Thursday, May 29, 2014

Calcium Entry in Toxoplasma gondii and its Enhancing Effect of Invasion-linked Traits

2014 May 27. pii: jbc.M114.565390. [Epub ahead of print]

Calcium Entry in Toxoplasma gondii and its Enhancing Effect of Invasion-linked Traits

Abstract

During invasion and egress from their host cells Apicomplexan parasites face sharp changes in the surrounding calcium ion (Ca2+) concentration. Our work with Toxoplasma gondii provides evidence for Ca2+ influx from the extracellular milieu leading to cytosolic Ca2+ increase and enhancement of virulence traits, such as gliding motility, conoid extrusion, microneme secretion, and host cell invasion. Assays of Mn2+ and Ba2+ uptake do not support a canonical store regulated Ca2+ entry mechanism. Ca2+ entry was blocked by the L-type Ca2+ channel inhibitor nifedipine and stimulated by the increase in cytosolic Ca2+ and by the specific L-type Ca2+ channel agonist Bay K-8644. Our results demonstrate that Ca2+ entry is critical for parasite virulence. We propose a regulated Ca2+ entry mechanism activated by cytosolic Ca2+ that has an enhancing effect on invasion-linked traits.
Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

KEYWORDS:

Conoid Extrusion; Gliding motility; Toxoplasma gondii; calcium; cell invasion; fluorescence; nifedipine; parasite; protozoan; signaling
PMID:
24867952
[PubMed - as supplied by publisher]

Complex immune cell interplay in the IFN-γ response during Toxoplasma gondii infection

2014 May 27. pii: IAI.01722-14. [Epub ahead of print]

Complex immune cell interplay in the IFN-γ response during Toxoplasma gondii infection

Abstract

Toxoplasma gondii is an obligate intracellular parasite of clinical importance, especially in immunocompromised patients. Investigations into the immune response to the parasite found that T cells are the primary effector cells regulating IFN-γ-mediated host resistance. However, recent studies have revealed a critical role for the innate immune system in mediating host defense independently of the T cell responses to the parasite. This body of knowledge is put into perspective by the unifying theme that immunity to the protozoan parasite requires a strong IFN-γ host response. In the following review, we discuss the role of IFN-γ-producing cells and the signals that regulate IFN-γ production during T. gondii infection.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
24866795
[PubMed - as supplied by publisher]

Toxoplasma gondii- Derived Profilin Triggers Human Toll-Like Receptor 5-Dependent Cytokine Production

2014 May 23. [Epub ahead of print]

Toxoplasma gondii- Derived Profilin Triggers Human Toll-Like Receptor 5-Dependent Cytokine Production

Abstract

Up to a third of the world's population is infected with Toxoplasma gondii. Natural infection in humans can be life threatening during pregnancy and in immunocompromised individuals. Toll-like receptor (TLR) 11 is the mouse innate sensor that recognizes T. gondii profilin; however, in humans the TLR11 gene leads to transcription of no functional protein. Herein, by using a multiple sequence alignment phylogenetic analysis program between human and mouse species, we found that human TLR5 seems to be the evolutionarily closest member of the TLR gene family to mouse tlr11. We therefore asked whether human TLR5 could mediate IL-6, IL-8 and IL-12p70 production in response to the T. gondii profilin. We found that this was the case both in human cell lines as well as peripheral blood monocytes. Moreover, TLR5 neutralization and gene silencing mediated specific ablation of cytokine production after profilin exposure. Finally, peripheral blood monocytes carrying the TLR5 R392X mutation failed to produce cytokines in response to stimulation with profilin. Taken together, the results presented herein reveal a previously unappreciated cross-recognition of a relevant human pathogen-derived pathogen-associated molecular pattern. © 2014 S. Karger AG, Basel.
PMID:
24861338
[PubMed - as supplied by publisher]

Tuesday, May 27, 2014

Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses

2014 May 21. pii: S2211-1247(14)00333-7. doi: 10.1016/j.celrep.2014.04.031. [Epub ahead of print]

Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses

Abstract

CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.
Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
PMID:
24857659
[PubMed - as supplied by publisher]

Characterisation of the Chloroquine Resistance Transporter homologue in Toxoplasma

2014 May 23. pii: EC.00027-14. [Epub ahead of print]

Characterisation of the Chloroquine Resistance Transporter homologue in Toxoplasma gondii

Abstract

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the anti-malarial drug chloroquine. PfCRT localises to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter (CRT) family. CRT family proteins are found throughout the Apicomplexa, in some protists and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) co-localizes with markers for the vacuolar compartment (VAC) in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT-family proteins in apicomplexan parasites.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
PMID:
24859994
[PubMed - as supplied by publisher]

Astrocytic TGF-β Signaling Limits Inflammation and Reduces Neuronal Damage during Central Nervous System Toxoplasma Infection

2014 May 23. pii: 1303284. [Epub ahead of print]

Astrocytic TGF-β Signaling Limits Inflammation and Reduces Neuronal Damage during Central Nervous System Toxoplasma Infection

Abstract

The balance between controlling infection and limiting inflammation is particularly precarious in the brain because of its unique vulnerability to the toxic effects of inflammation. Astrocytes have been implicated as key regulators of neuroinflammation in CNS infections, including infection with Toxoplasma gondii, a protozoan parasite that naturally establishes a chronic CNS infection in mice and humans. In CNS toxoplasmosis, astrocytes are critical to controlling parasite growth. They secrete proinflammatory cytokines and physically encircle parasites. However, the molecular mechanisms used by astrocytes to limit neuroinflammation during toxoplasmic encephalitis have not yet been identified. TGF-β signaling in astrocytes is of particular interest because TGF-β is universally upregulated during CNS infection and serves master regulatory and primarily anti-inflammatory functions. We report in this study that TGF-β signaling is activated in astrocytes during toxoplasmic encephalitis and that inhibition of astrocytic TGF-β signaling increases immune cell infiltration, uncouples proinflammatory cytokine and chemokine production from CNS parasite burden, and increases neuronal injury. Remarkably, we show that the effects of inhibiting astrocytic TGF-β signaling are independent of parasite burden and the ability of GFAP+ astrocytes to physically encircle parasites.
Copyright © 2014 by The American Association of Immunologists, Inc.
PMID:
24860191
[PubMed - as supplied by publisher]

Thursday, May 22, 2014

Identifying Novel Cell Cycle Proteins in Apicomplexa Parasites through Co-Expression Decision Analysis

2014 May 19;9(5):e97625. doi: 10.1371/journal.pone.0097625. eCollection 2014.

Identifying Novel Cell Cycle Proteins in Apicomplexa Parasites through Co-Expression Decision Analysis

 

Abstract

Hypothetical proteins comprise roughly half of the predicted gene complement of Toxoplasma gondii and Plasmodium falciparum and represent the largest class of uniquely functioning proteins in these parasites. Following the idea that functional relationships can be informed by the timing of gene expression, we devised a strategy to identify the core set of apicomplexan cell division cycling genes with important roles in parasite division, which includes many uncharacterized proteins. We assembled an expanded list of orthologs from the T. gondii and P. falciparum genome sequences (2781 putative orthologs), compared their mRNA profiles during synchronous replication, and sorted the resulting set of dual cell cycle regulated orthologs (744 total) into protein pairs conserved across many eukaryotic families versus those unique to the Apicomplexa. The analysis identified more than 100 ortholog gene pairs with unknown function in T. gondii and P. falciparum that displayed co-conserved mRNA abundance, dynamics of cyclical expression and similar peak timing that spanned the complete division cycle in each parasite. The unknown cyclical mRNAs encoded a diverse set of proteins with a wide range of mass and showed a remarkable conservation in the internal organization of ordered versus disordered structural domains. A representative sample of cyclical unknown genes (16 total) was epitope tagged in T. gondii tachyzoites yielding the discovery of new protein constituents of the parasite inner membrane complex, key mitotic structures and invasion organelles. These results demonstrate the utility of using gene expression timing and dynamic profile to identify proteins with unique roles in Apicomplexa biology.
PMID:
24841368
[PubMed - in process]

Wednesday, May 21, 2014

John Boothroyd: Disease Detective

Last week, John Boothroyd, PhD, kicked off Stanford’s first Disease Detective lecture series with a fascinating tale about how his lab invented a simple biochemical “secret sauce” that revolutionized the detection of viral and bacterial infections like HIV, Hepatitis C and gonorrhea.

“It mostly started as a sketch on a piece of paper, then later became Gen-Probe’s core technology, which won them the 2004 National Medal of Technology,” explained Boothroyd, a Stanford professor of microbiology and immunology.

What Boothroyd invented, in collaboration with postdoctoral researchers James Burg and Philippe Pouletty, is called Transcription-Mediated Amplification.

Before this discovery, detecting a snippet of disease-specific DNA in a sample of cells was like finding a needle in a haystack. To increase a test’s accuracy, a lab technician would try to coax the target DNA into replicating itself through hours of tedious time-and- temperature-sensitive steps.

Boothroyd and his team’s new process consisted of a simple recipe of primers and enzymes that, after optimization by Gen-Probe, tricked a target snippet of DNA into automatically creating 10 billion copies of itself in less than an hour. This ultimately enabled the development of cheaper and faster disease tests.

In 2012 Boothroyd was ushered into the Stanford Inventor’s Hall of Fame because of this patent, which is among the top-ten revenue-generating inventions Stanford. He has six other patented inventions, including one that makes antigen production for the testing of toxoplasmosis infections far more efficient. Another detects toxoplasmosis in the amniotic fluid of pregnant women. He describes this research in the video above.

Looking back on his career choices, one thing that Boothroyd is grateful for is being able to combine his two loves at Stanford — basic research and teaching — while leaving the business of running a company to his patent licensees.

To the lecture hall filled with student researchers worried about the “postdocalypse,” the shortage of tenure-track research positions in academia, he gave this advice:

“I think the [postdocalypse] negativity is overstated. You have to have faith in yourself. You have to do what you want to do. If you’re enjoying your work and it’s a stepping stone to where you’re going, relax and see what happens.”

The next Disease Detective lecture will be held during fall quarter 2014. Watch for details on the Stanford Predictives and Diagnostics Accelerator webpage.

Check out the video here.

Monday, May 19, 2014

Investigation of anti-Toxocara and anti-toxoplasma antibodies in patients with schizophrenia disorder

 2014;2014:230349. doi: 10.1155/2014/230349. Epub 2014 Apr 16.

Investigation of anti-Toxocara and anti-toxoplasma antibodies in patients with schizophrenia disorder

Abstract

Objective. The aim of the present study was to examine the relationship between Toxoplasma gondii and Toxocara spp. infections in patients with schizophrenia disorder. Method. A total of 100 patients with schizophrenia disorder and 95 healthy individuals participated in the study. Participants were tested for the presence of anti-T. gondii and anti-Toxocara spp. antibodies by ELISA and Western blotting. Data were analyzed using Chi-square test and Fisher 9 s exact test. Results. There were no differences in T. gondii IgG seroprevalence between patients with schizophrenia and healthy individuals (P = 0.1), but there were differences in seroprevalence between males and females with schizophrenia (P = 0.009). In contrast, Toxocara spp. IgG seroprevalence was greater in patients with schizophrenia disorder than in healthy individuals (P = 0.02), but there were no differences in seroprevalence between men and women with schizophrenia (P = 0.5). Finally, there were no differences in seroprevalence of T. gondii or Toxocara spp. IgG among different subtypes of schizophrenia, various age groups, residential area, or clinical course of treatment (P > 0.05). Conclusion. The present study suggests that patients with schizophrenia disorder are at elevated risk of Toxocara spp. infection. Moreover, contamination with T. gondii is a risk factor for schizophrenia in women.
PMID:
 
24834353
 
[PubMed] 

Silencing of GRA10 Protein Expression Inhibits Toxoplasma gondii Intracellular Growth and Development

 2014 May 12. pii: S1383-5769(14)00059-2. doi: 10.1016/j.parint.2014.05.001. [Epub ahead of print]

Silencing of GRA10 Protein Expression Inhibits Toxoplasma gondii Intracellular Growth and Development

Abstract

Toxoplasma gondii dense granule proteins (GRAs) are secreted abundantly in both the tachyzoite and bradyzoite stages of the parasite and are known to localize to various compartments of the parasitophorous vacuole (PV) that interfaces with the host cell milieu. Thus, GRAs may play significant roles in the biogenesis of the PV that is important for survival of intracellular T. gondii. GRA10 is a dense granule protein whose role in T. gondii has not yet been characterized. Therefore, in this study, we endeavored to determine the role of GRA10 in the growth and survival of intracellular T. gondii by using phosphorodiamidate morpholino oligomers (PPMOs) antisense knockdown approach to disrupt the translation of GRA10 mRNA in the parasites. We expressed and purified a truncated recombinant GRA10 protein to generate anti-GRA10 polyclonal antibodies that we used to characterize GRA10 in T. gondii. We found that GRA10 is a soluble, dense granule-associated protein that is secreted into the parasite cytosol and the parasitophorous vacuole milieu. Using in vitro cultures, we found that knockdown of GRA10 results in severe inhibition of T. gondii growth in human fibroblasts and in ovine monocytic cells. Together, our findings define GRA10 as a dense granule protein that plays a significant role in the growth and propagation of intracellular T. gondii in human fibroblasts and in ovine monocytic cells.
Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

KEYWORDS:

GRA10 knockdown, Toxoplasma gondii, dense granule proteins, morpholino oligomers
PMID:
 
24832208
 
[PubMed - as supplied by publisher]

The Toxoplasma Pseudokinase ROP5 Forms Complexes with ROP18 and ROP17 Kinases that Synergize to Control Acute Virulence in Mice

 2014 May 14;15(5):537-50. doi: 10.1016/j.chom.2014.04.002.

The Toxoplasma Pseudokinase ROP5 Forms Complexes with ROP18 and ROP17 Kinases that Synergize to Control Acute Virulence in Mice

Abstract

Polymorphic rhoptry-secreted kinases (ROPs) are essential virulence factors of Toxoplasma gondii. In particular, the pseudokinase ROP5 is the major determinant of acute virulence in mice, but the underlying mechanisms are unclear. We developed a tandem affinity protein tagging and purification approach in T. gondii and used it to show that ROP5 complexes with the active kinases ROP18 and ROP17. Biochemical analyses indicate that ROP18 and ROP17 have evolved to target adjacent and essential threonine residues in switch region I of immunity-related guanosine triphosphatases (GTPases) (IRGs), a family of host defense molecules that function to control intracellular pathogens. The combined activities of ROP17 and ROP18 contribute to avoidance of IRG recruitment to the intracellular T. gondii-containing vacuole, thus protecting the parasite from clearance in interferon-activated macrophages. These studies reveal an intricate, multilayered parasite survival strategy involving pseudokinases that regulate multiple active kinase complexes to synergistically thwart innate immunity.
Copyright © 2014 Elsevier Inc. All rights reserved.
PMID:
 
24832449
 
[PubMed - in process]

Friday, May 16, 2014

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

2014 May 13;5(3). pii: e01114-14. doi: 10.1128/mBio.01114-14.

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

Abstract

Toxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.

IMPORTANCE:

Genetic approaches have proven very powerful for studying the biology of organisms, including microbes. However, ease of genetic manipulation varies widely among isolates, with common lab isolates often being the most amenable to such approaches. Unfortunately, such common lab isolates have also been passaged frequently in vitro and have thus lost many of the attributes of wild isolates, often affecting important traits, like virulence. On the other hand, wild isolates are often not amenable to standard genetic approaches, thus limiting inquiry about the genetic basis of biological diversity. Here we imported a new genetic system based on CRISPR/CAS9, which allows high efficiency of targeted gene disruption in natural isolates of T. gondii. This advance promises to bring the power of genetics to bear on the broad diversity of T. gondii strains that have been described recently.
Copyright © 2014 Shen et al.
PMID:
24825012
[PubMed - in process]

Evaluation of anti-coccidial effects of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one and identification of its potential target proteins in Toxoplasma gondii

2014 May 15. [Epub ahead of print]

Evaluation of anti-coccidial effects of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one and identification of its potential target proteins in Toxoplasma gondii

 

Abstract

Coccidiosis affects many vertebrates worldwide, but treatment with known anti-coccidial drugs causes several adverse side effects. There is a critical need for the development and evaluation of new drugs. The anti-coccidial effect of 1-[4-(4-nitrophenoxy)phenyl]propane-1-one (NPPP), a synthetic compound, was studied in vitro and in vivo. Treatment with NPPP showed anti-Toxoplasma activity in vitro with a lower EC50 value than pyrimethamine. In ICR mice infected with Toxoplasma gondii, oral administration of NPPP for 4 days showed statistically significant anti-Toxoplasma activity with lower numbers of tachyzoite than those of the negative control (p < 0.01). NPPP also exhibited strong anti-Eimeria activity in Eimeria tenella-infected chickens when treated for 4 days with orally administered NPPP at a dose of 100 mg/kg. Potential target proteins of NPPP were analyzed by proteomic profiles of T. gondii tachyzoites. Two hypothetical proteins were identified as possible targets of NPPP, a putative ortholog of vacuolar ATP synthase subunit C and a class I S-adenosylmethionine-dependent methyltransferase. Our data show that the NPPP might be an anti-coccidial drug candidate for clinical application against coccidial infections. Future investigations will focus on identifying the function of proteins regulated by NPPP.
PMID:
24824336
[PubMed - as supplied by publisher]

Spontaneous cystogenesis of Toxoplasma gondii in feline epithelial cells in vitro

2014 Apr;61(2):113-9.

Spontaneous cystogenesis of Toxoplasma gondii in feline epithelial cells in vitro

Abstract

Toxoplasma gondii Nicolle et Manceaux, 1908 is an obligate intracellular parasite with the ability to infect mammals and birds. The only definitive hosts for T. gondii are felids, as the parasites form immature oocysts that are shed in the faeces. Here we introduce cat cells as a model for the study of experimental toxoplasmosis. We selected epithelial cells derived from cat kidneys (CRFK) as a target to determine the intracellular fate ofbradyzoites of the T. gondii ME49 strain. In parallel, we compared this infection using epithelial cells from the rat intestine (IEC-6), considering the enteroepithelial development that occurs in the cat. Different ratios of parasites to host cells were assayed over the course of a 14-day-infection. The intracellular development of T. gondii was dependent on the source of the epithelial cells and also on the parasite/host cell ratio. Cystogenesis was well established in the CRFK cell line at a ratio of 1:10 after 10-14 days of infection. This cellular model system opens a new field of investigation into the molecular aspects of the interactions between T. gondii and feline epithelial cells. The CRFK cell line appears to be a potential cellular model for large scale cyst production in vitro, which would allow a reduction in the number of animals used and/or replacement of animals by in vitro cultures.
PMID:
24822317
[PubMed - in process]

Saturday, May 10, 2014

Neglected parasitic infections in the United States: toxoplasmosis


2014 May;90(5):794-9. doi: 10.4269/ajtmh.13-0722.

Neglected parasitic infections in the United States: toxoplasmosis

Abstract

Abstract. Toxoplasma gondii is a leading cause of severe foodborne illness in the United States. Population-based studies have found T. gondii infection to be more prevalent in racial/ethnic minority and socioeconomically disadvantaged groups. Soil contaminated with cat feces, undercooked meat, and congenital transmission are the principal sources of infection. Toxoplasmosis-associated illnesses include congenital neurologic and ocular disease; acquired illness in immunocompetent persons, most notably ocular disease; and encephalitis or disseminated disease in immunosuppressed persons. The association of T. gondii infection with risk for mental illness is intriguing and requires further research. Reduction of T. gondii in meat, improvements in hygiene and food preparation practices, and reduction of environmental contamination can prevent toxoplasmosis, but more research is needed on how to implement these measures. In addition, screening and treatment may help prevent toxoplasmosis or reduce the severity of disease in some settings.
PMID:
24808246
[PubMed - in process]

Thursday, May 08, 2014

The Role of MACPF Proteins in the Biology of Malaria and Other Apicomplexan Parasites

2014;80:241-53. doi: 10.1007/978-94-017-8881-6_12.

The Role of MACPF Proteins in the Biology of Malaria and Other Apicomplexan Parasites

Abstract

Apicomplexans are eukaryotic parasites of major medical and veterinary importance. They have complex life cycles through frequently more than one host, interact with many cell types in their hosts, and can breach host cell membranes during parasite traversal of, or egress from, host cells. Some of these parasites make a strikingly heavy use of the pore-forming MACPF domain, and encode up to 10 different MACPF domain-containing proteins. In this chapter, we focus on the two most studied and medically important apicomplexans, Plasmodium and Toxoplasma, and describe the known functions of their MACPF polypeptide arsenal. Apicomplexan MACPF proteins appear to be involved in a variety of membrane-damaging events, making them an attractive model to dissect the structure-function relationships of the MACPF domain.
PMID:
24798015
[PubMed - in process]

Toxoplasma gondii 70 kDa Heat Shock Protein: Increased Expression in the Brain Is Associated with Parasite Replication

2014 May 6;9(5):e96527. doi: 10.1371/journal.pone.0096527. eCollection 2014.

Toxoplasma gondii 70 kDa Heat Shock Protein: Systemic Detection Is Associated with the Death of the Parasites by the Immune Response and Its Increased Expression in the Brain Is Associated with Parasite Replication

Abstract

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.
PMID:
24801069
[PubMed - in process]

Saturday, May 03, 2014

Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response

 2014 Apr 29;12(4):e1001845. doi: 10.1371/journal.pbio.1001845. eCollection 2014.

Toxoplasma Effector MAF1 Mediates Recruitment of Host Mitochondria and Impacts the Host Response

Abstract

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria.
PMID:
 
24781109
 
[PubMed - in process] 

Assessment of phosphorylation in Toxoplasma glideosome assembly and function

 2014 Apr 29. doi: 10.1111/cmi.12307. [Epub ahead of print]

Assessment of phosphorylation in Toxoplasma glideosome assembly and function

Abstract

Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated Gliding-Associated Protein GAP45 that recruits the motor complex composed of myosin A and two associated Myosin Light Chains (TgMLC1 and TgELC1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP50 and GAP40. To determine if the previously mapped phosphorylation sites on TgGAP45 and TgMLC1 have a direct significance for glideosome assembly and function, a series of phospho-mimetic and phospho-null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC1 with phospho-mutants impacted on glideosome assembly and parasite motility. TgGAP45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP45 inducible knockout background. The loss of interaction with TgGAP50 by one previously reported GAP45-mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC1 and TgGAP45, this posttranslational modification does not appear to be critical for the assembly and function of the glideosome.
This article is protected by copyright. All rights reserved.
PMID:
 
24779470
 
[PubMed - as supplied by publisher]