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 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 the secretory profile and exosomes of limbal stem cells in the canine species

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244327
Author(s):  
Antonio J. Villatoro ◽  
Cristina Alcoholado ◽  
María del Carmen Martín-Astorga ◽  
Gustavo Rico ◽  
Viviana Fernández ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ocular Surface ◽  
Inhibitory Effect ◽  
Proteomic Profile ◽  
Limbal Stem Cells ◽  
High Concentrations ◽  
First Time

Limbal stem cells (LSCs) are a quiescent cell population responsible for the renewal of the corneal epithelium. Their deficiency is responsible for the conjunctivization of the cornea that is seen in different ocular pathologies, both in humans and in the canine species. The canine species represents an interesting preclinical animal model in ocular surface pathologies. However, the role of LSCs in physiological and pathological conditions in canine species is not well understood. Our objective was to characterize for the first time the soluble factors and the proteomic profile of the secretome and exosomes of canine LSCs (cLSCs). In addition, given the important role that fibroblasts play in the repair of the ocular surface, we evaluated the influence of the secretome and exosomes of cLSCs on their proliferation in vitro. Our results demonstrated a secretory profile of cLSCs with high concentrations of MCP-1, IL-8, VEGF-A, and IL-10, as well as significant production of exosomes. Regarding the proteomic profile, 646 total proteins in the secretome and 356 in exosomes were involved in different biological processes. Functionally, the cLSC secretome showed an inhibitory effect on the proliferation of fibroblasts in vitro, which the exosomes did not. These results open the door to new studies on the possible use of the cLSC secretome or some of its components to treat certain pathologies of the ocular surface in canine species.

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 The Toxoplasma gondii Cyst Wall Interactome

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Vincent Tu ◽  
Tadakimi Tomita ◽  
Tatsuki Sugi ◽  
Joshua Mayoral ◽  
Bing Han ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Interactions ◽  
Cyst Wall ◽  
Affinity Purification ◽  
Dense Granule ◽  
Hypothetical Proteins ◽  
Content Type

ABSTRACT A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation. IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

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.

Calcium-ions are involved in erythrocyte invasion by equine Babesia parasites

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 289-294 ◽  
Author(s):  
K. OKUBO ◽  
P. WILAWAN ◽  
S. BORK ◽  
M. OKAMURA ◽  
N. YOKOYAMA ◽  
...  
Keyword(s):  
Calcium Ion ◽  
Chelating Agent ◽  
Inhibitory Effect ◽  
Protozoan Parasites ◽  
Babesia Caballi ◽  
Babesia Equi ◽  
Erythrocyte Invasion ◽  
High Concentrations ◽  
Positively Charged

Ethylene glycol bis (β-aminoethylether)-N,N,N,N-tetraacetic acid (EGTA) is a chelating agent capable of binding to positively-charged metal ions, including a calcium-ion (Ca2+). Here, we demonstrated the inhibitory effect of the chemical on the in vitro asexual growth of the equine protozoan parasites, Babesia caballi and Babesia equi. The growth of both B. caballi and B. equi was significantly inhibited in the presence of EGTA (IC50=1·27 and 2·25 mM, respectively). Under microscopical observation, increased percentages of extracellular merozoites in the total parasites were detected in both of the cultures treated with high concentrations of EGTA. In contrast, further addition of Ca2+ to the EGTA-treated cultures prevented the parasites from clearing and the percentages of extracellular merozoites from increasing. As for B. caballi, an invasion test using high-voltage pulsing proved that EGTA has an inhibitory effect to their erythrocyte invasion. These results suggest that Ca2+ is involved in erythrocyte invasion by equine Babesia parasites.

scholarly journals Glucose Metabolism byEscherichia coliInhibitsVibrio choleraeIntestinal Colonization of Zebrafish

2018 ◽  
Vol 86 (12) ◽  
Author(s):  
Dhrubajyoti Nag ◽  
Paul Breen ◽  
Saumya Raychaudhuri ◽  
Jeffrey H. Withey
Keyword(s):  
Carbon Source ◽  
Sugar Transport ◽  
Inhibitory Effect ◽  
Content Type ◽  
Oral Rehydration ◽  
E Coli ◽  
Similar Inhibitory Effect ◽  
Neutral Product ◽  
El Tor

ABSTRACTTheVibrio choleraeO1 serogroup is responsible for pandemic cholera and is divided into the classical and El Tor biotypes. ClassicalV. choleraeproduces acid when using glucose as a carbon source, whereas El TorV. choleraeproduces the neutral product acetoin when using glucose as a carbon source. An earlier study demonstrated thatEscherichia colistrains that metabolize glucose to acidic by-products drastically reduced the survival ofV. choleraestrainsin vitro. In the present study, zebrafish were fed 1% glucose and either inoculated with singleV. choleraeorE. colistrains or coinfected with bothV. choleraeandE. coli. A significant decrease in classical biotype colonization was observed after glucose feeding due to acid production in the zebrafish intestine. El Tor colonization was unaffected by glucose alone. However, the El Tor strain exhibited significantly lower colonization of the zebrafish when either of the acid-producingE. colistrains was coinoculated in the presence of glucose. AnE. colisugar transport mutant had no effect onV. choleraecolonization even in presence of glucose. Glucose andE. coliproduced a prophylactic effect on El Tor colonization in zebrafish whenE. coliwas inoculated beforeV. choleraeinfection. Thus, the probiotic feeding ofE. coliinhibitsV. choleraecolonization in a natural host. This suggests that a similar inhibitory effect could be seen in cholera patients, especially if a glucose-based oral rehydration solution (ORS) is administered in combination with probioticE. coliduring cholera treatment.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

1982 ◽  
Vol 47 (02) ◽  
pp. 150-153 ◽  
Author(s):  
P Han ◽  
C Boatwright ◽  
N G Ardlie
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Antiarrhythmic Drugs ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Second Phase ◽  
Ionophore A23187 ◽  
High Concentrations ◽  
Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

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.

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