scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals New Method for the Orthogonal Labeling and Purification of Toxoplasma gondii Proteins While Inside the Host Cell

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Gregory M. Wier ◽  
Erica M. McGreevy ◽  
Mark J. Brown ◽  
Jon P. Boyle
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Click Reaction ◽  
Severe Disease ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Content Type ◽  
Fluorescent Tags

ABSTRACTToxoplasma gondiiis an obligate intracellular protozoan parasite that is capable of causing severe disease in immunocompromised humans. How T. gondii is able to modulate the host cell to support itself is still poorly understood. Knowledge pertaining to the host-parasite interaction could be bolstered by developing a system to specifically label parasite proteins while the parasite grows inside the host cell. For this purpose, we have created a strain of T. gondii that expresses a mutant Escherichia coli methionyl-tRNA synthetase (MetRSNLL) that allows methionine tRNA to be loaded with the azide-containing methionine analog azidonorleucine (Anl). Anl-containing proteins are susceptible to a copper-catalyzed “click” reaction to attach affinity tags for purification or fluorescent tags for visualization. The MetRSNLL-Anl system labels nascent T. gondii proteins in an orthogonal fashion, labeling proteins only in MetRSNLL-expressing parasites. This system should be useful for nonradioactive pulse-chase studies and purification of nascently translated proteins. Although this approach allows labeling of a diverse array of parasite proteins, secreted parasite proteins appear to be only minimally labeled in MetRSNLL-expressing T. gondii. The minimal labeling of secreted proteins is likely a consequence of the selective charging of the initiator tRNA (and not the elongator methionine tRNA) by the heterologously expressed bacterial MetRS.IMPORTANCEStudying how T. gondii modifies the host cell to permit its survival is complicated by the complex protein environment of the host cell. The approach presented in this article provides the first method for specific labeling of T. gondii proteins while the parasite grows inside the host cell. We show that this approach is useful for pulse-chase labeling of parasite proteins duringin vitrogrowth. It should also be applicable duringin vivoinfections and in other apicomplexan parasites, including Plasmodium spp.

scholarly journals Transcriptional Profiling Suggests T Cells Cluster around Neurons Injected with Toxoplasma gondii Proteins

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Emily F. Merritt ◽  
Hannah J. Johnson ◽  
Zhee Sheen Wong ◽  
Adam S. Buntzman ◽  
Austin C. Conklin ◽  
...  
Keyword(s):  
T Cells ◽  
Toxoplasma Gondii ◽  
Immune Cells ◽  
Drug Targets ◽  
Transcriptional Profiling ◽  
Effector Proteins ◽  
Content Type ◽  
Symptomatic Disease

ABSTRACT Toxoplasma gondii’s tropism for and persistence in the central nervous system (CNS) underlies the symptomatic disease that T. gondii causes in humans. Our recent work has shown that neurons are the primary CNS cell with which Toxoplasma interacts and which it infects in vivo. This predilection for neurons suggests that T. gondii’s persistence in the CNS depends specifically upon parasite manipulation of the host neurons. Yet, most work on T. gondii-host cell interactions has been done in vitro and in nonneuronal cells. We address this gap by utilizing our T. gondii-Cre system that allows permanent marking and tracking of neurons injected with parasite effector proteins in vivo. Using laser capture microdissection (LCM) and RNA sequencing using RNA-seq, we isolated and transcriptionally profiled T. gondii-injected neurons (TINs), Bystander neurons (nearby non-T. gondii-injected neurons), and neurons from uninfected mice (controls). These profiles show that TIN transcriptomes significantly differ from the transcriptomes of Bystander and control neurons and that much of this difference is driven by increased levels of transcripts from immune cells, especially CD8+ T cells and monocytes. These data suggest that when we used LCM to isolate neurons from infected mice, we also picked up fragments of CD8+ T cells and monocytes clustering in extreme proximity around TINs and, to a lesser extent, Bystander neurons. In addition, we found that T. gondii transcripts were primarily found in the TIN transcriptome, not in the Bystander transcriptome. Collectively, these data suggest that, contrary to common perception, neurons that directly interact with or harbor parasites can be recognized by CD8+ T cells. IMPORTANCE Like other persistent intracellular pathogens, Toxoplasma gondii, a protozoan parasite, has evolved to evade the immune system and establish a chronic infection in specific cells and organs, including neurons in the CNS. Understanding T. gondii’s persistence in neurons holds the potential to identify novel, curative drug targets. The work presented here offers new insights into the neuron-T. gondii interaction in vivo. By transcriptionally profiling neurons manipulated by T. gondii, we unexpectedly revealed that immune cells, and specifically CD8+ T cells, appear to cluster around these neurons, suggesting that CD8+ T cells specifically recognize parasite-manipulated neurons. Such a possibility supports evidence from other labs that questions the long-standing dogma that neurons are often persistently infected because they are not directly recognized by immune cells such as CD8+ T cells. Collectively, these data suggest we reconsider the broader role of neurons in the context of infection and neuroinflammation.

scholarly journals AMA1-Deficient Toxoplasma gondii Parasites Transiently Colonize Mice and Trigger an Innate Immune Response That Leads to Long-Lasting Protective Immunity

2015 ◽  
Vol 83 (6) ◽  
pp. 2475-2486 ◽  
Author(s):  
Vanessa Lagal ◽  
Márcia Dinis ◽  
Dominique Cannella ◽  
Daniel Bargieri ◽  
Virginie Gonzalez ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Distinct Type ◽  
Genetically Engineered ◽  
Host Cells ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Immunocompromised Mice

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera,PlasmodiumandToxoplasma, until we genetically engineered viable parasites lackingAMA1. The reduction in invasiveness of theToxoplasma gondiiRH-AMA1 knockout (RH-AMA1KO) tachyzoite population,in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1KOtachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1KOpopulation amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type IIT. gondiigenotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite populationin vivo.

scholarly journals Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Reetika Manhas ◽  
Smriti Tandon ◽  
Shib Sankar Sen ◽  
Neha Tiwari ◽  
Manoj Munde ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Fungal Pathogens ◽  
Leishmania Donovani ◽  
Protozoan Parasite ◽  
Gene Replacement ◽  
Trna Synthetase ◽  
Content Type

ABSTRACT Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani. Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro. The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

scholarly journals TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

2010 ◽  
Vol 9 (6) ◽  
pp. 866-877 ◽  
Author(s):  
John C. Fisk ◽  
Cecilia Zurita-Lopez ◽  
Joyce Sayegh ◽  
Danielle L. Tomasello ◽  
Steven G. Clarke ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Arginine Methylation ◽  
Nuclear Pore ◽  
Type I ◽  
Content Type ◽  
Morphological Defects ◽  
Flagellar Proteins

ABSTRACT Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.

scholarly journals Sphingosine 1-phosphate in inflammation

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Lijuan Li ◽  
Lixia An ◽  
Lifang Li ◽  
Yongjuan Zhao
Keyword(s):  
Plasma Membrane ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Cell Types ◽  
In Vivo Models ◽  
Integral Role ◽  
Sphingosine 1 Phosphate ◽  
And Migration

Sphingolipids are formed via the metabolism of sphingomyelin, aconstituent of the plasma membrane, or by denovosynthesis. Enzymatic pathways result in the formation of several different lipid mediators, which are known to have important roles in many cellular processes, including proliferation, apoptosis and migration. Several studies now suggest that these sphingolipid mediators, including ceramide, ceramide 1-phosphate and sphingosine 1-phosphate (S1P), are likely to have an integral role in in?ammation. This can involve, for example, activation of pro-in?ammatory transcription factors in different cell types and induction of cyclooxygenase-2, leading to production of pro-in?ammatory prostaglandins. The mode of action of each sphingolipid is different. Increased ceramide production leads to the formation of ceramide-rich areas of the membrane, which may assemble signalling complexes, whereas S1P acts via high-af?nity G-protein-coupled S1P receptors on the plasma membrane. Recent studies have demonstrated that in vitro effects of sphingolipids on in?ammation can translate into in vivo models. This review will highlight the areas of research where sphingolipids are involved in in?ammation and the mechanisms of action of each mediator. In addition, the therapeutic potential of drugs that alter sphingolipid actions will be examined with reference to disease states, such as asthma and in?ammatory bowel disease, which involve important in?ammatory components. A signi?cant body of research now indicates that sphingolipids are intimately involved in the in?ammatory process and recent studies have demonstrated that these lipids, together with associated enzymes and receptors, can provide effective drug targets for the treatment of pathological in?ammation.

scholarly journals Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

2018 ◽  
Vol 115 (20) ◽  
pp. 5253-5258 ◽  
Author(s):  
Hideyuki Yanai ◽  
Shiho Chiba ◽  
Sho Hangai ◽  
Kohei Kometani ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cell Types ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Gene Ablation ◽  
Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

scholarly journals ROP18-Mediated Transcriptional Reprogramming of HEK293T Cell Reveals New Roles of ROP18 in the Interplay Between Toxoplasma gondii and the Host Cell

2020 ◽  
Vol 10 ◽  
Author(s):  
Jie-Xi Li ◽  
Jun-Jun He ◽  
Hany M. Elsheikha ◽  
Jun Ma ◽  
Xiao-Pei Xu ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Hek293t Cell ◽  
Effector Proteins ◽  
Host Cells ◽  
Pathway Enrichment Analysis ◽  
Type I ◽  
Human Embryonic Kidney Cells ◽  
Pathway Enrichment

Toxoplasma gondii secretes a number of virulence-related effector proteins, such as the rhoptry protein 18 (ROP18). To further broaden our understanding of the molecular functions of ROP18, we examined the transcriptional response of human embryonic kidney cells (HEK293T) to ROP18 of type I T. gondii RH strain. Using RNA-sequencing, we compared the transcriptome of ROP18-expressing HEK293T cells to control HEK293T cells. Our analysis revealed that ROP18 altered the expression of 750 genes (467 upregulated genes and 283 downregulated genes) in HEK293T cells. Gene ontology (GO) and pathway enrichment analyses showed that differentially expressed genes (DEGs) were significantly enriched in extracellular matrix– and immune–related GO terms and pathways. KEGG pathway enrichment analysis revealed that DEGs were involved in several disease-related pathways, such as nervous system diseases and eye disease. ROP18 significantly increased the alternative splicing pattern “retained intron” and altered the expression of 144 transcription factors (TFs). These results provide new insight into how ROP18 may influence biological processes in the host cells via altering the expression of genes, TFs, and pathways. More in vitro and in vivo studies are required to substantiate these findings.

scholarly journals Two Phosphoglucomutase Paralogs Facilitate Ionophore-Triggered Secretion of the Toxoplasma Micronemes

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Sudeshna Saha ◽  
Bradley I. Coleman ◽  
Rashmi Dubey ◽  
Ira J. Blader ◽  
Marc-Jan Gubbels
Keyword(s):  
Toxoplasma Gondii ◽  
Phosphatidic Acid ◽  
Host Cell ◽  
Lytic Cycle ◽  
Ionophore A23187 ◽  
Host Cell Invasion ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Genetic Deletion

ABSTRACT Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice. Paralogs of the widely prevalent phosphoglucomutase (PGM) protein called parafusin function in calcium (Ca2+)-mediated exocytosis across eukaryotes. In Toxoplasma gondii, the parafusin-related protein 1 (PRP1) has been associated with Ca2+-dependent microneme organelle secretion required for essential processes like host cell invasion and egress. Using reverse genetics, we observed PRP1 to be dispensable for completion of the lytic cycle, including host cell invasion and egress by the parasite. However, the absence of the gene affected increased microneme release triggered by A23187, a Ca2+ ionophore used to raise the cytoplasmic Ca2+ concentration mimicking the physiological role of Ca2+ during invasion and egress. The basal levels of constitutive microneme release in extracellular parasites and phosphatidic acid-triggered microneme secretion were unaffected in the mutant. The phenotype of the deletion mutant of the second PGM-encoding gene in Toxoplasma, PGM2, was similar to the phenotype of the PRP1 deletion mutant. Furthermore, the ability of the tachyzoites to induce acute infection in the mice remained normal in the absence of both PGM paralogs. Our data thus reveal that the microneme secretion upon high Ca2+ flux is facilitated by the Toxoplasma PGM paralogs, PRP1 and PGM2. However, this protein-mediated release is neither essential for lytic cycle completion nor for acute virulence of the parasite. IMPORTANCE Ca2+-dependent exocytosis is essential for the life cycle of apicomplexan parasites. Toxoplasma gondii harbors a phosphoglucomutase (PGM) ortholog, PRP1, previously associated with Ca2+-dependent microneme secretion. Here it is shown that genetic deletion of either PRP1, its PGM2 ortholog, or both genes is dispensable for the parasite’s lytic cycle, including host cell egress and invasion. Depletion of the proteins abrogated high Ca2+-mediated microneme secretion induced by the ionophore A23187; however, the constitutive and phosphatidic acid-mediated release remained unaffected. Secretion mediated by the former pathway is not essential for tachyzoite survival or acute in vivo infection in the mice.

scholarly journals The Protozoan Parasite Toxoplasma gondii Selectively Reprograms the Host Cell Translatome

2018 ◽  
Vol 86 (9) ◽  
Author(s):  
Louis-Philippe Leroux ◽  
Julie Lorent ◽  
Tyson E. Graber ◽  
Visnu Chaparro ◽  
Laia Masvidal ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Activation ◽  
Immune Cell ◽  
Mrna Translation ◽  
Protozoan Parasite ◽  
Translational Efficiency ◽  
Type I ◽  
Content Type ◽  
Infection Inhibition

ABSTRACT The intracellular parasite Toxoplasma gondii promotes infection by targeting multiple host cell processes; however, whether it modulates mRNA translation is currently unknown. Here, we show that infection of primary murine macrophages with type I or II T. gondii strains causes a profound perturbation of the host cell translatome. Notably, translation of transcripts encoding proteins involved in metabolic activity and components of the translation machinery was activated upon infection. In contrast, the translational efficiency of mRNAs related to immune cell activation and cytoskeleton/cytoplasm organization was largely suppressed. Mechanistically, T. gondii bolstered mechanistic target of rapamycin (mTOR) signaling to selectively activate the translation of mTOR-sensitive mRNAs, including those with a 5′-terminal oligopyrimidine (5′ TOP) motif and those encoding mitochondrion-related proteins. Consistent with parasite modulation of host mTOR-sensitive translation to promote infection, inhibition of mTOR activity suppressed T. gondii replication. Thus, selective reprogramming of host mRNA translation represents an important subversion strategy during T. gondii infection.

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