scholarly journals Host Cells Participate in the In Vitro Effects of Novel Diamidine Analogues against Tachyzoites of the Intracellular Apicomplexan Parasites Neospora caninum and Toxoplasma gondii

2008 ◽  
Vol 52 (6) ◽  
pp. 1999-2008 ◽  
Author(s):  
Angela Leepin ◽  
Angela Stüdli ◽  
Reto Brun ◽  
Chad E. Stephens ◽  
David W. Boykin ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Neospora Caninum ◽  
Host Cells ◽  
Active Metabolites ◽  
Apicomplexan Parasites ◽  
Functional Activities ◽  
Drug Treatments ◽  
In Vitro Effects ◽  
Uninfected Host

ABSTRACT The in vitro effects of 19 dicationic diamidine derivatives against the proliferative tachyzoite stages of the apicomplexan parasites Neospora caninum and Toxoplasma gondii were investigated. Four compounds (DB811, DB786, DB750, and DB766) with similar structural properties exhibited profound inhibition of tachyzoite proliferation. The lowest 50% inhibitory concentrations were found for DB786 (0.21 μM against Neospora and 0.22 μM against Toxoplasma) and DB750 (0.23 μM against Neospora and 0.16 μM against Toxoplasma), with complete proliferation inhibition at 1.7 μM for both drugs against both species. DB750 and DB786 were chosen for further studies. Electron microscopy of N. caninum-infected human foreskin fibroblast (HFF) cultures revealed distinct alterations and damage of parasite ultrastructure upon drug treatment, while host cells remained unaffected. For true parasiticidal efficacy against N. caninum, a treatment duration of 3 h at 1.7 μM was sufficient for DB750, while a longer treatment period (24 h) was necessary for DB786. Pretreatment of tachyzoites for 1 h prior to host cell exposure had no effect on infectivity. However, pretreatment of uninfected host cells had a significant adverse effect on N. caninum proliferation: exposure of HFFs to 1.7 μM DB750 for 6, 12, or 24 h, followed by infection with N. caninum tachyzoites and subsequent culture in the absence of DB750, resulted in significantly delayed parasite proliferation. This suggests that either (i) these compounds or their respective active metabolites were still present after the removal of the drugs or (ii) the drug treatments reversibly impaired some functional activities in HFFs that were essential for parasite proliferation and/or survival.

scholarly journals Systematic Analysis of Clemastine, a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

2021 ◽  
Vol 23 (1) ◽  
pp. 68
Author(s):  
Izra Abbaali ◽  
Danny A. Truong ◽  
Shania D. Day ◽  
Nancy Haro-Ramirez ◽  
Naomi S. Morrissette
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cells ◽  
Proof Of Concept ◽  
Systematic Analysis ◽  
Apicomplexan Parasites ◽  
Microtubule Disruption ◽  
Antiparasitic Drugs ◽  
General Response ◽  
Babesia Spp

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC50 values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.

scholarly journals In vitro effects of arylimidamides against Besnoitia besnoiti infection in Vero cells

Parasitology ◽  
2011 ◽  
Vol 138 (5) ◽  
pp. 583-592 ◽  
Author(s):  
H. C. E. CORTES ◽  
N. MULLER ◽  
D. BOYKIN ◽  
C. E. STEPHENS ◽  
A. HEMPHILL
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Neospora Caninum ◽  
Vero Cells ◽  
Besnoitia Besnoiti ◽  
Drug Treatments ◽  
Important Addition ◽  
In Vitro Effects ◽  
Intracellular Proliferation

SUMMARYThe in vitro effects of 4 arylimidamides (DB811, DB786, DB750 and DB766) against the proliferative tachyzoite stage of the apicomplexan parasite Besnoitia besnoiti were investigated. These four compounds had been shown earlier to exhibit in vitro activities in the nanomolar range against the related apicomplexans Neospora caninum and Toxoplasma gondii. Real-time-PCR was used to assess B. besnoiti intracellular proliferation in vitro. Preliminary assessment by light microscopy identified DB811 and DB750 as the most promising compounds, while DB786 and DB766 were much less effective. Three-day-growth assays and quantitative real-time PCR was used for IC50 determination of DB811 (0·079 μM) and DB750 (0·56 μM). Complete growth inhibition was observed at 1·6 μM for DB 811 and 1·7 μM for DB750. However, when infected cultures were treated for 14 days, proliferation of parasites occurred again in cultures treated with DB750 from day 4 onwards, while the proliferation of DB811-treated tachyzoites remained inhibited. Electron microscopy of B. besnoiti-infected fibroblast cultures fixed and processed at different time-points following the initiation of drug treatments revealed that DB811 exerted a much higher degree of ultrastructural alterations compared to DB750. These results show that arylimidamides such as DB811 could potentially become an important addition to the anti-parasitic arsenal for food animal production, especially in cattle.

Cytologic detection of Toxoplasma gondii in the cerebrospinal fluid of a dog and in vitro isolation of a unique mouse-virulent recombinant strain

2021 ◽  
pp. 104063872199668
Author(s):  
Waléria Borges-Silva ◽  
Mariana M. Rezende-Gondim ◽  
Gideão S. Galvão ◽  
Daniele S. Rocha ◽  
George R. Albuquerque ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Toxoplasma Gondii ◽  
Recombinant Strain ◽  
Neospora Caninum ◽  
Clinical Signs ◽  
Species Identity ◽  
Cytologic Examination ◽  
Pcr Rflp ◽  
Tissue Homogenates

Parasites resembling Neospora caninum or Toxoplasma gondii were detected by cytologic examination of cerebrospinal fluid (CSF) from a dog with neurologic disease. The dog became severely ill and was euthanized. Canine tissue homogenates were used for direct parasite isolation in cell culture, bioassay in 2 mouse lineages, and PCR. T. gondii was isolated in monkey kidney cells, and species identity was confirmed by PCR. Inoculated parasites were highly virulent for mice, which developed clinical signs and were euthanized immediately. PCR-RFLP for T. gondii using the cultured isolate (TgDgBA22) was conducted with 12 genetic markers, and a unique recombinant strain was identified. Detection of T. gondii by CSF cytology, although described in humans, had not been reported previously in dogs, to our knowledge, and was crucial for the diagnosis of toxoplasmosis in the examined dog.

Cellular and immunological basis of the host-parasite relationship during infection withNeospora caninum

Parasitology ◽  
2006 ◽  
Vol 133 (3) ◽  
pp. 261-278 ◽  
Author(s):  
A. HEMPHILL ◽  
N. VONLAUFEN ◽  
A. NAGULESWARAN
Keyword(s):  
Host Cell ◽  
Cell Biology ◽  
Neospora Caninum ◽  
Host Cells ◽  
Term Survival ◽  
Prevention Of Infection ◽  
Parasite Relationship ◽  
Potential Applications

Neospora caninumis an apicomplexan parasite that is closely related toToxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast toT. gondii, N. caninumrepresents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused byN. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cellsin vitroandin vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite orvice versa). Thirdly, by analogy withT. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features ofN. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

Identification and characterization of a novel Neospora caninum immune mapped protein 1

Parasitology ◽  
2012 ◽  
Vol 139 (8) ◽  
pp. 998-1004 ◽  
Author(s):  
X. CUI ◽  
T. LEI ◽  
D. Y. YANG ◽  
P. HAO ◽  
Q. LIU
Keyword(s):  
Neospora Caninum ◽  
Polyclonal Antibodies ◽  
Functional Protein ◽  
Reading Frame ◽  
Protein Motifs ◽  
Apicomplexan Parasites ◽  
Potential Vaccine ◽  
Palmitoylation Site

SUMMARYImmune mapped protein 1 (IMP1) is a newly discovered protein in Eimeria maxima. It is recognized as a potential vaccine candidate against E. maxima and a highly conserved protein in apicomplexan parasites. Although the Neospora caninum IMP1 (NcIMP1) orthologue of E. maxima IMP1 was predicted in the N. caninum genome, it was still not identified and characterized. In this study, cDNA sequence encoding NcIMP1 was cloned by RT-PCR from RNA isolated from Nc1 tachyzoites. NcIMP1 was encoded by an open reading frame of 1182 bp, which encoded a protein of 393 amino acids with a predicted molecular weight of 42·9 kDa. Sequence analysis showed that there was neither a signal peptide nor a transmembrane region present in the NcIMP1 amino acid sequence. However, several kinds of functional protein motifs, including an N-myristoylation site and a palmitoylation site were predicted. Recombinant NcIMP1 (rNcIMP1) was expressed in Escherichia coli and then purified rNcIMP1 was used to prepare specific antisera in mice. Mouse polyclonal antibodies raised against the rNcIMP1 recognized an approximate 43 kDa native IMP1 protein. Immunofluorescence analysis showed that NcIMP1 was localized on the membrane of N. caninum tachyzoites. The N-myristoylation site and the palmitoylation site were found to contribute to the localization of NcIMP1. Furthermore, the rNcIMP1-specific antibodies could inhibit cell invasion by N. caninum tachyzoites in vitro. All the results indicate that NcIMP1 is likely to be a membrane protein of N. caninum and may be involved in parasite invasion.

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 Toxoplasma gondii Rhoptry Discharge Correlates with Activation of the Early Growth Response 2 Host Cell Transcription Factor

2008 ◽  
Vol 76 (10) ◽  
pp. 4703-4712 ◽  
Author(s):  
Eric D. Phelps ◽  
Kristin R. Sweeney ◽  
Ira J. Blader
Keyword(s):  
Transcription Factor ◽  
Toxoplasma Gondii ◽  
Host Cell ◽  
Growth Response ◽  
Neospora Caninum ◽  
Early Growth ◽  
Apicomplexan Parasite ◽  
Host Cells ◽  
Early Growth Response ◽  
Cell Extracts

ABSTRACT Toxoplasma gondii is a ubiquitous apicomplexan parasite that can cause severe disease in fetuses and immune-compromised patients. Rhoptries, micronemes, and dense granules, which are secretory organelles unique to Toxoplasma and other apicomplexan parasites, play critical roles in parasite growth and virulence. To understand how these organelles modulate infected host cells, we sought to identify host cell transcription factors triggered by their release. Early growth response 2 (EGR2) is a host cell transcription factor that is rapidly upregulated and activated in Toxoplasma-infected cells but not in cells infected with the closely related apicomplexan parasite Neospora caninum. EGR2 upregulation occurred only when live parasites were in direct contact with the host cell and not from exposure to cell extracts that contain dense granule or micronemal proteins. When microneme-mediated attachment was blocked by pretreating parasites with a calcium chelator, EGR2 expression was significantly reduced. In contrast, when host cells were infected with parasites in the presence of cytochalasin D, which allows rhoptry secretion but prevents parasite invasion, EGR2 was activated. Finally, we demonstrate that Toxoplasma activation of host p38 mitogen-activated protein kinase is necessary but not sufficient for EGR2 activation. Collectively, these data indicate that EGR2 is specifically upregulated by a parasite-derived secreted factor that is most likely a resident rhoptry protein.

scholarly journals Systematic Identification of Thiosemicarbazides for Inhibition of Toxoplasma gondii Growth In Vitro

Molecules ◽  
2019 ◽  
Vol 24 (3) ◽  
pp. 614 ◽  
Author(s):  
Agata Paneth ◽  
Lidia Węglińska ◽  
Adrian Bekier ◽  
Edyta Stefaniszyn ◽  
Monika Wujec ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Small Molecules ◽  
High Selectivity ◽  
High Specificity ◽  
Host Cells ◽  
Structural Requirements ◽  
New Therapies ◽  
Cns Penetration ◽  
Systematic Identification

One of the key stages in the development of new therapies in the treatment of toxoplasmosis is the identification of new non-toxic small molecules with high specificity to Toxoplasma gondii. In the search for such structures, thiosemicarbazide-based compounds have emerged as a novel and promising leads. Here, a series of imidazole-thiosemicarbazides with suitable properties for CNS penetration was evaluated to determine the structural requirements needed for potent anti-Toxoplasma gondii activity. The best 4-arylthiosemicarbazides 3 and 4 showed much higher potency when compared to sulfadiazine at concentrations that are non-toxic to the host cells, indicating a high selectivity of their anti-toxoplasma activity.

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 Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii

2004 ◽  
Vol 165 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Elizabeth Gaskins ◽  
Stacey Gilk ◽  
Nicolette DeVore ◽  
Tara Mann ◽  
Gary Ward ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Complex ◽  
Integral Membrane Protein ◽  
Gliding Motility ◽  
Host Cells ◽  
Inner Membrane ◽  
Apicomplexan Parasites ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Two Stages

Apicomplexan parasites exhibit a unique form of substrate-dependent motility, gliding motility, which is essential during their invasion of host cells and during their spread between host cells. This process is dependent on actin filaments and myosin that are both located between the plasma membrane and two underlying membranes of the inner membrane complex. We have identified a protein complex in the apicomplexan parasite Toxoplasma gondii that contains the class XIV myosin required for gliding motility, TgMyoA, its associated light chain, TgMLC1, and two novel proteins, TgGAP45 and TgGAP50. We have localized this complex to the inner membrane complex of Toxoplasma, where it is anchored in the membrane by TgGAP50, an integral membrane glycoprotein. Assembly of the protein complex is spatially controlled and occurs in two stages. These results provide the first molecular description of an integral membrane protein as a specific receptor for a myosin motor, and further our understanding of the motile apparatus underlying gliding motility in apicomplexan parasites.

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