scholarly journals The Vacuolar Zinc Transporter TgZnT Protects Toxoplasma gondii from Zinc Toxicity

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
Nathan M. Chasen ◽  
Andrew J. Stasic ◽  
Beejan Asady ◽  
Isabelle Coppens ◽  
Silvia N. J. Moreno
Keyword(s):  
Toxoplasma Gondii ◽  
Metal Ion ◽  
Essential Element ◽  
Lytic Cycle ◽  
Zinc Toxicity ◽  
Content Type ◽  
Zinc Concentrations ◽  
High Concentrations ◽  
Yeast Mutants

ABSTRACT Zinc (Zn2+) is the most abundant biological metal ion aside from iron and is an essential element in numerous biological systems, acting as a cofactor for a large number of enzymes and regulatory proteins. Zn2+ must be tightly regulated, as both the deficiency and overabundance of intracellular free Zn2+ are harmful to cells. Zn2+ transporters (ZnTs) play important functions in cells by reducing intracellular Zn2+ levels by transporting the ion out of the cytoplasm. We characterized a Toxoplasma gondii gene (TgGT1_251630, TgZnT), which is annotated as the only ZnT family Zn2+ transporter in T. gondii. TgZnT localizes to novel vesicles that fuse with the plant-like vacuole (PLV), an endosome-like organelle. Mutant parasites lacking TgZnT exhibit reduced viability in in vitro assays. This phenotype was exacerbated by increasing zinc concentrations in the extracellular media and was rescued by media with reduced zinc. Heterologous expression of TgZnT in a Zn2+-sensitive Saccharomyces cerevisiae yeast strain partially restored growth in media with higher Zn2+ concentrations. These results suggest that TgZnT transports Zn2+ into the PLV and plays an important role in the Zn2+ tolerance of T. gondii extracellular tachyzoites. IMPORTANCE Toxoplasma gondii is an intracellular pathogen of human and animals. T. gondii pathogenesis is associated with its lytic cycle, which involves invasion, replication, egress out of the host cell, and invasion of a new one. T. gondii must be able to tolerate abrupt changes in the composition of the surrounding milieu as it progresses through its lytic cycle. We report the characterization of a Zn2+ transporter of T. gondii (TgZnT) that is important for parasite growth. TgZnT restored Zn2+ tolerance in yeast mutants that were unable to grow in media with high concentrations of Zn2+. We propose that TgZnT plays a role in Zn2+ homeostasis during the T. gondii lytic cycle.

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 Characterization of Plasmodium Atg3-Atg8 Interaction Inhibitors Identifies Novel Alternative Mechanisms of Action in Toxoplasma gondii

2017 ◽  
Vol 62 (2) ◽  
Author(s):  
Joseph M. Varberg ◽  
Kaice A. LaFavers ◽  
Gustavo Arrizabalaga ◽  
William J. Sullivan
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Inhibitory Effect ◽  
Protozoan Parasites ◽  
Conjugation System ◽  
Content Type ◽  
Similar Inhibitory Effect ◽  
High Concentrations

ABSTRACT Protozoan parasites, including the apicomplexan pathogens Plasmodium falciparum (which causes malaria) and Toxoplasma gondii (which causes toxoplasmosis), infect millions of people worldwide and represent major human disease burdens. Despite their prevalence, therapeutic strategies to treat infections caused by these parasites remain limited and are threatened by the emergence of drug resistance, highlighting the need for the identification of novel drug targets. Recently, homologues of the core autophagy proteins, including Atg8 and Atg3, were identified in many protozoan parasites. Importantly, components of the Atg8 conjugation system that facilitate the lipidation of Atg8 are required for both canonical and parasite-specific functions and are essential for parasite viability. Structural characterization of the P. falciparum Atg3-Atg8 (PfAtg3-Atg8) interaction has led to the identification of compounds that block this interaction. Additionally, many of these compounds inhibit P. falciparum growth in vitro, demonstrating the viability of this pathway as a drug target. Given the essential role of the Atg8 lipidation pathway in Toxoplasma, we sought to determine whether three PfAtg3-Atg8 interaction inhibitors identified in the Medicines for Malaria Venture Malaria Box exerted a similar inhibitory effect in Toxoplasma. While all three inhibitors blocked Toxoplasma replication in vitro at submicromolar concentrations, they did not inhibit T. gondii Atg8 (TgAtg8) lipidation. Rather, high concentrations of two of these compounds induced TgAtg8 lipidation and fragmentation of the parasite mitochondrion, similar to the effects seen following starvation and monensin-induced autophagy. Additionally, we report that one of the PfAtg3-Atg8 interaction inhibitors induces Toxoplasma egress and provide evidence that this is mediated by an increase in intracellular calcium in response to drug treatment.

scholarly journals Molecular Characterization of Toxoplasma gondii Formin 3, an Actin Nucleator Dispensable for Tachyzoite Growth and Motility

2011 ◽  
Vol 11 (3) ◽  
pp. 343-352 ◽  
Author(s):  
Wassim Daher ◽  
Natacha Klages ◽  
Marie-France Carlier ◽  
Dominique Soldati-Favre
Keyword(s):  
Toxoplasma Gondii ◽  
Actin Polymerization ◽  
Life Stage ◽  
Gliding Motility ◽  
Lytic Cycle ◽  
Host Cells ◽  
Content Type ◽  
Parasite Motility ◽  
Actin Nucleator

ABSTRACT Toxoplasma gondii belongs to the phylum Apicomplexa, a group of obligate intracellular parasites that rely on gliding motility to enter host cells. Drugs interfering with the actin cytoskeleton block parasite motility, host cell invasion, and egress from infected cells. Myosin A, profilin, formin 1, formin 2, and actin-depolymerizing factor have all been implicated in parasite motility, yet little is known regarding the importance of actin polymerization and other myosins for the remaining steps of the parasite lytic cycle. Here we establish that T. gondii formin 3 (TgFRM3), a newly described formin homology 2 domain (FH2)-containing protein, binds to Toxoplasma actin and nucleates rabbit actin assembly in vitro . TgFRM3 expressed as a transgene exhibits a patchy localization at several distinct structures within the parasite. Disruption of the TgFRM3 gene by double homologous recombination in a ku80-ko strain reveals no vital function for tachyzoite propagation in vitro , which is consistent with its weak level of expression in this life stage. Conditional stabilization of truncated forms of TgFRM3 suggests that different regions of the molecule contribute to distinct localizations. Moreover, expression of TgFRM3 lacking the C-terminal domain severely affects parasite growth and replication. This work provides a first insight into how this specialized formin, restricted to the group of coccidia, completes its actin-nucleating activity.

scholarly journals The Extracellular Milieu of Toxoplasma 's Lytic Cycle Drives Lab Adaptation, Primarily by Transcriptional Reprogramming

mSystems ◽  
2021 ◽  
Author(s):  
Vincent A. Primo ◽  
Yasaman Rezvani ◽  
Andrew Farrell ◽  
Connor Q. Murphy ◽  
Jingjing Lou ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Virulence Factors ◽  
Lytic Cycle ◽  
Content Type ◽  
Extracellular Milieu ◽  
Transcriptional Reprogramming

It has been well established that prolonged in vitro cultivation of Toxoplasma gondii augments progression of the lytic cycle. This lab adaptation results in increased capacities to divide, migrate, and survive outside a host cell, all of which are considered host-independent virulence factors.

Prostacyclin and cerebral vessel relaxation

1982 ◽  
Vol 57 (3) ◽  
pp. 334-340 ◽  
Author(s):  
Kamal S. Paul ◽  
Eric T. Whalley ◽  
Christine Forster ◽  
Richard Lye ◽  
John Dutton
Keyword(s):  
Angiotensin Ii ◽  
Arterial Spasm ◽  
Cerebral Vessel ◽  
Content Type ◽  
Wide Range ◽  
Potent Vasodilator ◽  
Dose Dependent Fashion ◽  
Basilar Arteries ◽  
High Concentrations

✓ The authors have studied the ability of prostacyclin to reverse contractions of human basilar arteries in vitro that were induced by a wide range of substances implicated in the etiology of cerebral arterial spasm. Prostacyclin (10−10 to 10−6M) caused a dose-related reversal of contractions induced by 5-hydroxytryptamine, noradrenaline, angiotensin II, prostaglandin (PG)F2α, and U-46619 (a thromboxane-A2 mimetic). These agents were tested at concentrations or volumes that produced almost maximum or maximum responses and those that produced approximately 50% of the maximum response. Contractions induced by maximum concentrations of angiotensin II and U-46619 were least affected by prostacyclin. In addition, contractions induced by thromboxane-A2 generated from guinea-pig lung were reversed in a dose-dependent fashion by prostacyclin. This ability of prostacyclin to physiologically antagonize contractions of the human basilar artery in vitro induced by high concentrations of various spasmogenic agents suggests that such a potent vasodilator agent or more stable analogue may be of value in the treatment of such disorders as cerebral arterial spasm following subarachnoid hemorrhage.

scholarly journals Downregulation of the Central Noradrenergic System by Toxoplasma gondii Infection

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
Isra Alsaady ◽  
Ellen Tedford ◽  
Mohammad Alsaad ◽  
Greg Bristow ◽  
Shivali Kohli ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Infection Intensity ◽  
Noradrenergic System ◽  
Neural Cells ◽  
Mrna Levels ◽  
Female Rat ◽  
Motor Impairments ◽  
Content Type ◽  
General Response

ABSTRACT Toxoplasma gondii is associated with physiological effects in the host. Dysregulation of catecholamines in the central nervous system has previously been observed in chronically infected animals. In the study described here, the noradrenergic system was found to be suppressed with decreased levels of norepinephrine (NE) in brains of infected animals and in infected human and rat neural cells in vitro. The mechanism responsible for the NE suppression was found to be downregulation of dopamine β-hydroxylase (DBH) gene expression, encoding the enzyme that synthesizes norepinephrine from dopamine, with downregulation observed in vitro and in infected brain tissue, particularly in the dorsal locus coeruleus/pons region. The downregulation was sex specific, with males expressing reduced DBH mRNA levels whereas females were unchanged. Rather, DBH expression correlated with estrogen receptor in the female rat brains for this estrogen-regulated gene. DBH silencing was not a general response of neurons to infection, as human cytomegalovirus did not downregulate DBH expression. The noradrenergic-linked behaviors of sociability and arousal were altered in chronically infected animals, with a high correlation between DBH expression and infection intensity. A decrease in DBH expression in noradrenergic neurons can elevate dopamine levels, which provides a possible explanation for mixed observations of changes in this neurotransmitter with infection. Decreased NE is consistent with the loss of coordination and motor impairments associated with toxoplasmosis. Further, the altered norepinephrine synthesis observed here may, in part, explain behavioral effects of infection and associations with mental illness.

scholarly journals Identification of Zinc-Dependent Mechanisms Used by Group B Streptococcus To Overcome Calprotectin-Mediated Stress

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Lindsey R. Burcham ◽  
Yoann Le Breton ◽  
Jana N. Radin ◽  
Brady L. Spencer ◽  
Liwen Deng ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Metal Ion ◽  
Invasive Disease ◽  
Female Reproductive Tract ◽  
Zinc Uptake ◽  
Wild Type ◽  
Metal Transporters ◽  
High Concentrations ◽  
Group B

ABSTRACT Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease. IMPORTANCE Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease.

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 Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Geetha Kannan ◽  
Manlio Di Cristina ◽  
Aric J. Schultz ◽  
My-Hang Huynh ◽  
Fengrong Wang ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Chloroquine Resistance ◽  
Congenital Defects ◽  
Functional Link ◽  
Content Type ◽  
Chloroquine Resistance Transporter

ABSTRACT Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii. To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection. IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite’s lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

scholarly journals Treatment with Bumped Kinase Inhibitor 1294 Is Safe and Leads to Significant Protection against Abortion and Vertical Transmission in Sheep Experimentally Infected with Toxoplasma gondii during Pregnancy

2019 ◽  
Vol 63 (7) ◽  
Author(s):  
Roberto Sánchez-Sánchez ◽  
Ignacio Ferre ◽  
Michela Re ◽  
Juan José Ramos ◽  
Javier Regidor-Cerrillo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Vertical Transmission ◽  
Kinase Inhibitor ◽  
Plasma Concentrations ◽  
Systemic Exposure ◽  
Fetal Mortality ◽  
Maximum Plasma ◽  
Content Type ◽  
Pregnant Sheep

ABSTRACT Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 μM and trough concentrations of 0.4 μM at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy.

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