Wednesday, July 01, 2015

Role of CD4+Foxp3+ regulatory T cells in protection induced by a live, attenuated, replicating Type-I vaccine strain of Toxoplasma

2015 Jun 29. pii: IAI.00217-15. [Epub ahead of print]
 
 
Vaccination with the live attenuated Toxoplasma gondii Mic1.3KO strain induced long-lasting immunity against challenge with Toxoplasma gondii Type I and Type II strains. The involvement of T regulatory (Treg) cells in the protection mechanism was investigated. Intraperitoneal injection of Mic1.3KO induced a weak and transient influx of CD4+Foxp3+ T regulatory cells followed by recruitment/expansion of CD4+Foxp3-CD25+ effector cells and control of the parasite at the site of infection. The local and systemic cytokine responses associated with this recruitment of Treg were of TH1/Treg-like type. In contrast, injection of RH, the wild-type strain from which the vaccinal strain is derived, induced a low CD4+Foxp3+ cell influx and uncontrolled multiplication of the parasites at this local site, followed by death of the mice. The associated local and systemic cytokine responses were of TH1/TH17-like type.In addition, in vivo Treg induction in the RH-infected mice with IL2/anti-IL2 complexes induced control of the parasite and a TH1/Treg cytokine response similar to the response after Mic1.3KO vaccination. These results suggest that Treg cells may contribute to the protective response after vaccination with Mic1.3KO.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PMID:
26123802
[PubMed - as supplied by publisher]

Tuesday, June 30, 2015

Nitric Oxide Production Increases During Toxoplasma gondii Encephalitis in Mice

2015 Jun 23. pii: S0014-4894(15)00169-1. doi: 10.1016/j.exppara.2015.06.009. [Epub ahead of print]
 

Abstract

Toxoplasma gondii is a intracellular parasite with the potential of causing severe encephalitis among immunocompromised human and animals. The aim of this experimental study was to investigate the immunomodulatory and immunopathological role of nitric oxide (NO) in central nervous systems and to identify any correlation between toxoplasmosis neuropathology and investigate the consequences of the cellular responses protect against Toxoplasma gondii. Mice were infected with ME49 strain Toxoplasma gondii and levels of endothelial, neuronal and inducible nitric oxide synthase (eNOS, nNOS, iNOS), glial fibrillary acidic protein (GFAP) and neurofilament (NF) were examined in brain tissues by immunohistochemistry, during the development and establishment of a chronic infection at 10 30 and 60 days post infection.

KEYWORDS:

Toxoplasma gondii; glial fibrillary acidic protein; neurofilament; neuropathology; nitric oxide
PMID:
26115941
[PubMed - as supplied by publisher]

Parasite Calcineurin Regulates Host Cell Recognition and Attachment by Apicomplexans

2015 Jun 24. pii: S1931-3128(15)00251-6. doi: 10.1016/j.chom.2015.06.003. [Epub ahead of print]
 
Apicomplexans invade a variety of metazoan host cells through mechanisms involving host cell receptor engagement and secretion of parasite factors to facilitate cellular attachment. We find that the parasite homolog of calcineurin, a calcium-regulated phosphatase complex central to signal transduction in eukaryotes, also contributes to host cell invasion by the malaria parasite Plasmodium falciparum and related Toxoplasma gondii. Using reverse-genetic and chemical-genetic approaches, we determine that calcineurin critically regulates and stabilizes attachment of extracellular P. falciparum to host erythrocytes before intracellular entry and has similar functions in host cell engagement by T. gondii. Calcineurin-mediated Plasmodium invasion is strongly associated with host receptors required for host cell recognition, and calcineurin function distinguishes this form of receptor-mediated attachment from a second mode of host-parasite adhesion independent of host receptors. This specific role of calcineurin in coordinating physical interactions with host cells highlights an ancestral mechanism for parasitism used by apicomplexans.
Copyright © 2015 Elsevier Inc. All rights reserved.
PMID:
26118996
[PubMed - as supplied by publisher]

Sunday, June 28, 2015

Comparative Analysis of Apicoplast-Targeted Protein Extension Lengths in Apicomplexan Parasites

 2015;2015:452958. doi: 10.1155/2015/452958. Epub 2015 May 31.

Abstract

In general, the mechanism of protein translocation through the apicoplast membrane requires a specific extension of a functionally important region of the apicoplast-targeted proteins. The corresponding signal peptides were detected in many apicomplexans but not in the majority of apicoplast-targeted proteins in Toxoplasma gondii. In T. gondii signal peptides are either much diverged or their extension region is processed, which in either case makes the situation different from other studied apicomplexans. We propose a statistic method to compare extensions of the functionally important regions of apicoplast-targeted proteins. More specifically, we provide a comparison of extension lengths of orthologous apicoplast-targeted proteins in apicomplexan parasites. We focus on results obtained for the model species T. gondii, Neospora caninum, and Plasmodium falciparum. With our method, cross species comparisons demonstrate that, in average, apicoplast-targeted protein extensions in T. gondii are 1.5-fold longer than in N. caninum and 2-fold longer than in P. falciparum. Extensions in P. falciparum less than 87 residues in size are longer than the corresponding extensions in N. caninum and, reversely, are shorter if they exceed 88 residues. 
PMID:
 
26114107
 
[PubMed - in process]

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

 2015 Jun 22. pii: S1096-4959(15)00119-0. doi: 10.1016/j.cbpb.2015.06.006. [Epub ahead of print]

Abstract

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ■ 0.0002 mM, KATP = 0.05 ■ 0.008 mM, and Vmax = 920 ■ 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5'-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS: 

Adenosine kinase; Enzyme kinetics; Parasite; Purine metabolism; Toxoplasma gondii
PMID:
 
26112826
 
[PubMed - as supplied by publisher]

Could miltefosine be used as a therapy for toxoplasmosis?

 2015 Jun 23. pii: S0014-4894(15)00165-4. doi: 10.1016/j.exppara.2015.06.005. [Epub ahead of print]

Abstract

Toxoplasmosis is a zoonotic protozoal disease affecting more than a billion people worldwide. The shortfalls of the current treatment options necessitate the development of non-toxic and well-tolerated, efficient alternatives especially against the cyst form. The current study was undertaken to investigate, for the first time, the potential potency of miltefosine against Toxoplasma gondii infection in acute and chronic experimental toxoplasmosis. Results showed that there is no evidence of anti-parasitic activity of miltefosine against T. gondii tachyzoites in acute experimental toxoplasmosis. However, anti-parasitic activity of miltefosine against T. gondii cyst stage in chronic experimental toxoplasmosis could not be excluded as demonstrated by significant reduction in brain cyst burden. Moreover, considerable morphological changes in the cysts were revealed by light and electron microscopy study and also by amelioration of pathological changes in the brain. Future studies should focus on enhancement of anti-toxoplasma activity of miltefosine against chronic toxoplasmosis using formulation based nanotechnology. To the best of our knowledge, this is the first study highlighting efficacy of miltefosine against chronic toxoplasmosis, thus, increasing the list of diseases that can be targeted by this drug.
Copyright © 2015. Published by Elsevier Inc.

KEYWORDS: 

Chronic toxoplasmosis; Electron microscopy; Me49 strain; Miltefosine; RH strain; Toxoplasma gondii
PMID:
 
26112396
 
[PubMed - as supplied by publisher]

Thursday, June 25, 2015

siRNA Screening Identifies the Host Hexokinase 2 (HK2) Gene as an Important Hypoxia-Inducible Transcription Factor 1 (HIF-1) Target Gene in Toxoplasma gondii-Infected Cells

2015 Jun 23;6(3). pii: e00462-15. doi: 10.1128/mBio.00462-15.
 
 
Although it is established that oxygen availability regulates cellular metabolism and growth, little is known regarding how intracellular pathogens use host factors to grow at physiological oxygen levels. Therefore, large-scale human small interfering RNA screening was performed to identify host genes important for growth of the intracellular protozoan parasite Toxoplasma gondii at tissue oxygen tensions. Among the genes identified by this screen, we focused on the hexokinase 2 (HK2) gene because its expression is regulated by hypoxia-inducible transcription factor 1 (HIF-1), which is important for Toxoplasma growth. Toxoplasma increases host HK2 transcript and protein levels in a HIF-1-dependent manner. In addition, parasite growth at 3% oxygen is restored in HIF-1-deficient cells transfected with HK2 expression plasmids. Both HIF-1 activation and HK2 expression were accompanied by increases in host glycolytic flux, suggesting that enhanced HK2 expression in parasite-infected cells is functionally significant. Parasite dependence on host HK2 and HIF-1 expression is not restricted to transformed cell lines, as both are required for parasite growth in nontransformed C2C12 myoblasts and HK2 is upregulated in vivo following infection. While HK2 is normally associated with the cytoplasmic face of the outer mitochondrial membrane at physiological O2 levels, HK2 relocalizes to the host cytoplasm following infection, a process that is required for parasite growth at 3% oxygen. Taken together, our findings show that HIF-1-dependent expression and relocalization of HK2 represent a novel mechanism by which Toxoplasma establishes its replicative niche at tissue oxygen tensions.

IMPORTANCE:

Little is known regarding how the host cell contributes to the survival of the intracellular parasite Toxoplasma gondii at oxygen levels that mimic those found in tissues. Our previous work showed that Toxoplasma activates the expression of an oxygen-regulated transcription factor that is required for growth. Here, we report that Toxoplasma regulates the abundance and activity of a key host metabolic enzyme, hexokinase 2, by activating HIF-1 and by promoting dissociation of hexokinase 2 from the mitochondrial membrane. Collectively, our data reveal HIF-1/hexokinase 2 as a novel target for an intracellular pathogen that acts by reprograming the host cell's metabolism to create an environment conducive for parasite replication at physiological oxygen levels.
Copyright © 2015 Menendez et al.
PMID:
26106078
[PubMed - in process]

Host-parasite interactions; an intimate epigenetic relationship

2015 Jun 11. doi: 10.1111/cmi.12471. [Epub ahead of print]
 
 
The epigenetics of host-pathogen interactions is emerging as an interesting angle from which to study how parasites have evolved sophisticated strategies to manipulate host gene transcription and protein expression. In this review we discuss the application of an operational framework to investigate the host cell signalling pathways that are induced by intracellular parasites and the epigenomic consequences in the host nucleus. To illustrate this conceptual approach, we have focused on examples from two eukaryotic intracellular parasites of the apicomplexa phylum: Theileria and Toxoplasma. We review recent findings on intracellular parasitism strategies for hijacking host nuclear functions and discuss how we might think of the parasite and its proteome as an intracellular epigenator.
PMID:
26096716
[PubMed - as supplied by publisher]

Toxoplasma prophylaxis in haematopoietic cell transplant recipients: a review of the literature and recommendations

2015 Aug;28(4):283-92. doi: 10.1097/QCO.0000000000000169.
 

PURPOSE OF REVIEW:

Toxoplasmosis in haematopoietic cell transplant (HCT) recipients is associated with high morbidity and mortality rates. Prophylaxis following HCT is recommended for high-risk pre-HCT toxoplasma-seropositive (pre-HCTSP) recipients. However, there is no agreement or consistency among programmes on whether to adopt prophylaxis or not, or if used, on the chosen antitoxoplasma prophylactic regimen. This review discusses the role of prophylaxis, and preemptive treatment, for toxoplasmosis in the setting of HCT.

RECENT FINDINGS:

Approximately two-thirds of toxoplasmosis cases following HCT are reported in allogeneic pre-HCTSP (allo pre-HCTSP) patients. This finding confirms a major role of reactivation of latent infection in the pathogenesis of toxoplasmosis in this patient population. Toxoplasma disease-related mortality in allo pre-HCTSP patients was reported at 62%, but it can be significantly decreased with early detection and treatment of toxoplasma infection. There are no randomized trials comparing the efficacy of different prophylactic agents to prevent toxoplasmosis after HCT. Several observational studies have demonstrated the efficacy of trimethoprim-sulfamethoxazole (TMP/SMX) in decreasing the incidence of toxoplasmosis following HCT. There is limited information regarding efficacy of other prophylactic agents. Preemptive treatment using routine blood PCR monitoring seems to be beneficial in detecting infection early and preventing disease in several observational studies and has been adopted for allo pre-HCTSP HCT patients when universal prophylaxis is not possible.

SUMMARY:

Universal prophylaxis with TMP/SMX in allo pre-HCTSP patients should be implemented by all transplant programmes. Preemptive treatment with routine blood PCR monitoring is an option if prophylaxis cannot be used.
PMID:
26098500
[PubMed - in process]

Monday, June 22, 2015

Toxoplasma Actin Is Required for Efficient Host Cell Invasion

 2015 Jun 16;6(3). pii: e00557-15. doi: 10.1128/mBio.00557-15.

Abstract

Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of Δact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible Δact1 parasites as long as 5 days after ACT1 deletion. We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in Δact1 parasites being responsible for their invasive ability. Furthermore, invasion by the Δact1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by Δact1 parasites. However, Δact1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion.

IMPORTANCE: 

The prevailing model for apicomplexan invasion has recently been suggested to require major revision, based on studies where core components of the invasion machinery were genetically disrupted using a Cre-Lox-based inducible knockout system. For the myosin component of the motor thought to power invasion, an alternative parasite myosin was recently demonstrated to functionally compensate for loss of the primary myosin involved in invasion. Here, we highlight a second mechanism that can account for the surprising ability of parasites to invade after genetic disruption of core invasion machinery. Specifically, residual actin protein present in inducible knockout parasites appears able to support their limited invasion of host cells. Our results have important implications for the interpretation of the apicomplexan invasion model and also highlight significant considerations when analyzing the phenotypes of inducible knockout parasites generated using Cre-Lox technology.
Copyright © 2015 Drewry and Sibley.
PMID:
 
26081631
 
[PubMed - in process] 
PMCID:
 
PMC4471557
 

The Organellar Genomes of Chromera and Vitrella, the Phototrophic Relatives of Apicomplexan Parasites

 2015 Jun 18. [Epub ahead of print]

Abstract

Apicomplexa are known to contain greatly reduced organellar genomes. Their mitochondrial genome carries only three protein-coding genes, and their plastid genome is reduced to a 35-kb-long circle.The discovery of coralendosymbiotic algae Chromera velia and Vitrella brassicaformis, which share a common ancestry with Apicomplexa, provided an opportunity to study possibly ancestral forms of organellar genomes, a unique glimpse into the evolutionary history of apicomplexan parasites. The structurally similar mitochondrial genomes of Chromera and Vitrella differ in gene content, which is reflected in the composition of their respiratory chains. Thus, Chromera lacks respiratory complexes I and III, whereas Vitrella and apicomplexan parasites are missing only complex I. Plastid genomes differ substantially between these algae, particularly in structure: The Chromera plastid genome is a linear, 120-kb molecule with large and divergent genes, whereas the plastid genome of Vitrella is a highly compact circle that is only 85 kb long but nonetheless contains more genes than that of Chromera. It appears that organellar genomes have already been reduced in free-living phototrophic ancestors of apicomplexan parasites, and such reduction is not associated with parasitism. Expected final online publication date for the Annual Review of Microbiology Volume 69 is September 08, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
PMID:
 
26092225
 
[PubMed - as supplied by publisher]

Cutting Edge: IFN-γ Produced by Brain-Resident Cells Is Crucial To Control Cerebral Infection with Toxoplasma gondii

 2015 Jun 19. pii: 1500814. [Epub ahead of print]

Abstract

In vitro studies demonstrated that microglia and astrocytes produce IFN-γ in response to various stimulations, including LPS. However, the physiological role of IFN-γ production by brain-resident cells, including glial cells, in resistance against cerebral infections remains unknown. We analyzed the role of IFN-γ production by brain-resident cells in resistance to reactivation of cerebral infection with Toxoplasma gondii using a murine model. Our study using bone marrow chimeric mice revealed that IFN-γ production by brain-resident cells is essential for upregulating IFN-γ-mediated protective innate immune responses to restrict cerebral T. gondii growth. Studies using a transgenic strain that expresses IFN-γ only in CD11b+ cells suggested that IFN-γ production by microglia, which is the only CD11b+ cell population among brain-resident cells, is able to suppress the parasite growth. Furthermore, IFN-γ produced by brain-resident cells is pivotal for recruiting T cells into the brain to control the infection. These results indicate that IFN-γ produced by brain-resident cells is crucial for facilitating both the protective innate and T cell-mediated immune responses to control cerebral infection with T. gondii.
Copyright © 2015 by The American Association of Immunologists, Inc.
PMID:
 
26091720
 
[PubMed - as supplied by publisher]

Conditional U1 Gene Silencing in Toxoplasma gondii

 2015 Jun 19;10(6):e0130356. doi: 10.1371/journal.pone.0130356.

Abstract

The functional characterisation of essential genes in apicomplexan parasites, such as Toxoplasma gondii or Plasmodium falciparum, relies on conditional mutagenesis systems. Here we present a novel strategy based on U1 snRNP-mediated gene silencing. U1 snRNP is critical in pre-mRNA splicing by defining the exon-intron boundaries. When a U1 recognition site is placed into the 3'-terminal exon or adjacent to the termination codon, pre-mRNA is cleaved at the 3'-end and degraded, leading to an efficient knockdown of the gene of interest (GOI). Here we describe a simple method that combines endogenous tagging with DiCre-mediated positioning of U1 recognition sites adjacent to the termination codon of the GOI which leads to a conditional knockdown of the GOI upon rapamycin-induction. Specific knockdown mutants of the reporter gene GFP and several endogenous genes of T. gondii including the clathrin heavy chain gene 1 (chc1), the vacuolar protein sorting gene 26 (vps26), and the dynamin-related protein C gene (drpC) were silenced using this approach and demonstrate the potential of this technology. We also discuss advantages and disadvantages of this method in comparison to other technologies in more detail.
PMID:
 
26090798
 
[PubMed - as supplied by publisher] 

Wednesday, June 10, 2015

Lack of a Functioning P2X7 Receptor Leads to Increased Susceptibility to Toxoplasmic Ileitis

2015 Jun 8;10(6):e0129048. doi: 10.1371/journal.pone.0129048. eCollection 2015.
 

BACKGROUND:

Oral infection of C57BL/6J mice with the protozoan parasite Toxoplasma gondii leads to a lethal inflammatory ileitis.

PRINCIPAL FINDINGS:

Mice lacking the purinergic receptor P2X7R are acutely susceptible to toxoplasmic ileitis, losing significantly more weight than C57BL/6J mice and exhibiting much greater intestinal inflammatory pathology in response to infection with only 10 cysts of T. gondii. This susceptibility is not dependent on the ability of P2X7R-deficient mice to control the parasite, which they accomplish just as efficiently as C57BL/6J mice. Rather, susceptibility is associated with elevated ileal concentrations of pro-inflammatory cytokines, reactive nitrogen intermediates and altered regulation of elements of NFκB activation in P2X7R-deficient mice.

CONCLUSIONS:

Our data support the thesis that P2X7R, a well-documented activator of pro-inflammatory cytokine production, also plays an important role in the regulation of intestinal inflammation.
PMID:
26053862
[PubMed - in process]

Monday, June 08, 2015

From the blood to the brain: avenues of eukaryotic pathogen dissemination to the central nervous system

2015 Jun 2;26:53-59. doi: 10.1016/j.mib.2015.05.006. [Epub ahead of print]
 
 
Infection of the central nervous system (CNS) is a significant cause of morbidity and mortality, and treatments available to combat the highly debilitating symptoms of CNS infection are limited. The mechanisms by which pathogens in the circulation overcome host immunity and breach the blood-brain barrier are active areas of investigation. In this review, we discuss recent work that has significantly advanced our understanding of the avenues of pathogen dissemination to the CNS for four eukaryotic pathogens of global health importance: Toxoplasma gondii, Plasmodium falciparum, Trypanosoma brucei, and Cryptococcus neoformans. These studies highlight the remarkable diversity of pathogen strategies for trafficking to the brain and will ultimately contribute to an improved ability to combat life-threatening CNS disease.
Copyright © 2015. Published by Elsevier Ltd.
PMID:
26048316
[PubMed - as supplied by publisher]