Friday, September 30, 2016

Characterization of aspartyl aminopeptidase from Toxoplasma gondii

 2016 Sep 28;6:34448. doi: 10.1038/srep34448.

Abstract

Aminopeptidases have emerged as new promising drug targets for the development of novel anti-parasitic drugs. An aspartyl aminopeptidase-like gene has been identified in the Toxoplasma gondii genome (TgAAP), although its function remains unknown. In this study, we characterized TgAAP and performed functional analysis of the gene product. Firstly, we expressed a functional recombinant TgAAP (rTgAAP) protein in Escherichia coli, and found that it required metal ions for activity and showed a substrate preference for N-terminal acidic amino acids Glu and Asp. Then, we evaluated the function and drug target potential of TgAAP using the CRISPR/Cas9 knockout system. Western blotting demonstrated the deletion of TgAAP in the knockout strain. Indirect immunofluorescence analysis showed that TgAAP was localized in the cytoplasm of the wild-type parasite, but was not expressed in the knockout strain. Phenotype analysis revealed that TgAAP knockout inhibited the attachment/invasion, replication, and substrate-specific activity in T. gondii. Finally, the activity of drug CID 23724194, previously described as targeting Plasmodium and malarial parasite AAP, was tested against rTgAAP and the parasite. Overall, TgAAP knockout affected the growth of T. gondii but did not completely abolish parasite replication and growth. Therefore, TgAAP may comprise a useful adjunct drug target of T. gondii.
PMID:
 
27678060
 
DOI:
 
10.1038/srep34448

Wednesday, September 28, 2016

Developmental change in translation initiation alters the localization of a common microbial protein necessary for Toxoplasma chronic infection

2016 Sep 27. doi: 10.1111/mmi.13538. [Epub ahead of print]


The Toxoplasma gondii cyst stage is resistant to drug therapy. To identify potential targets for new therapeutics, we screened insertional mutants of T. gondii for a reduced ability to form cysts in the brains of mice. In one of these mutants, named 38C3, the mutagenesis plasmid inserted into the mRNA of a protein that is highly conserved in microbes but is not present in humans. The mutation in 38C3 causes reduced brain cyst production during chronic infection, but does not affect acute virulence, so the disrupted gene and protein are called T. gondii Brain Colonization Protein 1 (TgBCP1). TgBCP1 has three potential in frame start codons that produce either 51, 33 or 25 kDa proteins. In rapidly replicating tachyzoites, translation initiates at the third methionine, producing the 25 kDa form that is conserved in many bacteria and protozoans. Brain cysts exclusively express the 51 kDa form of TgBCP1, which is secreted from the parasites and localizes to the cyst wall. Only expression of the long form of TgBCP1 restored cyst formation in the 38C3 mutant. TgBCP1 is essential for cyst formation and is the first example of a developmental regulation in translation initiation site preference for a T. gondii protein. This article is protected by copyright. All rights reserved.
© 2016 John Wiley & Sons Ltd.

KEYWORDS:

Apicomplexa; Toxoplasma; cyst development; parasite; translation initiation

Tuesday, September 27, 2016

O-fucosylated glycoproteins form assemblies in close proximity to the nuclear pore complexes of Toxoplasma gondii

 2016 Sep 23. pii: 201613653. [Epub ahead of print]

Abstract

Toxoplasma gondii is an intracellular parasite that causes disseminated infections in fetuses and immunocompromised individuals. Although gene regulation is important for parasite differentiation and pathogenesis, little is known about protein organization in the nucleus. Here we show that the fucose-binding Aleuria aurantia lectin (AAL) binds to numerous punctate structures in the nuclei of tachyzoites, bradyzoites, and sporozoites but not oocysts. AAL also binds to Hammondia and Neospora nuclei but not to more distantly related apicomplexans. Analyses of the AAL-enriched fraction indicate that AAL binds O-linked fucose added to Ser/Thr residues present in or adjacent to Ser-rich domains (SRDs). Sixty-nine Ser-rich proteins were reproducibly enriched with AAL, including nucleoporins, mRNA-processing enzymes, and cell-signaling proteins. Two endogenous SRDs-containing proteins and an SRD-YFP fusion localize with AAL to the nuclear membrane. Superresolution microscopy showed that the majority of the AAL signal localizes in proximity to nuclear pore complexes. Host cells modify secreted proteins with O-fucose; here we describe the O-fucosylation pathway in the nucleocytosol of a eukaryote. Furthermore, these results suggest O-fucosylation is a mechanism by which proteins involved in gene expression accumulate near the NPC.

KEYWORDS: 

fucose; nuclear glycosylation; nuclear pore complex; toxoplasma
[PubMed - as supplied by publisher]

Transcriptional profile of SH-SY5Y human neuroblastoma cells transfected by Toxoplasma rhoptry protein 16

 2016 Sep 21. doi: 10.3892/mmr.2016.5758. [Epub ahead of print]

Abstract

Toxoplasma rhoptry protein 16 (ROP16) is crucial in the host-pathogen interaction by acting as a virulent factor during invasion. To improve understanding of the molecular function underlying the effect of ROP16 on host cells, the present study analyzed the transcriptional profile of genes in the ROP16‑transfected SH‑SY5Y human neuroblastoma cell line. The transcriptional profile of the SH‑SY5Y human neuroblastoma cell line overexpressing ROP16 were determined by microarray analysis in order to determine the host neural cell response to the virulent factor. Functional analysis was performed using the Protein Analysis Through Evolutionary Relationships classification system. The ToppGene Suite was used to select candidate genes from the differentially expressed genes. A protein‑protein interaction network was constructed using Cytoscape software according to the interaction associations determined using the Search Tool for the Retrieval of Interacting Genes/Proteins. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis of the selected genes confirmed the results of the microarray. The results showed that 383 genes were differentially expressed in response to ROP16 transfection, of which 138 genes were upregulated and 245 genes were downregulated. Functional analysis indicated that the differentially expressed genes (DEGs) were involved in several biological processes, including developmental process, biological regulation and apoptotic process. A total of 15 candidate genes from the DEGs were screened using the ToppGene Suite. No significant differences in expression were observed between the RT‑qPCR data and the microarray data. Transfection with ROP16 resulted in alterations of several biological processes, including nervous system development, apoptosis and transcriptional regulation. Several genes, including CXCL12, BAI1, ZIC2, RBMX, RASSF6, MAGE‑A6 and HOX, were identified as significant DEGs. Taken together, these results may contribute to understanding the mechanisms underlying the functions of ROP16 and provide scope for further investigation of the pathogenesis of Toxoplasma gondii.
PMID:
 
27666388
 
DOI:
 
10.3892/mmr.2016.5758
[PubMed - as supplied by publisher]

Thursday, September 22, 2016

Peroxiredoxin 3 promotes IL-12 production from macrophages and partially protects mice against infection with Toxoplasma gondii

 2016 Sep 16. pii: S1383-5769(16)30208-2. doi: 10.1016/j.parint.2016.09.008. [Epub ahead of print]

Abstract

Toxoplasmosis remains a life-threatening infection of humans and various domestic and wild animals worldwide. It is caused by the obligatory intracellular protozoan parasite Toxoplasma gondii. Peroxiredoxins (Prxs) are a family of antioxidant enzymes that protect cells from oxidative stress from hydroperoxides. In the recent years, several studies have reported the potential use of T. gondii-derived enzymes in triggering protective immunity against T. gondii infection. Therefore, this study was conducted to investigate the immunogenicity and protective efficacy of TgPrx3. In vitro stimulation of peritoneal macrophages with recombinant TgPrx3 protein fused to glutathione-S transferase (TgPrx3-GST) enhanced IL-12p40 production, indicating the immune-stimulating potentials of TgPrx3. Next, protective efficacy was investigated by subcutaneous inoculation of mice with TgPrx3-GST (25pmol), and recombinant GST or PBS were used as the controls. Mice immunized with TgPrx3-GST exhibited a significant elevation of specific antibodies in terms of IgG1 and IgG2c isotypes. Moreover, interferon-gamma production and spleen cell proliferation dramatically increased in the TgPrx3-GST-sensitized cells from mice immunized with the same antigen. The severity of the T. gondii infections tended to be attenuated in the TgPrx3-GST-immunized mice, as evidenced by their higher survival rates and lower parasite burdens in the brain. Altogether, TgPrx3 immunization induced specific humoral and cellular immune responses and partially protected the mice against lethal toxoplasmosis. Our results suggest the possible use of TgPrx3 as a vaccine candidate against T. gondii infections.
Copyright © 2016. Published by Elsevier Ireland Ltd.

KEYWORDS: 

Immunization; Peroxiredoxin 3; Toxoplasma gondii; Vaccine

Evaluation of Propranolol Effect on Experimental Acute and Chronic Toxoplasmosis using Q-PCR

 2016 Sep 19. pii: AAC.01323-16. [Epub ahead of print]

Abstract

Current therapies against toxoplasmosis are limited and drugs have significant side effects and low efficacies. We evaluated the potential anti-Toxoplasma activity of propranolol 2, 3 mg/kg/day in vivo in acute and chronic phases. Propranolol as the stabilizing cell membrane is a suitable drug for inhibiting the entrance of Toxoplasma gondii (T. gondii) tachyzoites into cells. The acute phase was performed using propranolol, pyrimethamine, propranolol plus pyrimethamine before (pre-treatment) and after (post-treatment) intraperitoneally challenge with 1×103 tachyzoites of the virulent RH strain of T. gondii in Balb/c mice. Also in the chronic phase, treatment was performed 12 hours before intraperitoneally challenge with 1×106 tachyzoites of the virulent RH strain of T. gondii in rats. One week (in acute phase) and two months (in chronic phase) after post infection, tissues were isolated and DNA was extracted. Subsequently parasite load was calculated using Q-PCR. In acute phase, in both groups, significant anti-Toxoplasma activity was observed using propranolol (P < 0.001). Propranolol in pre-treatment group showed higher anti-Toxoplasma activity than propranolol in post-treatment in brain tissues displaying therapeutic efficiency on toxoplasmosis. Also, propranolol combined with pyrimethamine reduced the parasite load as well as significantly increased survival of mice in pre-treatment group. In the chronic phase, anti-Toxoplasma activity was observed with propranolol and decreased parasite load in tissues. In conclusion, the presented results demonstrate that propranolol, as an orally available drug, is effective against acute and latent murine toxoplasmosis at low doses and increase efficiency of drug in combination therapy with propranolol-pyrimethamine.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
PMID:
 
27645234
 
DOI:
 
10.1128/AAC.01323-16

Tuesday, September 20, 2016

Macrophages facilitate the excystation and differentiation of Toxoplasma gondii sporozoites into tachyzoites following oocyst internalisation

 2016 Sep 19;6:33654. doi: 10.1038/srep33654.

Abstract

Toxoplasma gondii is a common parasite of humans and animals, which is transmitted via oocysts in cat faeces or tissue cysts in contaminated meat. The robust oocyst and sporocyst walls protect the infective sporozoites from deleterious external attacks including disinfectants. Upon oocyst acquisition, these walls lose their integrity to let the sporozoites excyst and invade host cells following a process that remains poorly understood. Given the resistance of the oocyst wall to digestive enzymes and the ability of oocysts to cause parenteral infections, the present study investigated the possible contribution of macrophages in supporting sporozoite excystation following oocyst internalisation. By using single cell micromanipulations, real-time and time-point imaging techniques, we demonstrated that RAW macrophages could interact rapidly with oocysts and engulfed them by remodelling of their actin cytoskeleton. Internalised oocysts were associated to macrophage acidic compartments and showed evidences of wall disruption. Sporozoites were observed in macrophages containing oocyst remnants or in new macrophages, giving rise to dividing tachyzoites. All together, these results highlight an unexpected role of phagocytic cells in processing T. gondii oocysts, in line with non-classical routes of infection, and open new perspectives to identify chemical factors that lead to oocyst wall disruption under physiological conditions. 
PMID:
 
27641141
 
DOI:
 
10.1038/srep33654

Thursday, September 15, 2016

Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

 2016 Sep 13;7(5). pii: e00754-16. doi: 10.1128/mBio.00754-16.

Abstract

Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the "moving junction," a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.

IMPORTANCE: 

Nearly one-third of the world's population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.
Copyright © 2016 Krishnamurthy et al.
PMID:
 
27624124
 
DOI:
 
10.1128/mBio.00754-16
[PubMed - in process]

Tuesday, September 13, 2016

Huntingtons Disease Mice Infected with Toxoplasma gondii Demonstrate Early Kynurenine Pathway Activation, Altered CD8+ T-Cell Responses, and Premature Mortality

2016 Sep 9;11(9):e0162404. doi: 10.1371/journal.pone.0162404. eCollection 2016.


Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-repeat expansion in the huntingtin protein. Activation of the kynurenine pathway of tryptophan degradation is implicated in the pathogenesis of HD. Indoleamine-2,3-dioxygenase (IDO) catalyzes the oxidation of tryptophan to kynurenine, the first step in this pathway. The prevalent, neuroinvasive protozoal pathogen Toxoplasma gondii (T. gondii) results in clinically silent life-long infection in immune-competent individuals. T. gondii infection results in activation of IDO which provides some protection against the parasite by depleting tryptophan which the parasite cannot synthesize. The kynurenine pathway may therefore represent a point of synergism between HD and T. gondii infection. We show here that IDO activity is elevated at least four-fold in frontal cortex and striata of non-infected N171-82Q HD mice at 14-weeks corresponding to early-advanced HD. T. gondii infection at 5 weeks resulted in elevation of cortical IDO activity in HD mice. HD-infected mice died significantly earlier than wild-type infected and HD control mice. Prior to death, infected HD mice demonstrated decreased CD8+ T-lymphocyte proliferation in brain and spleen compared to wild-type infected mice. We demonstrate for the first time that HD mice have an altered response to an infectious agent that is characterized by premature mortality, altered immune responses and early activation of IDO. Findings are relevant to understanding how T. gondii infection may interact with pathways mediating neurodegeneration in HD.