9. doi: 10.1038/cmi.2016.21. [Epub ahead of print]

Xie Y1, Wen H1,2,3, Yan K4, Wang S5, Wang X5, Chen J1,6, Li Y1, Xu Y1, Zhong Z4, Shen J1,2, Chu D1.

Recent studies indicated that type II Toxoplasma gondii (Tg) GRA15II favored the generation of classically activated macrophages (M1), whereas type I/III TgROP16I/III promoted the polarization of alternatively activated macrophages (M2). A number of studies have demonstrated that M2 cells are involved in the pathogenesis of the liver fibrogenesis caused by Schistosoma japonicum. The purpose of the present study was to explore the inhibitory effect of Toxoplasma-derived TgGRA15II on mouse hepatic fibrosis with schistosomiasis. The gra15II and rop16I/III genes were amplified from strains T. gondii PRU and Chinese 1 Wh3, respectively. Lentiviral vectors containing the gra15II or rop16I/III plasmid were constructed and used to infect the RAW264.7 cell line. The polarization of the transfected cells was evaluated, followed by co-culture of the biased macrophages with mouse hepatic stellate JS1 cells. Then, mice were injected with GRA15II-driven macrophages via the tail vein and infected with S. japonicum cercariae. TgGRA15II induced a M1-biased response, whereas TgROP16I/III drove the macrophages to a M2-like phenotype. The in vitro experiments indicated that JS1 cell proliferation and collagen synthesis were decreased following co-culture with TgGRA15II-activated macrophages. Furthermore, mice inoculated with TgGRA15II-biased macrophages displayed a notable alleviation of collagen deposition and granuloma formation in their liver tissues. Our results suggest that TgGRA15II-induced M1 cells may dampen the M2 dominant pathogenesis of hepatic fibrosis and granulomatosis. These results provide insights into the use of parasite-derived immunomodulators as potential anti-fibrosis agents and to re-balance the schistosomiasis-induced immune response.Cellular & Molecular Immunology advance online publication, 9 May 2016; doi:10.1038/cmi.2016.21.
PMID:
27157496
[PubMed - as supplied by publisher]