scholarly journals 1,3,4-Thiadiazoles Effectively Inhibit Proliferation of Toxoplasma gondii

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1053
Author(s):  
Lidia Węglińska ◽  
Adrian Bekier ◽  
Katarzyna Dzitko ◽  
Barbara Pacholczyk-Sienicka ◽  
Łukasz Albrecht ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Direct Action ◽  
Human Fibroblasts ◽  
Imidazole Ring ◽  
Host Cells ◽  
In Vitro Assays ◽  
Host Cell Invasion

Congenital and acquired toxoplasmosis caused by the food- and water-born parasite Toxoplasma gondii (T. gondii) is one of the most prevalent zoonotic infection of global importance. T. gondii is an obligate intracellular parasite with limited capacity for extracellular survival, thus a successful, efficient and robust host cell invasion process is crucial for its survival, proliferation and transmission. In this study, we screened a series of novel 1,3,4-thiadiazole-2-halophenylamines functionalized at the C5 position with the imidazole ring (1b–12b) for their effects on T. gondii host cell invasion and proliferation. To achieve this goal, these compounds were initially subjected to in vitro assays to assess their cytotoxicity on human fibroblasts and then antiparasitic efficacy. Results showed that all of them compare favorably to control drugs sulfadiazine and trimethoprim in terms of T. gondii growth inhibition (IC50) and selectivity toward the parasite, expressed as selectivity index (SI). Subsequently, the most potent of them with meta-fluoro 2b, meta-chloro 5b, meta-bromo 8b, meta-iodo 11b and para-iodo 12b substitution were tested for their efficacy in inhibition of tachyzoites invasion and subsequent proliferation by direct action on established intracellular infection. All the compounds significantly inhibited the parasite invasion and intracellular proliferation via direct action on both tachyzoites and parasitophorous vacuoles formation. The most effective was para-iodo derivative 12b that caused reduction in the percentage of infected host cells by 44% and number of tachyzoites per vacuole by 93% compared to non-treated host cells. Collectively, these studies indicate that 1,3,4-thiadiazoles 1b–12b, especially 12b with IC50 of 4.70 µg/mL and SI of 20.89, could be considered as early hit compounds for future design and synthesis of anti-Toxoplasma agents that effectively and selectively block the invasion and subsequent proliferation of T. gondii into host cells.

scholarly journals Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Shruthi Krishnamurthy ◽  
Bin Deng ◽  
Roxana del Rio ◽  
Kerry R. Buchholz ◽  
Moritz Treeck ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Host Cells ◽  
Receptor Protein ◽  
Host Cell Invasion ◽  
Cell Binding ◽  
Apicomplexan Parasites

ABSTRACT Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T . gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the “moving junction,” a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion. IMPORTANCE Nearly one-third of the world’s population is infected with the protozoan parasite Toxoplasma gondii , which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

scholarly journals A Member of the Ferlin Calcium Sensor Family Is Essential for Toxoplasma gondii Rhoptry Secretion

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Bradley I. Coleman ◽  
Sudeshna Saha ◽  
Seiko Sato ◽  
Klemens Engelberg ◽  
David J. P. Ferguson ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Host Cells ◽  
Model Organisms ◽  
Host Cell Invasion ◽  
Content Type ◽  
Calcium Dependent ◽  
Calcium Sensing ◽  
Intracellular Ca

ABSTRACT Invasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility, and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+ independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms, such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. Here we focus on one ferlin that is conserved across the Apicomplexa, T. gondii FER2 (TgFER2). Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Knockdown of TgFER2 prevented rhoptry secretion, and these parasites failed to form the moving junction at the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate the requirement of TgFER2 for rhoptry secretion in Toxoplasma tachyzoites and suggest a possible Ca2+ dependence of rhoptry secretion. These findings provide the first mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion. IMPORTANCE Apicomplexan protozoan parasites, such as those causing malaria and toxoplasmosis, must invade the cells of their hosts in order to establish a pathogenic infection. Timely release of proteins from a series of apical organelles is required for invasion. Neither the vesicular fusion events that underlie secretion nor the observed reliance of the various processes on changes in intracellular calcium concentrations is completely understood. We identified a group of three proteins with strong homology to the calcium-sensing ferlin family, which are known to be involved in protein secretion in other organisms. Surprisingly, decreasing the amounts of one of these proteins (TgFER2) did not have any effect on the typically calcium-dependent steps in invasion. Instead, TgFER2 was essential for the release of proteins from organelles called rhoptries. These data provide a tantalizing first look at the mechanisms controlling the very poorly understood process of rhoptry secretion, which is essential for the parasite’s infection cycle.

High Resolution Low Voltage Field Emission Scanning Electron Microscopy on De-Embedded Thick Sections Reveals Structural Interactions of the Surface of the Apicomplexan Parasite Toxoplasma Gondii with the Host Cell in the Parasitophorous Vacuole

2000 ◽  
Vol 6 (S2) ◽  
pp. 648-649
Author(s):  
Heide Schatten ◽  
David Sibley ◽  
Hans Ris
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Biochemical Characterization ◽  
Low Voltage ◽  
Cytoskeletal Proteins ◽  
Host Cells ◽  
Farm Animals ◽  
Host Cell Invasion ◽  
Apicomplexan Parasites

The protozoan parasite Toxoplasma gondii represents a large group of Apicomplexan parasites with a highly unusual motility system that is crucial for cell locomotion and host cell invasion. Studying the motility system and interactions with host cells will increase our knowledge on how to prevent infection. Apicomplexan parasites can cause considerable health problems to animals including sheep, goats, pigs, and chicken. Eimeria is known as a pathogen of coccidiosis in chicken, and Cryptosporidium causes cryptospiridiosis in cattle and other farm animals. Toxoplasma gondii can cause infection that results in abortion, central nervous disorders and death of stressed and immunocompromised farm animals. Common to all Apicomplexan parasites is an unconventional motility system that is thought to be actively involved in host-cell invasion. However, the structural and biochemical characterization of Apicomplexan parasites has proven more difficult than previously thought because of genetic and behavioral differences of the parasites’ cytoskeletal proteins that differ from those in well studied mammalian systems.

scholarly journals Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

2019 ◽  
Author(s):  
Cong Li ◽  
Qiping Zhao ◽  
Shunhai Zhu ◽  
Qingjie Wang ◽  
Haixia Wang ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Apical Membrane ◽  
Specific Protein ◽  
Host Cells ◽  
Bimolecular Fluorescence Complementation ◽  
Host Cell Invasion ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

Abstract Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In this study, we characterized one putative Et AMA1-interacting protein, E. tenella Eimeria -specific protein ( Et Esp). The interaction between Et AMA1 and Et Esp was confirmed with bimolecular fluorescence complementation (BiFC) in vivo and by glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) in vitro . We showed that Et Esp is differentially expressed during distinct phases of the parasite life cycle by using qPCR and western blotting. Immunofluorescence analysis showed that the Et Esp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that Et Esp is a micronemal protein secreted by sporozoites. In inhibition tests, a polyclonal anti-r Et Esp antibody attenuated the capacity of E. tenella to invade host cells in vitro . These data have implications for the use of Et AMA1 or Et AMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

scholarly journals A member of the ferlin calcium sensor family is essential for Toxoplasma gondii rhoptry secretion

2018 ◽  
Author(s):  
Bradley I. Coleman ◽  
Sudeshna Saha ◽  
Seiko Sato ◽  
Klemens Engelberg ◽  
David J. P. Ferguson ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Membrane Skeleton ◽  
Host Cells ◽  
Model Organisms ◽  
Host Cell Invasion ◽  
Apicomplexan Parasites ◽  
Intracellular Ca ◽  
Related Proteins

AbstractInvasion of host cells by apicomplexan parasites such as Toxoplasma gondii is critical for their infectivity and pathogenesis. In Toxoplasma, secretion of essential egress, motility and invasion-related proteins from microneme organelles is regulated by oscillations of intracellular Ca2+. Later stages of invasion are considered Ca2+-independent, including the secretion of proteins required for host cell entry and remodeling from the parasite’s rhoptries. We identified a family of three Toxoplasma proteins with homology to the ferlin family of double C2 domain-containing Ca2+ sensors. In humans and model organisms such Ca2+ sensors orchestrate Ca2+-dependent exocytic membrane fusion with the plasma membrane. One ferlin that is conserved across the Apicomplexa, TgFER2, localizes to the parasite’s cortical membrane skeleton, apical end, and rhoptries. Unexpectedly, conditionally TgFER2-depleted parasites secreted their micronemes normally and were completely motile. However, these parasites were unable to invade host cells and were therefore not viable. Specifically, knockdown of TgFER2 prevented rhoptry secretion and these parasites failed to form the moving junction on the parasite-host interface necessary for host cell invasion. Collectively, these data demonstrate that the putative Ca2+ sensor TgFER2 is required for the secretion of rhoptries. These findings provide the first regulatory and mechanistic insights into this critical yet poorly understood aspect of apicomplexan host cell invasion.Graphical abstract

scholarly journals Two phosphoglucomutase paralogs regulate triggered secretion of the Toxoplasma micronemes

2017 ◽  
Author(s):  
Sudeshna Saha ◽  
Bradley I. Coleman ◽  
Tiffany Sansom ◽  
Rashmi Dubey ◽  
Ira J. Blader ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Lytic Cycle ◽  
Host Cell Invasion ◽  
Plain Language ◽  
Functional Interaction ◽  
Similar Phenotype

AbstractParafusin is a phosphoglucomutase (PGM) paralog that acts as a signaling scaffold protein in calcium mediated exocytosis across many eukaryotes. In Toxoplasma gondii the parafusin related protein 1 (PRP1) has been associated in indirect and heterologous studies with the regulated exocytosis of the micronemes, which are required for successful host cell invasion and egress. Here we directly assessed the role of PRP1 by deleting the gene from the parasite. We observed a specific defect in microneme secretion in response to high Ca2+ fluxes, but not to phosphatidic acid fluxes controlling microneme release. We observed no defect in constitutive microneme secretion which was sufficient to support completion of the lytic cycle. Furthermore, deletion of the other PGM in Toxoplasma, PGM2, as well as the double PRP1/PGM2 deletion resulted in a similar phenotype. This suggests a functional interaction between these two genes. Strikingly, tachyzoites without both paralogs are completely viable in vitro and during acute mice infections. This indicates that PGM activity is neither required for glycolysis. In conclusion, the PRP1-PGM2 pair is required for a burst in microneme secretion upon high Ca2+ fluxes, but this burst is not essential to complete the lytic cycle of the parasite.Plain Language SummaryCalcium mediated control of microneme secretion is essential for host cell invasion and egress of Toxoplasma gondii. Here it is shown that the two phosphoglucomutases in Toxoplasma both function in the translation of a spike in calcium into a burst in microneme secretion.

scholarly journals Evolution of Invasion in a Diverse Set of Fusobacterium Species

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Abigail Manson McGuire ◽  
Kyla Cochrane ◽  
Allison D. Griggs ◽  
Brian J. Haas ◽  
Thomas Abeel ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Preterm Birth ◽  
Host Cell ◽  
Cell Invasion ◽  
Unknown Function ◽  
Host Cells ◽  
Host Cell Invasion ◽  
Whole Genome ◽  
Large Expansion ◽  
Invading Species

ABSTRACTThe diverseFusobacteriumgenus contains species implicated in multiple clinical pathologies, including periodontal disease, preterm birth, and colorectal cancer. The lack of genetic tools for manipulating these organisms leaves us with little understanding of the genes responsible for adherence to and invasion of host cells. Actively invadingFusobacteriumspecies can enter host cells independently, whereas passively invading species need additional factors, such as compromise of mucosal integrity or coinfection with other microbes. We applied whole-genome sequencing and comparative analysis to study the evolution of active and passive invasion strategies and to infer factors associated with active forms of host cell invasion. The evolution of active invasion appears to have followed an adaptive radiation in which two of the three fusobacterial lineages acquired new genes and underwent expansions of ancestral genes that enable active forms of host cell invasion. Compared to passive invaders, active invaders have much larger genomes, encode FadA-related adhesins, and possess twice as many genes encoding membrane-related proteins, including a large expansion of surface-associated proteins containing the MORN2 domain of unknown function. We predict a role for proteins containing MORN2 domains in adhesion and active invasion. In the largest and most comprehensive comparison of sequencedFusobacteriumspecies to date, we have generated a testable model for the molecular pathogenesis ofFusobacteriuminfection and illuminate new therapeutic or diagnostic strategies.IMPORTANCEFusobacteriumspecies have recently been implicated in a broad spectrum of human pathologies, including Crohn’s disease, ulcerative colitis, preterm birth, and colorectal cancer. Largely due to the genetic intractability of member species, the mechanisms by whichFusobacteriumcauses these pathologies are not well understood, although adherence to and active invasion of host cells appear important. We examined whole-genome sequence data from a diverse set ofFusobacteriumspecies to identify genetic determinants of active forms of host cell invasion. Our analyses revealed that actively invadingFusobacteriumspecies have larger genomes than passively invading species and possess a specific complement of genes—including a class of genes of unknown function that we predict evolved to enable host cell adherence and invasion. This study provides an important framework for future studies on the role ofFusobacteriumin pathologies such as colorectal cancer.

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