Toxoplasma gondii Requires Glycogen Phosphorylase for Balancing Amylopectin Storage and for Efficient Production of Brain Cysts

MBio. 2017 Aug 29;8(4). pii: e01289-17. doi: 10.1128/mBio.01289-17.

Sugi T¹, Tu V¹, Ma Y¹, Tomita T¹, Weiss LM^2,3.

In immunocompromised hosts, latent infection with Toxoplasma gondii can reactivate from tissue cysts, leading to encephalitis. A characteristic of T. gondii bradyzoites in tissue cysts is the presence of amylopectin granules. The regulatory mechanisms and role of amylopectin accumulation in this organism are not fully understood. The T. gondii genome encodes a putative glycogen phosphorylase (TgGP), and mutants were constructed to manipulate the activity of TgGP and to evaluate the function of TgGP in amylopectin storage. Both a stop codon mutant (Pru/TgGP^S25stop [expressing a Ser-to-stop codon change at position 25 in TgGP]) and a phosphorylation null mutant (Pru/TgGP^S25A [expressing a Ser-to-Ala change at position 25 in TgGp]) mutated at Ser25 displayed amylopectin accumulation, while the phosphorylation-mimetic mutant (Pru/TgGP^S25E [expressing a Ser-to-Glu change at position 25 in TgGp]) had minimal amylopectin accumulation under both tachyzoite and bradyzoite growth conditions. The expression of active TgGP^S25S or TgGP^S25E restored amylopectin catabolism in Pru/TgGP^S25A To understand the relation between GP and calcium-dependent protein kinase 2 (CDPK2), which was recently reported to regulate amylopectin consumption, we knocked out CDPK2 in these mutants. PruΔcdpk2/TgGP^S25E had minimal amylopectin accumulation, whereas the Δcdpk2 phenotype in the other GP mutants and parental lines displayed amylopectin accumulation. Both the inactive S25A and hyperactive S25E mutant produced brain cysts in infected mice, but the numbers of cysts produced were significantly less than the number produced by the S25S wild-type GP parasite. Complementation that restored amylopectin regulation restored brain cyst production to the control levels seen in infected mice. These data suggest that T. gondii requires tight regulation of amylopectin expression for efficient production of cysts and persistent infections and that GP phosphorylation is a regulatory mechanism involved in amylopectin storage and utilization.


IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that causes disease in immune-suppressed individuals, as well as a fetopathy in pregnant women who acquire infection for the first time during pregnancy. This parasite can differentiate between tachyzoites (seen in acute infection) and bradyzoites (seen in latent infection), and this differentiation is associated with disease relapse. A characteristic of bradyzoites is that they contain cytoplasmic amylopectin granules. The regulatory mechanisms and the roles of amylopectin granules during latent infection remain to be elucidated. We have identified a role of T. gondii glycogen phosphorylase (TgGP) in the regulation of starch digestion and a role of posttranslational modification of TgGP, i.e., phosphorylation of Ser25, in the regulation of amylopectin digestion. By manipulating TgGP activity in the parasite with genome editing, we found that the digestion and storage of amylopectin due to TgGP activity are both important for latency in the brain.

Copyright © 2017 Sugi et al.

KEYWORDS:

Toxoplasma gondii; amylopectin; bradyzoite; energy utilization; glycogen metabolism; glycogen phosphorylase; latency; metabolism; posttranslational modification; protein phosphorylation

PMID:

28851850

DOI:

10.1128/mBio.01289-17

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

RNA. 2017 Aug 29. pii: rna.062794.117. doi: 10.1261/rna.062794.117. [Epub ahead of print]

Nakamoto MA¹, Lovejoy AF², Cygan AM¹, Boothroyd JC³.

RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψs in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψs being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many of the Ψs in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψs are enriched in developmentally regulated transcripts. RNA-Seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψs play an important biological role.

Published by Cold Spring Harbor Laboratory Press for the RNA Society.

KEYWORDS:

Pseudouridine; RNA Modification; Toxoplasma gondii

PMID:

28851751

DOI:

10.1261/rna.062794.117

Efficient invasion by Toxoplasma depends on the subversion of host protein networks

Nat Microbiol. 2017 Aug 28. doi: 10.1038/s41564-017-0018-1. [Epub ahead of print]

Guérin A¹, Corrales RM¹, Parker ML², Lamarque MH¹, Jacot D³, El Hajj H⁴, Soldati-Favre D³, Boulanger MJ², Lebrun M⁵.

Apicomplexan parasites are important pathogens of humans and domestic animals, including Plasmodium species (the agents of malaria) and Toxoplasma gondii, which is responsible for toxoplasmosis. They replicate within the cells of their animal hosts, to which they gain access using a unique parasite-driven invasion process. At the core of the invasion machine is a structure at the interface between the invading parasite and host cell called the moving junction (MJ) ¹ . The MJ serves as both a molecular doorway to the host cell and an anchor point enabling the parasite to engage its motility machinery to drive the penetration of the host cell ² , ultimately yielding a protective vacuole ³ . The MJ is established through self-assembly of parasite proteins at the parasite-host interface ⁴ . However, it is unknown whether host proteins are subverted for MJ formation. Here, we show that Toxoplasma parasite rhoptry neck proteins (RON2, RON4 and RON5) cooperate to actively recruit the host CIN85, CD2AP and the ESCRT-I components ALIX and TSG101 to the MJ during invasion. We map the interactions in detail and demonstrate that the parasite mimics and subverts conserved binding interfaces with remarkable specificity. Parasite mutants unable to recruit these host proteins show inefficient host cell invasion in culture and attenuated virulence in mice. This study reveals molecular mechanisms by which parasites subvert widely conserved host machinery to force highly efficient host cell access. Toxoplasma gondii uses its proteins RON2, RON4 and RON5 to recruit host proteins, including the ESCRT-I components ALIX and TSG101 to the moving junction, a multimolecular structure that enables invasion.

PMID:

28848228

DOI:

10.1038/s41564-017-0018-1

The Lymphotoxin β Receptor Is Essential for Upregulation of IFN-Induced Guanylate-Binding Proteins and Survival after Toxoplasma gondii Infection

Mediators Inflamm. 2017;2017:7375818. doi: 10.1155/2017/7375818. Epub 2017 Aug 6.

Behnke K^1,2, Sorg UR¹, Gabbert HE³, Pfeffer K¹.

Lymphotoxin β receptor (LTβR) signaling plays an important role in efficient initiation of host responses to a variety of pathogens, encompassing viruses, bacteria, and protozoans via induction of the type I interferon response. The present study reveals that after Toxoplasma gondii infection, LTβR^-/- mice show a substantially reduced survival rate when compared to wild-type mice. LTβR^-/- mice exhibit an increased parasite load and a more pronounced organ pathology. Also, a delayed increase of serum IL-12p40 and a failure of the protective IFNγ response in LTβR^-/- mice were observed. Serum NO levels in LTβR^-/- animals rose later and were markedly decreased compared to wild-type animals. At the transcriptional level, LTβR^-/- animals exhibited a deregulated expression profile of several cytokines known to play a role in activation of innate immunity in T. gondii infection. Importantly, expression of the IFNγ-regulated murine guanylate-binding protein (mGBP) genes was virtually absent in the lungs of LTβR^-/- mice. This demonstrates clearly that the LTβR is essential for the induction of a type II IFN-mediated immune response against T. gondii. The pronounced inability to effectively upregulate host defense effector molecules such as GBPs explains the high mortality rates of LTβR^-/- animals after T. gondii infection.

PMID:

28845089

PMCID:

PMC5563413

DOI:

10.1155/2017/7375818

The Toxoplasma Centrocone Houses Cell Cycle Regulatory Factors

MBio. 2017 Aug 22;8(4). pii: e00579-17. doi: 10.1128/mBio.00579-17.

Naumov A¹, Kratzer S¹, Ting LM², Kim K², Suvorova ES¹, White MW³.

Our knowledge of cell cycle regulatory mechanisms in apicomplexan parasites is very limited. In this study, we describe a novel Toxoplasma gondii factor that has a vital role in chromosome replication and the regulation of cytoplasmic and nuclear mitotic structures, and we named this factor ECR1 for essential for chromosome replication 1. ECR1 was discovered by complementation of a temperature-sensitive (ts) mutant that suffers lethal, uncontrolled chromosome replication at 40°C similar to a ts mutant carrying a defect in topoisomerase. ECR1 is a 52-kDa protein containing divergent RING and TRAF-Sina-like zinc binding domains that are dynamically expressed in the tachyzoite cell cycle. ECR1 first appears in the unique spindle compartment of the Apicomplexa (centrocone) of the nuclear envelope in early S phase and then in the nucleus in late S phase where it reaches maximum expression. Following nuclear division, but before daughter parasites separate from the mother parasite, ECR1 is downregulated and is absent in new daughter parasites. The proteomics of ECR1 identified interactions with the ubiquitin-mediated protein degradation machinery and the minichromosome maintenance complex, and the loss of ECR1 led to increased stability of a key member of this complex, MCM2. ECR1 also forms a stable complex with the cyclin-dependent kinase (CDK)-related kinase, Tgondii Crk5 (TgCrk5), which displays a similar cell cycle expression and localization during tachyzoite replication. Importantly, the localization of ECR1/TgCrk5 in the centrocone indicates that this Apicomplexa-specific spindle compartment houses important regulatory factors that control the parasite cell cycle.


IMPORTANCE Parasites of the apicomplexan family are important causes of human disease, including malaria, toxoplasmosis, and cryptosporidiosis. Parasite growth is the underlying cause of pathogenesis, yet despite this importance, the molecular basis for parasite replication is poorly understood. Filling this knowledge gap cannot be accomplished by mining recent whole-genome sequencing data because apicomplexan cell cycles differ substantially and lack many of the key regulatory factors of well-studied yeast and mammalian cell division models. We have utilized forward genetics to discover essential factors that regulate cell division in these parasites using the Toxoplasma gondii model. An example of this approach is described here with the discovery of a putative E3 ligase/protein kinase mechanism involved in regulating chromosome replication and mitotic processes of asexual stage parasites.

Copyright © 2017 Naumov et al.

KEYWORDS:

E3 ligase; Toxoplasma gondii; apicomplexan parasites; cell cycle; chromosome replication; cyclin-dependent kinases

PMID:

28830940

DOI:

10.1128/mBio.00579-17

Evaluation of gene expression levels for cytokines in ocular toxoplasmosis

Parasite Immunol. 2017 Aug 24. doi: 10.1111/pim.12462. [Epub ahead of print]

Maia MM¹, Meira-Strejevitch CS¹, Pereira-Chioccola VL¹, de Hippólito DDC¹, Silva VO¹, Brandão de Mattos CC², Frederico FB³, Siqueira RC², de Mattos LC²; FAMERP and IAL Toxoplasma Research Group.

This study evaluated levels for mRNA expression of 7 cytokines in ocular toxoplasmosis. Peripheral blood mononuclear cells (PBMC) of ocular toxoplasmosis patients (OT Group, n= 23) and chronic toxoplasmosis individuals (CHR Group, n= 9) were isolated and stimulated in vitro with T. gondii antigen. Negative controls (NC) were constituted of 7 PBMC samples from individuals seronegative for toxoplasmosis. mRNA expression for cytokines was determined by qPCR. Results showed a significant increase of mRNA levels from antigen stimulated PBMCs derived from OT Group for expressing IL-6 (at p0.005 and p  0.0005 for CHR and NC groups, respectively), IL-10 (at p0.0005 and p  0.005 for CHR and NC groups, respectively) and TGF-β (at p  0.005) for NC group. mRNA levels for TNF-α and IL-12 were also up-regulated in OT patients compared to CHR and NC individuals, although without statistical significance. Additionally, mRNA levels for IL-27 and IFN-γ in PBMC of OT patients were up-regulated in comparison with NC individuals. Differences between OT and NC groups were statistically significant at p0.05 and p  0.0005, respectively. This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

KEYWORDS:

PBMC ; Cytokines; Gene expression; Ocular toxoplasmosis

PMID:

28836673

DOI:

10.1111/pim.12462

Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens

Elife. 2017 Aug 18;6. pii: e29865. doi: 10.7554/eLife.29865. [Epub ahead of print]

Amberg-Johnson K¹, Hari SB², Ganesan SM³, Lorenzi HA⁴, Sauer RT², Niles JC³, Yeh E¹.

The malaria parasite Plasmodium falciparum and related apicomplexan pathogens contain an essential plastid organelle, the apicoplast, which is a key anti-parasitic target. Derived from secondary endosymbiosis, the apicoplast depends on novel, but largely cryptic, mechanisms for protein/lipid import and organelle inheritance during parasite replication. These critical biogenesis pathways present untapped opportunities to discover new parasite-specific drug targets. We used an innovative screen to identify actinonin as having a novel mechanism-of-action inhibiting apicoplast biogenesis. Resistant mutation, chemical-genetic interaction, and biochemical inhibition demonstrate that the unexpected target of actinonin in P. falciparum and Toxoplasma gondii is FtsH1, a homolog of a bacterial membrane AAA+ metalloprotease. PfFtsH1 is the first novel factor required for apicoplast biogenesis identified in a phenotypic screen. Our findings demonstrate that FtsH1 is a novel and, importantly, druggable antimalarial target. Development of FtsH1 inhibitors will have significant advantages with improved drug kinetics and multistage efficacy against multiple human parasites.

KEYWORDS:

P. falciparum; biochemistry; infectious disease; microbiology

PMID:

28826494

DOI:

10.7554/eLife.29865

Dissecting Amyloid Beta Deposition Using Distinct Strains of the Neurotropic Parasite Toxoplasma gondii as a Novel Tool

ASN Neuro. 2017 Jul-Aug;9(4):1759091417724915. doi: 10.1177/1759091417724915.

Cabral CM1, McGovern KE1, MacDonald WR2, Franco J1, Koshy AA1,3,4.

Author information

Abstract

Genetic and pathologic data suggest that amyloid beta (Aβ), produced by processing of the amyloid precursor protein, is a major initiator of Alzheimer's disease (AD). To gain new insights into Aβ modulation, we sought to harness the power of the coevolution between the neurotropic parasite Toxoplasma gondii and the mammalian brain. Two prior studies attributed Toxoplasma-associated protection against Aβ to increases in anti-inflammatory cytokines (TGF-β and IL-10) and infiltrating phagocytic monocytes. These studies only used one Toxoplasma strain making it difficult to determine if the noted changes were associated with Aβ protection or simply infection. To address this limitation, we infected a third human amyloid precursor protein AD mouse model (J20) with each of the genetically distinct, canonical strains of Toxoplasma (Type I, Type II, or Type III). We then evaluated the central nervous system (CNS) for Aβ deposition, immune cell responses, global cytokine environment, and parasite burden. We found that only Type II infection was protective against Aβ deposition despite both Type II and Type III strains establishing a chronic CNS infection and inflammatory response. Compared with uninfected and Type I-infected mice, both Type II- and Type III-infected mice showed increased numbers of CNS T cells and microglia and elevated pro-inflammatory cytokines, but neither group showed a >2-fold elevation of TGF-β or IL-10. These data suggest that we can now use our identification of protective (Type II) and nonprotective (Type III) Toxoplasma strains to determine what parasite and host factors are linked to decreased Aβ burden rather than simply with infection.

KEYWORDS: 

Alzheimer’s disease; Toxoplasma; amyloid beta; neurodegeneration; neuroprotection

PMID:

 

28817954

 

DOI:

 

10.1177/1759091417724915

Concordance of Toxoplasma gondii Infection Markers Among Heterosexual Couples

J Clin Med Res. 2017 Sep;9(9):788-792. doi: 10.14740/jocmr3117w. Epub 2017 Jul 27.

Alvarado-Esquivel C1, Pacheco-Vega SJ2, Hernandez-Tinoco J3, Berumen-Segovia LO1, Sanchez-Anguiano LF3, Estrada-Martinez S3, Sandoval-Carrillo AA3, Salas-Pacheco JM3, Antuna-Salcido EI3.

Author information

Abstract

BACKGROUND: 

Very little is known about concordance of Toxoplasma gondii (T. gondii) infection markers among couples. Through a cross-sectional study, we sought to determine the correlation of T. gondii infection in a sample of 119 heterosexual couples in Durango State, Mexico.

METHODS: 

Participants were examined for the presence of anti-T. gondii IgG and IgM antibodies using enzyme-linked immunoassays. IgG and IgM seropositive couples were further analyzed for the presence of T. gondii DNA by using polymerase chain reaction (PCR).

RESULTS: 

Anti-T. gondii IgG antibodies were found in 71 (59.7%) men and in 63 (52.9%) women (odds ratio (OR) = 1.31; 95% confidence interval (CI): 0.78 - 2.19; P = 0.29). Of the 71 seropositive men, 40 (56.3%) had a seropositive couple; in contrast, of the 63 seropositive women, 40 (63.5%) had a seropositive couple (OR = 0.74; 95% CI: 0.37 - 1.48; P = 0.39). In total, 65 (54.6%) couples had concordant results (both IgG positive or both IgG negative), and 54 (45.4%) had discordant results (a seropositive man with a seronegative woman, or a seropositive woman with a seronegative man) (kappa index = 0.08; 95% CI: -0.09 - 0.26). With respect to high (> 150 IU/mL) levels of anti-T. gondii IgG antibodies, 79 (66.4%) of the 119 couples had concordant results and 40 (33.6%) had discordant results (kappa index = -0.15; 95% CI: -0.03 - 0.33). Forty couples were positive for anti-T. gondii IgM antibodies. Of them, 21 (52.5%) had concordant results, and 19 (47.5%) had discordant results (kappa index = -0.01; 95% CI: -0.39 - 0.28). Concerning PCR, eight (50%) of 16 couples were positive for T. gondii DNA. Of them, 11 (68.8%) had concordant results, and five (31.2%) had discordant results (kappa index = 0.31; 95% CI: -0.17 - 0.79).

CONCLUSIONS: 

Results suggest a poor concordance of serological and molecular markers of T. gondii infection among heterosexual couples. Further studies to confirm our results should be conducted.

KEYWORDS: 

Concordance; Couples; Epidemiology; Infection markers; Mexico; Seroprevalence; Toxoplasma gondii

PMID:

 

28811857

 

PMCID:

 

PMC5544485

 

DOI:

 

10.14740/jocmr3117w

Cryo-EM structures of the 80S ribosomes from human parasites Trichomonas vaginalis and Toxoplasma gondii

Cell Res. 2017 Aug 15. doi: 10.1038/cr.2017.104. [Epub ahead of print]

Li Z1,2, Guo Q1, Zheng L3, Ji Y4, Xie YT5, Lai DH5, Lun ZR5, Suo X6, Gao N1,3.

Author information

Abstract

As an indispensable molecular machine universal in all living organisms, the ribosome has been selected by evolution to be the natural target of many antibiotics and small-molecule inhibitors. High-resolution structures of pathogen ribosomes are crucial for understanding the general and unique aspects of translation control in disease-causing microbes. With cryo-electron microscopy technique, we have determined structures of the cytosolic ribosomes from two human parasites, Trichomonas vaginalis and Toxoplasma gondii, at resolution of 3.2-3.4 Å. Although the ribosomal proteins from both pathogens are typical members of eukaryotic families, with a co-evolution pattern between certain species-specific insertions/extensions and neighboring ribosomal RNA (rRNA) expansion segments, the sizes of their rRNAs are sharply different. Very interestingly, rRNAs of T. vaginalis are in size comparable to prokaryotic counterparts, with nearly all the eukaryote-specific rRNA expansion segments missing. These structures facilitate the dissection of evolution path for ribosomal proteins and RNAs, and may aid in design of novel translation inhibitors.Cell Research advance online publication 15 August 2017; doi:10.1038/cr.2017.104.

PMID:

 

28809395

 

DOI:

 

10.1038/cr.2017.104

Targeted overexpression of cyclic AMP-dependent protein kinase subunit in Toxoplasma gondii promotes replication and virulence in host cells

Vet Parasitol. 2017 Aug 30;243:248-255. doi: 10.1016/j.vetpar.2017.06.002. Epub 2017 Jun 3.

Sun H1, Wang S2, Zhao X3, Yao C4, Zhuang H1, Huang Y1, Chen X1, Yang Y1, Du A5.

Author information

Abstract

Toxoplasma gondii (T. gondii) is one of the most common parasite that can infect almost any warm-blooded animals including humans. The cyclic nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii. Here, T.gondii PKAC (TgPKAC) overexpression strain (TgPKAC-OE) was constructed. The growth of the TgPKAC-OE, RH△Ku80, and TgPKAC inhibition strains (TgPKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of TgPKAC in Vero cells by laser scanning microscope. We found that TgPKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro, and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in TgPKAC-H89 parasite compared to RH△Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in TgPKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that TgPKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we hypothesized that inhibition of TgPKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. TgPKAC plays an important role in parasite virulence in vivo, and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of TgPKAC function and help screen drug targets of T. gondii.

Copyright © 2017. Published by Elsevier B.V.

KEYWORDS: 

Autophagy; Growth; Protein kinase catalytic subunit; Subcellular localization; Toxoplasma gondii; Virulence

PMID:

 

28807302

 

DOI:

 

10.1016/j.vetpar.2017.06.002

Azithromycin treatment is able to control the infection by two genotypes of Toxoplasma gondii in human trophoblast BeWo cells

Exp Parasitol. 2017 Aug 10. pii: S0014-4894(17)30006-1. doi: 10.1016/j.exppara.2017.08.004. [Epub ahead of print]

Ribeiro M¹, Franco PS¹, Lopes-Maria JB¹, Angeloni MB¹, de Freitas Barbosa B¹, de Oliveira Gomes A², Castro AS¹, da Silva RJ¹, de Oliveira FC¹, Balga Milian IC¹, Martins-Filho OA³, Ietta F⁴, Mineo JR⁵, Ferro EAV⁶.

Trophoblast infection by Toxoplasma gondii plays a pivotal role in the vertical transmission of toxoplasmosis. Here, we investigate whether the antibiotic therapy with azithromycin, spiramycin and sulfadiazine/pyrimethamine are effective to control trophoblast infection by two Brazilian T. gondii genotypes, TgChBrUD1 or TgChBrUD2. Two antibiotic protocols were evaluated, as follow: i) pre-treatment of T. gondii-tachyzoites with selected antibiotics prior trophoblast infection and ii) post-treatment of infected trophoblasts. The infection index/replication and the impact of the antibiotic therapy on the cytokine milieu were characterized. It was observed that TgChBrUD2 infection induced lower infection index/replication as compared to TgChBrUD1. Regardless the therapeutic protocol, azithromycin was more effective to control the trophoblast infection with both genotypes when compared to conventional antibiotics. Azithromycin induced higher IL-12 production in TgChBrUD1-infected cells that may synergize the anti-parasitic effect. In contrast, the effectiveness of azithromycin to control the TgChBrUD2-infection was not associated with the IL-12 production. BeWo-trophoblasts display distinct susceptibility to T. gondii genotypes and the azithromycin treatment showed to be more effective than conventional antibiotics to control the T. gondii infection/replication regardless the parasite genotype.

Copyright © 2017. Published by Elsevier Inc.

KEYWORDS:

Azithromycin; Cytokines; Toxoplasma gondii atypical strains; Trophoblast cell

PMID:

28803905

DOI:

10.1016/j.exppara.2017.08.004

Toxoplasma gondii and Neospora caninum induce different host cell responses at proteome-wide phosphorylation events

Parasitol Res. 2017 Aug 12. doi: 10.1007/s00436-017-5579-7. [Epub ahead of print]

Al-Bajalan MMM^1,2, Xia D^3,4, Armstrong S³, Randle N³, Wastling JM^3,5.

Toxoplasma gondii and Neospora caninum are closely related intracellular protozoan parasites and tissue cyst-forming Coccidia of the phylum Apicomplexa. There are remarkable similarities between the morphology, genomes and transcriptomes of both parasites. Toxoplasma is zoonotic, with a wide host range and is mainly transmitted horizontally between its definitive host, the cat, and its intermediate hosts. Neospora causes disease within a narrow host range and with reduced virulence potential to the hosts. The dog is the definitive host of Neospora and its epidemiology in cattle mainly depends on vertical transmission. What causes these biological differences is not well understood. Since these parasites secrete an array of secretory proteins, including kinases, during infection to manipulate host cell responses. Host-parasite interactions due to phosphorylation of host cell proteins by T. gondii kinases enhance virulence and maintenance of infection. In this study, proteome-wide phosphorylation events of host cell proteins were investigated in response to infection with T. gondii and N. caninum using phosphoproteomic analyses, followed by pathway analysis on host signalling pathways. A few interesting differences in host responses at both the qualitative and quantitative levels were identified between the two infections; about one third of the phosphoproteomes, approximately 21% of the phospho-motifs and several pathways such as glycolysis/gluconeogenesis and mTOR pathways of the host cell were found differentially enriched between infection with these parasites. Identifying the differences in host-parasite interactions represents a promising step forward for uncovering the biological dissimilarities between both parasites.

KEYWORDS:

Biological differences; Neospora caninum; Phosphopeptides enrichment; Proteome-wide phosphorylation; Tandem mass spectrometry; Toxoplasma gondii

PMID:

28803361

DOI:

10.1007/s00436-017-5579-7

Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions

Sci Rep. 2017 Aug 3;7(1):7229. doi: 10.1038/s41598-017-07838-w.

Swierzy IJ1, Händel U2, Kaever A3, Jarek M4, Scharfe M4, Schlüter D2,5, Lüder CGK6.

Author information

Abstract

The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.

PMID:

 

28775382

 

DOI:

 

10.1038/s41598-017-07838-w

Activity of rosuvastatin in tachyzoites of Toxoplasma gondii (RH strain) in HeLa cells

Exp Parasitol. 2017 Jul 31. pii: S0014-4894(17)30166-2. doi: 10.1016/j.exppara.2017.07.009. [Epub ahead of print]

Sanfelice RA1, Machado LF1, Bosqui LR1, Miranda-Sapla MM1, Tomiotto-Pellissier F1, de Alcântara Dalevedo G1, Ioris D1, Reis GF2, Panagio LA2, Navarro IT3, Bordignon J4, Conchon-Costa I1, Pavanelli WR1, Almeida RS2, Costa IN5.

Author information

Abstract

Due to the toxicity of conventional medication in toxoplasmosis, some drugs are being studied for treating this infection, such as statins, especially rosuvastatin compound, which is efficient in inhibiting the initial isoprenoid biosynthesis processes in humans and the parasite. The goal of this studywas to assess the activity of rosuvastatin in HeLa cells infectedwiththe RH strain of T. gondii. In the experiment, HeLa cells (1 × 105) were infected with tachyzoites of T. gondii (5 × 105). After the experimental infection, we assessed the number of infected cells and the amount of intracellular tachyzoites. In addition, culture supernatants were collected to determine the amount of cytokines by cytometric bead array. We observed that there was no cytotoxicity in the concentrations tested in this cell line. The effect of rosuvastatin showed a significant reduction in both the number of infected cells and the proliferation index of the intracellular parasite, when compared with the conventional treatment combining sulfadiazine and pyrimethamine for toxoplasmosis. There were also reduced levels of cytokines IL-6 and IL-17. Therefore, it was concluded that rosuvastatin exhibited antiproliferative activity. The data presented are significantto promote further studiesandthe search for alternative treatment for toxoplasmosis.

Copyright © 2017. Published by Elsevier Inc.

KEYWORDS: 

Rosuvastatin; Statins; Toxoplasmosis

PMID:

 

28774497

 

DOI:

 

10.1016/j.exppara.2017.07.009