Friday, December 09, 2016

Genetic Evidence for Cytochrome b Qi Site Inhibition by 4(1H)-quinolone-3-diarylethers and Antimycin in Toxoplasma gondii

2016 Dec 5. pii: AAC.01866-16. [Epub ahead of print]


Toxoplasma gondii is an apicomplexan parasite that causes fatal and debilitating brain and eye disease. Endochin-like-quinolones (ELQs) are preclinical compounds that are efficacious against apicomplexan-caused diseases including toxoplasmosis, malaria and babesiosis. Of the ELQs, ELQ-316 has demonstrated the greatest efficacy against acute and chronic experimental toxoplasmosis. Although genetic analyses in other organisms have highlighted the importance of the cytochrome bc1 complex Qi site for ELQ sensitivity, the mechanism of action of ELQs against T. gondii and the mechanism of ELQ-316 remains unknown. Here we describe the selection and genetic characterization of T. gondii clones resistant to ELQ-316. A T. gondii strain selected under ELQ-316 drug pressure was found to possess a Thr222-Pro amino acid substitution that confers 49-fold resistance to ELQ-316 and 19-fold resistance to antimycin, a well-characterized Qi site inhibitor. These findings provide further evidence for ELQ Qi site inhibition in T. gondii and greater insight into the interaction of Qi site inhibitors with the apicomplexan cytochrome bc1 complex.
PMID:
27919897
DOI:
10.1128/AAC.01866-16
[PubMed - as supplied by publisher]

Wednesday, November 30, 2016

Regulatory T Cells Promote Myositis and Muscle Damage in Toxoplasma gondii Infection

2016 Nov 28. pii: 1600914. [Epub ahead of print]


The coordination of macrophage polarization is essential for the robust regenerative potential of skeletal muscle. Repair begins with a phase mediated by inflammatory monocytes (IM) and proinflammatory macrophages (M1), followed by polarization to a proregenerative macrophage (M2) phenotype. Recently, regulatory T cells (Tregs) were described as necessary for this M1 to M2 transition. We report that chronic infection with the protozoan parasite Toxoplasma gondii causes a nonresolving Th1 myositis with prolonged tissue damage associated with persistent M1 accumulation. Surprisingly, Treg ablation during chronic infection rescues macrophage homeostasis and skeletal muscle fiber regeneration, showing that Tregs can directly contribute to muscle damage. This study provides evidence that the tissue environment established by the parasite could lead to a paradoxical pathogenic role for Tregs. As such, these findings should be considered when tailoring therapies directed at Tregs in inflammatory settings.
PMID:
27895180
DOI:
10.4049/jimmunol.1600914

Activation of chronic toxoplasmosis by transportation stress in a mouse model

2016 Nov 24. doi: 10.18632/oncotarget.13568. [Epub ahead of print]


Toxoplasma gondii is an obligate intracellular parasite infecting 25% of the world population and enormous number of animals. It can exist in two forms in intermediate hosts: the fast replicating tachyzoites responsible for acute infection and the slowly replicating bradyzoites responsible for life-long chronic infection. The interconversion between tachyzoites and bradyzoites plays critical roles in the transmission and pathogenesis of T. gondii. However, the molecular mechanisms that govern the interconversion are largely unknown. In this study, we established a chronic infection model in mice and examined the impact of transportation stress on the status of chronic infection. Our results demonstrated that, treating chronically infected mice with conditions mimicking transportation stress reduced the levels of several key cytokines that restrict the infection at chronic stage. Increased expression of the tachyzoite specific gene SAG1 (surface antigen 1) was detected in brain cysts of stress treated mice, indicating activation and conversion of bradyzoites to tachyzoites. Using this model, we identified fifteen toxoplasmic proteins that had significant abundance changes during stress induced cysts reactivation. These proteins serve as a basis for further investigation of the mechanisms governing bradyzoite conversion.

KEYWORDS:

Toxoplasma gondii; bradyzoite; chronic infection; reactivation; transportation stress
PMID:
27895319
DOI:
10.18632/oncotarget.13568
[PubMed - as supplied by publisher]

In Vitro and In Vivo Activity of Sulfur-Containing Linear Bisphosphonates against Apicomplexan Parasites

2016 Nov 28. pii: AAC.01590-16. [Epub ahead of print]


We tested a series of sulfur-containing linear bisphosphonates against Toxoplasma gondii, the etiologic agent of toxoplasmosis. The most potent compound ( 22: , 1-[(n-decylsulfonyl)ethyl]-1,1-biphosphonic acid) is a sulfone-containing compound, which had an EC50 of 0.11 ± 0.02 μM against intracellular tachyzoites. The compound showed low toxicity when tested in tissue culture with a selectivity index of >2,000. 22: also showed high activity in vivo in a toxoplasmosis mouse model. The compound inhibited the Toxoplasma farnesyl diphosphate synthase (TgFPPS) but the concentration needed to inhibit 50% of the enzymatic activity (IC50) was higher than the concentration that inhibited 50% of growth. We tested 22: against two other Apicomplexan parasites, Plasmodium falciparum (EC50 of 0.6 ± 0.01 μM), the agent of malaria, and Cryptosporidium parvum (EC50 of ∼65 μM), the agent of cryptosporidiosis. Our results suggest that 22: is an excellent novel compound that could lead to the development of potent agents against Apicomplexan parasites.
PMID:
27895021
DOI:
10.1128/AAC.01590-16
[PubMed - as supplied by publisher]

Molecular evidence for Toxoplasma gondii in feeding and questing Ixodes ricinus ticks

2016 Nov 21. pii: S1877-959X(16)30228-X. doi: 10.1016/j.ttbdis.2016.11.009. [Epub ahead of print]

The aim of the present study was to detect Toxoplasma gondii in ticks collected from ponies and field vegetation and to determine the role of Shetland ponies as a potential reservoir host for T. gondii. A total of 1737 feeding Ixodes ricinus collected from 49 horses and 371 questing ticks were tested by PCR and sequencing for the presence and genotyping of T. gondii. All ticks were examined in a previous study to detect and identify pathogenic bacterial species. The aim of this study was also to detect co-infection of ticks with these bacteria and T. gondii. Genotyping of the sequenced B1 gene revealed that detected T. gondii strains represented genotype I, which is pathogenic for humans. T. gondii genotype I was detected in 4.5% of all I. ricinus, including in 2.99% of feeding ticks and in 10.24% of questing ticks; this difference was statistically significant. Thus, the above results indicate that ponies probably are not an essential host for the detected sporozoan. Infections with more than one pathogenic species were rare and involved mostly T. gondii and Borrelia burgdorferi sensu lato. Our results confirmed the presence of T. gondii in I. ricinus and showed a new geographical habitat of T. gondii occurring in I. ricinus ticks in Poland.

KEYWORDS:

Feeding and questing Ixodes ricinus; PCR; Sequencing; Toxoplasma gondii
PMID:
27894863
DOI:
10.1016/j.ttbdis.2016.11.009

Monday, November 28, 2016

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist

 2016 Nov 14. pii: S0969-2126(16)30338-0. doi: 10.1016/j.str.2016.10.011. [Epub ahead of print]

Abstract

The causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.

KEYWORDS:

GRA24; MAPK p38; Toxoplasma effector; immunity; intrinsically disordered protein
PMID:
 
27889209
 
DOI:
 
10.1016/j.str.2016.10.011

Toxoplasma gondii in stranded marine mammals from the North Sea and Eastern Atlantic Ocean: Findings and diagnostic difficulties

 2016 Oct 30;230:25-32. doi: 10.1016/j.vetpar.2016.10.021. Epub 2016 Oct 24.

Abstract

The occurrence of the zoonotic protozoan parasite Toxoplasma gondii in marine mammals remains a poorly understood phenomenon. In this study, samples from 589 marine mammal species and 34 European otters (Lutra lutra), stranded on the coasts of Scotland, Belgium, France, The Netherlands and Germany, were tested for the presence of T. gondii. Brain samples were analysed by polymerase chain reaction (PCR) for detection of parasite DNA. Blood and muscle fluid samples were tested for specific antibodies using a modified agglutination test (MAT), a commercial multi-species enzyme-linked immunosorbent assay (ELISA) and an immunofluorescence assay (IFA). Out of 193 animals tested by PCR, only two harbour porpoise (Phocoena phocoena) cerebrum samples, obtained from animals stranded on the Dutch coast, tested positive. The serological results showed a wide variation depending on the test used. Using a cut-off value of 1/40 dilution in MAT, 141 out of 292 animals (41%) were positive. Using IFA, 30 out of 244 tested samples (12%) were positive at a 1/50 dilution. The commercial ELISA yielded 7% positives with a cut-off of the sample-to-positive (S/P) ratio≥50; and 12% when the cut-off was set at S/P ratio≥20. The high number of positives in MAT may be an overestimation due to the high degree of haemolysis of the samples and/or the presence of lipids. The ELISA results could be an underestimation due to the use of a multispecies conjugate. Our results confirm the presence of T. gondii in marine mammals in The Netherlands and show exposure to the parasite in both the North Sea and the Eastern Atlantic Ocean. We also highlight the limitations of the tests used to diagnose T. gondii in stranded marine mammals.

KEYWORDS:

ELISA; IFA; MAT; Marine mammals; PCR; Seroprevalence; Toxoplasma gondii
PMID:
 
27884438
 
DOI:
 
10.1016/j.vetpar.2016.10.021

Relationship between Toxoplasma gondii Seropositivity and Acoustic Startle Response in an Inner-City Population

 2016 Nov 21. pii: S0889-1591(16)30523-2. doi: 10.1016/j.bbi.2016.11.021. [Epub ahead of print]

Abstract

Toxoplasma gondii (TOXO) is a neuroinvasive protozoan parasite that induces the formation of persistent cysts in mammalian brains. It infects approximately 1.1 million people in the United States annually. Latent TOXO infection is implicated in the etiology of psychiatric disorders, especially schizophrenia (Scz), and has been correlated with modestly impaired cognition. The acoustic startle response (ASR) is a reflex seen in all mammals. It is mediated by a simple subcortical circuit, and provides an indicator of neural function. We previously reported the association of TOXO with slowed acoustic startle latency, an index of neural processing speed, in a sample of schizophrenia and healthy control subjects. The alterations in neurobiology with TOXO latent infection may not be specific to schizophrenia. Therefore we examined TOXO in relation to acoustic startle in an urban, predominately African American, population with mixed psychiatric diagnoses, and healthy controls. Physiological and diagnostic data along with blood samples were collected from 364 outpatients treated at an inner-city hospital. TOXO status was determined with an ELISA assay for TOXO-specific IgG. A discrete titer was calculated based on standard cut-points as an indicator of seropositivity, and the TOXO-specific IgG concentration served as serointensity. A series of linear regression models were used to assess the association of TOXO seropositivity and serointensity with ASR magnitude and latency in models adjusting for demographics and psychiatric diagnoses (PTSD, major depression, schizophrenia, psychosis, substance abuse). ASR magnitude was 11.5% higher in TOXO seropositive subjects compared to seronegative individuals (p=0.01). This effect was more pronounced in models with TOXO serointensity that adjusted for sociodemographic covariates (F=7.41, p=.0068; F=10.05, p=0.0017), and remained significant when psychiatric diagnoses were stepped into the models. TOXO showed no association with startle latency (t=0.49, p=0.63) in an unadjusted model, nor was TOXO associated with latency in models that included demographic factors. After stepping in individual psychiatric disorders, we found a significant association of latency with a diagnosis of PTSD (F=5.15, p=0.024), but no other psychiatric diagnoses, such that subjects with PTSD had longer startle latency. The mechanism by which TOXO infection is associated with high startle magnitude is not known, but possible mechanisms include TOXO cyst burden in the brain, parasite recrudescence, or molecular mimicry of a host epitope by TOXO. Future studies will focus on the neurobiology underlying the effects of latent TOXO infection as a potential inroad to the development of novel treatment targets for psychiatric disease.

KEYWORDS:

Acoustic Startle Response; Infection; Mental Health; PTSD; Schizophrenia; Substance Use Disorder; Toxoplasma gondii
PMID:
 
27884623
 
DOI:
 
10.1016/j.bbi.2016.11.021

Activity of Fluorine-Containing Analogues of WC-9 and Structurally Related Analogues against Two Intracellular Parasites: Trypanosoma cruzi and Toxoplasma gondii

 2016 Nov 25. doi: 10.1002/cmdc.201600505. [Epub ahead of print]

Abstract

Two obligate intracellular parasites, Trypanosoma cruzi, the agent of Chagas disease, and Toxoplasma gondii, an agent of toxoplasmosis, upregulate the mevalonate pathway of their host cells upon infection, which suggests that this host pathway could be a potential drug target. In this work, a number of compounds structurally related to WC-9 (4-phenoxyphenoxyethyl thiocyanate), a known squalene synthase inhibitor, were designed, synthesized, and evaluated for their effect on T. cruzi and T. gondii growth in tissue culture cells. Two fluorine-containing derivatives, the 3-(3-fluorophenoxy)- and 3-(4-fluorophenoxy)phenoxyethyl thiocyanates, exhibited half-maximal effective concentration (EC50 ) values of 1.6 and 4.9 μm, respectively, against tachyzoites of T. gondii, whereas they showed similar potency to WC-9 against intracellular T. cruzi (EC50 values of 5.4 and 5.7 μm, respectively). In addition, 2-[3- (phenoxy)phenoxyethylthio]ethyl-1,1-bisphosphonate, which is a hybrid inhibitor containing 3-phenoxyphenoxy and bisphosphonate groups, has activity against T. gondii proliferation at sub-micromolar levels (EC50 =0.7 μm), which suggests a combined inhibitory effect of the two functional groups.

KEYWORDS:

antiprotozoal agents; drug discovery; enzymes; fluorine; inhibitors
PMID:
 
27886451
 
DOI:
 
10.1002/cmdc.201600505

Toxoplasma gondii immune mapped protein 1 is anchored to the inner leaflet of the plasma membrane and adopts a novel protein fold

 2016 Nov 22. pii: S1570-9639(16)30247-3. doi: 10.1016/j.bbapap.2016.11.010. [Epub ahead of print]

Abstract

The immune mapped protein 1 (IMP1) was first identified as a protective antigen in Eimeria maxima and described as vaccine candidate and invasion factor in Toxoplasma gondii. We show here that TgIMP1 localizes to the inner leaflet of plasma membrane (PM) via dual acylation. Mutations either in the N-terminal myristoylation or palmitoylation sites (G2 and C5) cause relocalization of TgIMP1 to the cytosol. The first 11 amino acids are sufficient for PM targeting and the presence of lysine (K7) is critical. Disruption of TgIMP1 gene by double homologous recombination revealed no invasion defect or any measurable alteration in the lytic cycle of tachyzoites. Following immunization with TgIMP1 DNA vaccine, mice challenged with either wild type or IMP1-ko parasites showed no significant difference in protection. The sequence analysis identified a structured C-terminal domain that is present in a broader family of IMP1-like proteins conserved across the members of Apicomplexa. We present the solution structure of this domain determined from NMR data and describe a new protein fold not seen before.

KEYWORDS:

Acylation; Invasion; NMR; Protein structure; Toxoplasma gondii; Vaccine
PMID:
 
27888074
 
DOI:
 
10.1016/j.bbapap.2016.11.010

Is there any association between Toxoplasma gondii infection and bipolar disorder? A systematic review and meta-analysis

 2016 Nov 17;209:59-65. doi: 10.1016/j.jad.2016.11.016. [Epub ahead of print]

Abstract

BACKGROUND:

The relationship between Toxoplasma gondii infection and the development of bipolar disorder (BD) has long been investigated, yet to date it is still poorly understood and documented. The aim of this review is to derive a summary estimate of the strength of the association between infection with T. gondii and BD from the available published studies.

METHODS:

A systematic review was performed using PubMed, LILACS, PsycINFO, and Embase databases. Studies which included a proportion of seropositive BD patients and controls were further examined in a meta-analysis.

RESULTS:

One hundred eighteen citations were initially retrieved. Thirteen studies were included in our systematic review. Eight out of these thirteen studies were included in our meta-analysis. Statistical analyses showed that T. gondii infection is associated with with BD (OR=1.26).

LIMITATIONS:

Small sample size was the major limitation among the studies that carried out serological analyses. In addition, the available studies did not have enough information on disease status/severity or type of bipolar disorder. Also, it was not possible to analyze pregnancy status or perinatal infection. Future studies addressing the aforementioned topics are clearly needed.

CONCLUSIONS:

Despite heterogeneous results, patients with BD are more likely to be infected by T. gondii than controls. Early T. gondii infection might predispose the development of BD. T.gondii infection is becoming clinically relevant in psychiatric disorders and future mechanistic studies are required to elucidate the underlying pathophysiological mechanisms.

KEYWORDS:

Bipolar disorder; Immunology; Inflammation; Toxoplasma gondii
PMID:
 
27889597
 
DOI:
 
10.1016/j.jad.2016.11.016

Thursday, November 24, 2016

K63-Linked Ubiquitination Targets Toxoplasma gondii for Endo-lysosomal Destruction in IFNγ-Stimulated Human Cells

 2016 Nov 22;12(11):e1006027. doi: 10.1371/journal.ppat.1006027. eCollection 2016.

Abstract

Toxoplasma gondii is the most common protozoan parasitic infection in man. Gamma interferon (IFNγ) activates haematopoietic and non-haematopoietic cells to kill the parasite and mediate host resistance. IFNγ-driven host resistance pathways and parasitic virulence factors are well described in mice, but a detailed understanding of pathways that kill Toxoplasma in human cells is lacking. Here we show, that contrary to the widely held belief that the Toxoplasma vacuole is non-fusogenic, in an immune-stimulated environment, the vacuole of type II Toxoplasma in human cells is able to fuse with the host endo-lysosomal machinery leading to parasite death by acidification. Similar to murine cells, we find that type II, but not type I Toxoplasma vacuoles are targeted by K63-linked ubiquitin in an IFNγ-dependent manner in non-haematopoetic primary-like human endothelial cells. Host defence proteins p62 and NDP52 are subsequently recruited to the type II vacuole in distinct, overlapping microdomains with a loss of IFNγ-dependent restriction in p62 knocked down cells. Autophagy proteins Atg16L1, GABARAP and LC3B are recruited to 10% of parasite vacuoles and show no parasite strain preference, which is consistent with inhibition and enhancement of autophagy showing no effect on parasite replication. We demonstrate that this differs from HeLa human epithelial cells, where type II Toxoplasma are restricted by non-canonical autophagy leading to growth stunting that is independent of lysosomal acidification. In contrast to mouse cells, human vacuoles do not break. In HUVEC, the ubiquitinated vacuoles are targeted for destruction in acidified LAMP1-positive endo-lysosomal compartments. Consequently, parasite death can be prevented by inhibiting host ubiquitination and endosomal acidification. Thus, K63-linked ubiquitin recognition leading to vacuolar endo-lysosomal fusion and acidification is an important, novel virulence-driven Toxoplasma human host defence pathway.
PMID:
 
27875583
 
DOI:
 
10.1371/journal.ppat.1006027

Tuesday, November 22, 2016

Ocular toxoplasmosis: susceptibility in respect to the genes encoding the KIR receptors and their HLA class I ligands

2016 Nov 9;6:36632. doi: 10.1038/srep36632.


The objective of this study was to investigate the influence of the genes encoding the KIR receptors and their HLA ligands in the susceptibility of ocular toxoplasmosis. A total of 297 patients serologically-diagnosed with toxoplasmosis were selected and stratified according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping was performed by PCR-SSOP. Statistical analyses were conducted using the Chi-square test, and odds ratio with a 95% confidence interval was also calculated to evaluate the risk association. The activating KIR3DS1 gene was associated with increased susceptibility for ocular toxoplasmosis. The activating KIR together with their HLA ligands (KIR3DS1-Bw4-80Ile and KIR2DS1+/C2++ KIR3DS1+/Bw4-80Ile+) were associated with increased susceptibility for ocular toxoplasmosis and its clinical manifestations. KIR-HLA inhibitory pairs -KIR2DL3/2DL3-C1/C1 and KIR2DL3/2DL3-C1- were associated with decreased susceptibility for ocular toxoplasmosis and its clinical forms, while the KIR3DS1-/KIR3DL1+/Bw4-80Ile+ combination was associated as a protective factor against the development of ocular toxoplasmosis and, in particular, against recurrent manifestations. Our data demonstrate that activating and inhibitory KIR genes may influence the development of ocular toxoplasmosis.
PMID:
27827450
PMCID:
PMC5101474
DOI:
10.1038/srep36632