Saturday, June 24, 2017

Proteomic profiling of extracellular vesicles secreted from Toxoplasma gondii

 2017 Jun 23. doi: 10.1002/pmic.201600477. [Epub ahead of print]

Abstract

Toxoplasma gondii infects a wide range of hosts worldwide, including humans and domesticated animals causing toxoplasmosis disease. Recently, exosomes, small extracellular vesicles (EV) that contain nucleic acids, proteins, and lipids derived from their original cells were linked with disease protection. The effect of EVs derived from T. gondii on the immune response and its relevance in a physiological context is unknown. Here we disclose the first proteomic profiling of T. gondii EVs compared to EVs isolated from a human foreskin fibroblast infected cell line cultured in a vesicle- free medium. Our results reveal a broad range of canonical exosomes proteins. Data are available via ProteomeXchange with identifier PXD004895. This article is protected by copyright. All rights reserved.

KEYWORDS: 

Extracellular vesicles; MS; Toxoplasma gondii; Toxoplasma-infected cell
PMID:
 
28643940
 
DOI:
 
10.1002/pmic.201600477

Thursday, June 22, 2017

Parasites lacking the micronemal protein MIC2 are deficient in surface attachment and host cell egress, but remain virulent in vivo

 2017 May 19;2:32. doi: 10.12688/wellcomeopenres.11594.1. eCollection 2017.

Abstract

Background: Micronemal proteins of the thrombospondin-related anonymous protein (TRAP) family are believed to play essential roles during gliding motility and host cell invasion by apicomplexan parasites, and currently represent major vaccine candidates against Plasmodium falciparum, the causative agent of malaria. However, recent evidence suggests that they play multiple and different roles than previously assumed. Here, we analyse a null mutant for MIC2, the TRAP homolog in Toxoplasma gondiiMethods: We performed a careful analysis of parasite motility in a 3D-environment, attachment under shear stress conditions, host cell invasion and in vivo virulence. Results: We verified the role of MIC2 in efficient surface attachment, but were unable to identify any direct function of MIC2 in sustaining gliding motility or host cell invasion once initiated. Furthermore, we find that deletion of mic2causes a slightly delayed infection in vivo, leading only to mild attenuation of virulence; like with wildtype parasites, inoculation with even low numbers of mic2 KO parasites causes lethal disease in mice. However, deletion of mic2 causes delayed host cell egress in vitro, possibly via disrupted signal transduction pathways. Conclusions: We confirm a critical role of MIC2 in parasite attachment to the surface, leading to reduced parasite motility and host cell invasion. However, MIC2 appears to not be critical for gliding motility or host cell invasion, since parasite speed during these processes is unaffected. Furthermore, deletion of MIC2 leads only to slight attenuation of the parasite.

KEYWORDS: 

Gliding motility; Host cell invasion; MIC2; Microneme; Plasmodium; TRAP; Toxoplasma
PMID:
 
28630943
 
PMCID:
 
PMC5473411
 
DOI:
 
10.12688/wellcomeopenres.11594.1

New molecular tools in Neospora caninum for studying apicomplexan parasite proteins

 2017 Jun 19;7(1):3768. doi: 10.1038/s41598-017-03978-1.

Abstract

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. We have established a system specifically designed for Neospora caninum, and used this system as a heterologous platform for the expression of foreign genes. Plasmid constructs containing fluorescent proteins or targeted genes of Toxoplasma gondii, driven by N. caninum promoters, have yielded robust expression and correct trafficking of target gene products as assessed by immunofluorescence assays and Western blot analyses. Using this approach, we here demonstrated that N. caninum expressing T. gondii's GRA15 and ROP16 kinase are biologically active and induced immunological phenotypes consistent with T. gondii strains. N. caninum expressing TgGRA15 differentially disturbed the NF-κB pathway, inducing an increased IL-12 production. On the other hand, N. caninum expressing TgROP16 induced host STAT3 phosphorylation and consequent reduction of IL-12 synthesis. These results indicate that heterologous gene expression in N. caninum is a useful tool for the study of specific gene functions and may allow the identification of antigenic targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. Additionally, these observations may prove to be useful for the development of vaccine protocols to control toxoplasmosis and/or neosporosis.
PMID:
 
28630403
 
DOI:
 
10.1038/s41598-017-03978-1

CCR5 is Involved in Interruption of Pregnancy in Mice Infected with Toxoplasma gondii During Early Pregnancy

 2017 Jun 19. pii: IAI.00257-17. doi: 10.1128/IAI.00257-17. [Epub ahead of print]

Abstract

Toxoplasmosis can cause abortion in pregnant humans and other animals; however, the mechanism of abortion remains unknown. C-C chemokine receptor type 5 (CCR5) is essential for host defense in T. gondii infection. To investigate the relationship between CCR5 and abortion in toxoplasmosis, we inoculated wild-type and CCR5-deficient (CCR5-/-) mice with T. gondii tachyzoites intraperitoneally on day 3 of pregnancy (E3). The pregnancy rate decreased as pregnancy progressed in the infected wild-type mice. Histopathologically, no inflammatory lesions were observed in the fetoplacental tissues. Although the wild-type mice showed a higher parasite burden at the implantation sites than the CCR5-/- mice at E6 (3 days post-infection, dpi), T. gondii antigen was detected only in the uterine tissue, not in the fetoplacental tissues. At E8 (5 dpi), the embryos in the infected wild-type mice showed poor development compared with the infected CCR5-/- mice and apoptosis was observed in poorly developed embryos. Compared with the uninfected mice, the infected wild-type mice showed increased CCR5 expression at the implantation site at E6 and E8. Furthermore, analyses of mRNA expression in the uterus of non-pregnant and pregnant mice suggested that lack of the CCR5 gene and downregulation of TNF-α and CCL3 expression at E6 (3 dpi) are important factors for maintenance of pregnancy following T. gondii infection. These results suggested that CCR5 signaling is involved in embryo loss in T. gondii infection during early pregnancy, and that apoptosis is associated with embryo loss rather than direct damage to the fetoplacental tissues.
PMID:
 
28630065
 
DOI:
 
10.1128/IAI.00257-17

Association between Toxoplasma gondii types and outcomes of human infection: A meta-analysis

 2017 Jun 20:1-16. doi: 10.1556/030.64.2017.016. [Epub ahead of print]

Abstract

The virulence and pathogenicity of various types of Toxoplasma gondii differ considerably in mice. Recent studies have claimed that similar phenomenon was observed in humans, but no relevant studies have been performed to validate this finding. In addition, reports showing association between a given T. gondii type and outcomes of human infection yielded conflicting results. To provide a more precise estimation of the association and a more reliable conclusion on this subject, we performed this meta-analysis. Relevant literatures were identified in multiple databases and selected based on strict screening. T. gondii-type proportions among different severities of infection were calculated and compared using Fisher's exact test. Pooled odds ratios (OR) were calculated. Our results showed that the difference among T. gondii-type proportions was significant (p < 0.0001). In addition, significant associations were detected between Type I strains infection and congenital toxoplasmosis (OR: 1.91, p = 0.0009), Type III strains infection and pulmonary toxoplasmosis (OR: 5.15, p = 0.04). In our subgroup analysis, Type I strains were significantly associated with cerebral toxoplasmosis in offspring (OR: 1.81, p = 0.02). This result indicated that different types of T. gondii exhibited different virulence and caused different outcomes in humans.

KEYWORDS: 

Toxoplasma gondii; human infection; meta-analysis; type
PMID:
 
28629230
 
DOI:
 
10.1556/030.64.2017.016

Wednesday, June 21, 2017

Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection

 2017 Jun 19;2:17096. doi: 10.1038/nmicrobiol.2017.96.

Abstract

Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.
PMID:
 
28628099
 
DOI:
 
10.1038/nmicrobiol.2017.96

The heat shock protein 90 of Toxoplasma gondii is essential for invasion of host cells and tachyzoite growth

 2017;24:22. doi: 10.1051/parasite/2017023. Epub 2017 Jun 19.

Sun H1Zhuo X1Zhao X2Yang Y1Chen X1Yao C3Du A1.

Abstract

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells.
PMID:
 
28627357
 
DOI:
 
10.1051/parasite/2017023

Proteomic Differences between Developmental Stages of Toxoplasma gondii Revealed by iTRAQ-Based Quantitative Proteomics

 2017 Jun 2;8:985. doi: 10.3389/fmicb.2017.00985. eCollection 2017.

Wang ZX1Zhou CX1,2Elsheikha HM3He S1,4Zhou DH1Zhu XQ1,5.

Abstract

Toxoplasma gondii has a complex two-host life-cycle between intermediate host and definitive host. Understanding proteomic variations across the life-cycle stages of T. gondii may improve the understanding of molecular adaption mechanism of T. gondiiacross life-cycle stages, and should have implications for the development of new treatment and prevention interventions against T. gondii infection. Here, we utilized LC-MS/MS coupled with iTRAQ labeling technology to identify differentially expressed proteins (DEPs) specific to tachyzoite (T), bradyzoites-containing cyst (C) and sporulated oocyst (O) stages of the cyst-forming T. gondiiPrugniuad (Pru) strain. A total of 6285 proteins were identified in the three developmental stages of T. gondii. Our analysis also revealed 875, 656, and 538 DEPs in O vs. T, T vs. C, and C vs. O, respectively. The up- and down-regulated proteins were analyzed by Gene Ontology enrichment, KEGG pathway and STRING analyses. Some virulence-related factors and ribosomal proteins exhibited distinct expression patterns across the life-cycle stages. The virulence factors expressed in sporulated oocysts and the number of up-regulated virulence factors in the cyst stage were about twice as many as in tachyzoites. Of the 79 ribosomal proteins identified in T. gondii, the number of up-regulated ribosomal proteins was 33 and 46 in sporulated oocysts and cysts, respectively, compared with tachyzoites. These results support the hypothesis that oocyst and cystic stages are able to adapt to adverse environmental conditions and selection pressures induced by the host's immune response, respectively. These findings have important implications for understanding of the developmental biology of T. gondii, which may facilitate the discovery of novel therapeutic targets to better control toxoplasmosis.

KEYWORDS: 

Toxoplasma gondii; differentially expressed protein (DEP); iTRAQ; life-cycle; mass spectrometry; proteomics
PMID:
 
28626452
 
PMCID:
 
PMC5454076
 
DOI:
 
10.3389/fmicb.2017.00985

Monday, June 19, 2017

Schizogony and gametogony of oocyst-deficient T-263 strain of Toxoplasma gondii

 2017 May 27. pii: S0304-4017(17)30225-X. doi: 10.1016/j.vetpar.2017.05.024. [Epub ahead of print]

Abstract

Oocysts are important stage for the spread of Toxoplasma gondii because they are environmentally resistant. Among all hosts of T. gondii, only felids can excrete oocysts. Cats that have excreted T. gondii oocysts after primary infection develop immunity to re-excretion of oocysts, and this immunity appears to be long-lasting. It would be desirable to have a non-infectious vaccine for the prevention of T. gondii infection in cats and to understand mechanism of immunity to excretion of oocysts. An initial step will be to indentify stage/stages of the parasite for induction of immunity. A chemically-induced mutant of T. gondii, T-263, is immunogenic but lacks the capacity to form oocysts in cats. Cats fed live bradyzoites of T-263 do not excrete oocysts after challenge with oocyst producing strains. However, it is not known at what stage of the parasite development the oocyst formation is halted. Here, four cats were fed live tissue cysts of the T-263 strain and examined for enteroepithelial stages and oocyst production. Two cats were administered methyl prednisolone aceatate (20mg/kg) once intramuscularly and these cats were euthanized 5 and 7days post inoculation. No oocysts but immature and mature schizonts (types D and E), male, and female gamonts were detected in two cats euthanized. The remaining two cats did not excrete oocysts examined 3-14days post inoculation, but both seroconverted and developed antibody titers of 1:400 tested by the modification agglutination test, indicating exposure to the inocula. The results demonstrate that the T-263 strain is defective in oocyst formation and the observations should help future studies in identification of genes/factors responsible for oocyst formation in the intestine of cats.

KEYWORDS: 

Gamonts; Oocysts; T-263 strain; Toxoplasma gondii
PMID:
 
28624132
 
DOI:
 
10.1016/j.vetpar.2017.05.024

Wednesday, June 14, 2017

Transcriptional Responses in the Murine Spleen after Toxoplasma gondii Infection: Inflammasome and Mucus-Associated Genes

I 2017 Jun 10;18(6). pii: E1245. doi: 10.3390/ijms18061245.

Abstract

The spleen plays an important role in coordinating both adaptive and innate immune responses. Here, the transcriptional response to T. gondii infection in the murine spleen was characterized concerning inflammasome sensors (two different models: seven days after oral or four weeks after intraperitoneal infection). Additionally, Tff1KO and Tff3KO mice were investigated because TFF genes are often upregulated during inflammation. The expression of the pattern-recognition receptors Nlrp3, Nlrp12, and Nlrp1a was significantly increased after infection. This increase was diminished in Tff1KO and Tff3KO mice pointing towards a positive regulation of the inflammatory response by Tff1 and Tff3. Furthermore, the transcription of Tff1 (encoding a motogenic lectin) and other secretory genes was analyzed, i.e., gastrokines (Gkn), IgG Fc binding protein (Fcgbp), and the mucin Muc2. The corresponding gene products belong to an interactome protecting mucous epithelia. Tff1 was significantly induced after infection, which might increase the motility of immune cells. In contrast, Gkn3, Fcgbp, and Muc2 were downregulated seven days after oral infection; whereas four weeks after i.p. infection only Gkn3 remained downregulated. This might be an indication that Gkn3, Fcgbp, and Muc2 are involved in the transient disruption of the splenic architecture and its reorganization, which is characteristic after T. gondii infection.

KEYWORDS: 

IgG Fc binding protein; MUC2; TFF1; Toxoplasma gondii; gastrokine; inflammasome; inflammation; trefoil factor
PMID:
 
28604600
 
DOI:
 
10.3390/ijms18061245

Parkinson's disease and Toxoplasma gondii Infection: Sero-molecular Assess the Possible Link among Patients

 2017 Jun 8. pii: S0001-706X(16)30989-5. doi: 10.1016/j.actatropica.2017.06.002. [Epub ahead of print]

Abstract

We investigated the possible association between Parkinson's disease (PD), the second most common neurodegenerative disorder and Toxoplasma gondii infection, the most common neurotropic protozoan parasitic infection, using serological and molecular techniques. One hundred and fifteen patients with confirmed PD and 115 healthy subjects in the same age and sex distribution were enrolled in this study. Blood samples were taken from each participant and the sera were screened for anti-Toxoplasma antibodies (IgG and IgM). PCR assay was performed in duplicate using the primer pair targeting the B1 gene of Toxoplasma. Amplicons were directly sequenced to conduct the phylogenetic analysis. The prevalence of Toxoplasma infection based on IgG titer was 53% in case and 55.6% in the control groups, revealing no statistically significant association between Toxoplasma seropositivity and PD (OR=0.90; 95% CI=0.54-1.51; P=0.691). According to PCR assay, the prevalence of Toxoplasma infections was 19.3% in the case and 10.4% in control groups which the difference was statistically significant (OR=3.02; 95% CI=1.46-6.27; P=0.002). Multiple sequence alignment of Toxoplasma gondii isolates manifested a common haplotype by the identity: 93.6-100% and divergence: 0-6.7%. We concluded that T. gondii infection not only could not be a risk factor to PD, but even it could be concluded that patients with PD are in more risk to acquisition of infection. These results provide fresh insights into the ambiguous association between T. gondii infection and PD.

KEYWORDS: 

Parkinson’s disease; Sero-molecular assessment; Toxoplasma gondii infection
PMID:
 
28602836
 
DOI:
 
10.1016/j.actatropica.2017.06.002

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense


 2017 Jun 12. doi: 10.1038/ni.3767. [Epub ahead of print]

Abstract

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.
PMID:
 
28604719
 
DOI:
 
10.1038/ni.3767