In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites

ABSTRACT We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Vero cells and the separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 μM sodium nitroprusside for 8 days severely scaled down parasite proliferation, led to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite marker NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody MAbCC2. Transmission electron microscopy demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure, and the dense granule antigens NcGRA1, NcGRA2, and NcGRA7 were incorporated into the cyst wall. Adhesion-invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency than bradyzoites. However, removal of terminal sialic acid residues from either the host cell or the parasite surface increased the invasion of Vero cells by bradyzoites, but not tachyzoites.

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Molecular cloning and characterization ofNcROP2Fam-1, a member of the ROP2 family of rhoptry proteins inNeospora caninumthat is targeted by antibodies neutralizing host cell invasionin vitro

Parasitology ◽

10.1017/s0031182013000383 ◽

2013 ◽

Vol 140 (8) ◽

pp. 1033-1050 ◽

Cited By ~ 11

Author(s):

FERIAL ALAEDDINE ◽

ANDREW HEMPHILL ◽

KARIM DEBACHE ◽

CHRISTOPHE GUIONAUD

Keyword(s):

Host Cell ◽

Family Member ◽

Vaccine Candidate ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Host Cells ◽

General Belief ◽

Intracellular Parasites ◽

Promising Vaccine Candidate

SUMMARYRecent publications demonstrated that a fragment of aNeospora caninumROP2 family member antigen represents a promising vaccine candidate. We here report on the cloning of the cDNA encoding this protein,N. caninumROP2 family member 1 (NcROP2Fam-1), its molecular characterization and localization. The protein possesses the hallmarks of ROP2 family members and is apparently devoid of catalytic activity. NcROP2Fam-1 is synthesized as a pre-pro-protein that is matured to 2 proteins of 49 and 55 kDa that localize to rhoptry bulbs. Upon invasion the protein is associated with the nascent parasitophorous vacuole membrane (PVM), evacuoles surrounding the host cell nucleus and, in some instances, the surface of intracellular parasites. Staining was also observed within the cyst wall of ‘cysts’ producedin vitro. Interestingly, NcROP2Fam-1 was also detected on the surface of extracellular parasites entering the host cells and antibodies directed against NcROP2Fam-1-specific peptides partially neutralized invasionin vitro. We conclude that, in spite of the general belief that ROP2 family proteins are intracellular antigens, NcROP2Fam-1 can also be considered as an extracellular antigen, a property that should be taken into account in further experiments employing ROP2 family proteins as vaccines.

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

Parasitology ◽

10.1017/s0031182006000485 ◽

2006 ◽

Vol 133 (3) ◽

pp. 261-278 ◽

Cited By ~ 76

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.

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Attachment and invasion ofToxoplasma gondiiandNeospora caninumto epithelial and fibroblast cell linesin vitro

Parasitology ◽

10.1017/s0031182005008310 ◽

2005 ◽

Vol 131 (5) ◽

pp. 583-590 ◽

Cited By ~ 13

Author(s):

YING LEI ◽

M. DAVEY ◽

J. T. ELLIS

Keyword(s):

Cell Lines ◽

Neospora Caninum ◽

Fibroblast Cell ◽

Cell Types ◽

Vero Cells ◽

Host Cells ◽

Trypsin Treatment ◽

Epithelial Cell Lines ◽

Pre Treatment

Attachment and invasion ofToxoplasma gondiiandNeospora caninumto a cat and a dog fibroblast cell line and 2 epithelial cell lines (a cat kidney and Vero) were comparedin vitrousing fluorescence antibody methodology. In addition, trypsin treatment of tachyzoites was used to determine whether protein molecules were essential to the process of invasion. The results show that bothT. gondiiandN. caninuminvaded all 4 cell lines, and that pre-treatment ofT. gondiitachyzoites with trypsin caused an increase in the ability of the parasite to invade these host cells. FurthermoreT. gondii, in comparison toN. caninum, invaded all 4 cell lines at greater levels. The results here support the conclusion that bothT. gondiiandN. caninumhave the ability to invade a variety of cell types including both dog and cat cells, and questions the utility of Vero cells as an appropriate host cell forin vitrostudies on the biology of these taxa.

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Toxoplasma gondii reorganizes the host cell architecture during spontaneous cyst formation in vitro

Parasitology ◽

10.1017/s0031182017002050 ◽

2017 ◽

Vol 145 (8) ◽

pp. 1027-1038 ◽

Cited By ~ 5

Author(s):

T. C. Paredes-Santos ◽

E. S. Martins-Duarte ◽

W. de Souza ◽

M. Attias ◽

R. C. Vommaro

Keyword(s):

Toxoplasma Gondii ◽

Host Cell ◽

Cyst Wall ◽

Cyst Formation ◽

Acute Stage ◽

Host Cells ◽

Cell Cytoplasm ◽

Chronic Stage ◽

Host Cell Cytoplasm

AbstractToxoplasma gondii is an intracellular protozoan parasite that causes toxoplasmosis, a prevalent infection related to abortion, ocular diseases and encephalitis in immuno-compromised individuals. In the untreatable (and life-long) chronic stage of toxoplasmosis, parasitophorous vacuoles (PVs, containing T. gondii tachyzoites) transform into tissue cysts, containing slow-dividing bradyzoite forms. While acute-stage infection with tachyzoites involves global rearrangement of the host cell cytoplasm, focused on favouring tachyzoite replication, the cytoplasmic architecture of cells infected with cysts had not been described. Here, we characterized (by fluorescence and electron microscopy) the redistribution of host cell structures around T. gondii cysts, using a T. gondii strain (EGS) with high rates of spontaneous cystogenesis in vitro. Microtubules and intermediate filaments (but not actin microfilaments) formed a ‘cage’ around the cyst, and treatment with taxol (to inhibit microtubule dynamics) favoured cystogenesis. Mitochondria, which appeared adhered to the PV membrane, were less closely associated with the cyst wall. Endoplasmic reticulum (ER) profiles were intimately associated with folds in the cyst wall membrane. However, the Golgi complex was not preferentially localized relative to the cyst, and treatment with tunicamycin or brefeldin A (to disrupt Golgi or ER function, respectively) had no significant effect on cystogenesis. Lysosomes accumulated around cysts, while early and late endosomes were more evenly distributed in the cytoplasm. The endocytosis tracer HRP (but not BSA or transferrin) reached bradyzoites after uptake by infected host cells. These results suggest that T. gondii cysts reorganize the host cell cytoplasm, which may fulfil specific requirements of the chronic stage of infection.

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Tissue Culture and Explant Approaches to Studying and VisualizingNeospora caninumand Its Interactions with the Host Cell

Microscopy and Microanalysis ◽

10.1017/s1431927604040930 ◽

2004 ◽

Vol 10 (5) ◽

pp. 602-620 ◽

Cited By ~ 27

Author(s):

Andrew Hemphill ◽

Nathalie Vonlaufen ◽

Arunasalam Naguleswaran ◽

Nadine Keller ◽

Michele Riesen ◽

...

Keyword(s):

Tissue Culture ◽

Host Cell ◽

Neospora Caninum ◽

Final Host ◽

Host Cells ◽

Mammalian Host ◽

Molecular Characteristics ◽

Culture Models ◽

Cerebral Infection

Neospora caninumis an apicomplexan parasite first mentioned in 1984 as a causative agent of neuromuscular disease in dogs. It is closely related toToxoplasma gondiiandHammondia heydorni, and its subsequent description in 1988 has been, and still is, accompanied by discussions on the true phylogenetical status of the genusNeospora.N. caninumexhibits features that clearly distinguish this parasite from other members of the Apicomplexa, including distinct ultrastructural properties, genetic background, antigenic composition, host cell interactions, and the definition of the dog as a final host. Most importantly,N. caninumhas a particular significance as a cause of abortion in cattle.In vitroculture has been indispensable for the isolation of this parasite and for investigations on the ultrastructural, cellular, and molecular characteristics of the different stages ofN. caninum. Tissue culture systems include maintenance ofN. caninumtachyzoites, which represent the rapidly proliferating stage in a large number of mammalian host cells, culture of parasites in organotypic brain slice cultures as a tool to investigate cerebral infection byN. caninum, and the use of techniques to induce the stage conversion from the tachyzoite stage to the slowly proliferating and tissue cyst-forming bradyzoite stage. This review will focus on the use of these tissue culture models as well as light- and electron-microscopical techniques for studies onN. caninumtachyzoites and bradyzoites, and on the physical interactions between parasites and host cells.

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Identification and Characterization of a Neospora caninum Microneme-Associated Protein (NcMIC4) That Exhibits Unique Lactose-Binding Properties

Infection and Immunity ◽

10.1128/iai.72.8.4791-4800.2004 ◽

2004 ◽

Vol 72 (8) ◽

pp. 4791-4800 ◽

Cited By ~ 22

Author(s):

Nadine Keller ◽

Michèle Riesen ◽

Arunasalam Naguleswaran ◽

Nathalie Vonlaufen ◽

Rebecca Stettler ◽

...

Keyword(s):

Host Cell ◽

Gel Electrophoresis ◽

Neospora Caninum ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Vero Cells ◽

Host Cells ◽

Immunogold Electron Microscopy ◽

Binding Properties ◽

Microneme Protein

ABSTRACT Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M r but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.

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Molecular characterization ofNeospora caninumMAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

Parasitology ◽

10.1017/s0031182010000442 ◽

2010 ◽

Vol 137 (11) ◽

pp. 1605-1619 ◽

Cited By ~ 12

Author(s):

CHRISTOPHE GUIONAUD ◽

ANDREW HEMPHILL ◽

MEIKE MEVISSEN ◽

FERIAL ALAEDDINE

Keyword(s):

Amino Acid ◽

Cyst Wall ◽

Neospora Caninum ◽

Parasitophorous Vacuole ◽

Rabbit Antiserum ◽

Variable Region ◽

Vero Cells ◽

Amino Acid Sequences ◽

Variable Regions

SUMMARYInNeospora caninumandToxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, theN. caninumorthologue ofT. gondiiMAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of ~67 kDa in extracts ofN. caninumtachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to inducein vitrobradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein inT. gondiibut those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

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Neospora caninum and neosporosis — recent achievements in host and parasite cell biology and treatment

Acta Parasitologica ◽

10.2478/s11686-006-0002-z ◽

2006 ◽

Vol 51 (1) ◽

Cited By ~ 5

Author(s):

Andrew Hemphill ◽

Bruno Gottstein

Keyword(s):

Life Cycle ◽

Host Cell ◽

Cell Biology ◽

Neospora Caninum ◽

Host Cells ◽

Term Survival ◽

Parasite Cell ◽

Invasion Stage

AbstractNeospora caninum is an apicomplexan parasite, which owes its importance to the fact that it represents the major infectious cause of bovine abortion worldwide. Its life cycle is comprised of three distinct stages: Tachyzoites, representing the proliferative and disease-causing stage, bradyzoites, representing a slowly replicating, tissue cyst-forming stage, and sporozoites, which represent the end product of a sexual process taking place within the intestinal tissue of the final canine host. Tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo, while bradyzoites have been found mainly within the central nervous system. In order to survive, proliferate, and proceed in its life cycle, N. caninum has evolved some amazing features. First, the parasite profits immensely from its ability to interact with, and invade, a large number of host cell types. Secondly, N. caninum exploits its capability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, 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. These three key events (host cell invasion — stage conversion — host cell modulation) represent potential targets for intervention. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are extensively exploited. In this review, we will summarize the present knowledge and achievements related to host cell and parasite cell biology.

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Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

PLoS Pathogens ◽

10.1371/journal.ppat.1009127 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009127

Author(s):

Suelen Silva Gomes Dias ◽

Vinicius Cardoso Soares ◽

André C. Ferreira ◽

Carolina Q. Sacramento ◽

Natalia Fintelman-Rodrigues ◽

...

Keyword(s):

Lipid Droplets ◽

Intracellular Transport ◽

Energy Homeostasis ◽

Lipid Synthesis ◽

Vero Cells ◽

Multiple Roles ◽

Host Cells ◽

Intracellular Parasites ◽

Viral Particles

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.

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Revisiting Ehrlichia ruminantium Replication Cycle Using Proteomics: The Host and the Bacterium Perspectives

Microorganisms ◽

10.3390/microorganisms9061144 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1144

Author(s):

Isabel Marcelino ◽

Philippe Holzmuller ◽

Ana Coelho ◽

Gabriel Mazzucchelli ◽

Bernard Fernandez ◽

...

Keyword(s):

Endothelial Cells ◽

Host Cell ◽

Cell Metabolism ◽

Replication Cycle ◽

Host Cells ◽

Ehrlichia Ruminantium ◽

Host Interaction ◽

Infected Cells ◽

Related Proteins

The Rickettsiales Ehrlichia ruminantium, the causal agent of the fatal tick-borne disease Heartwater, induces severe damage to the vascular endothelium in ruminants. Nevertheless, E. ruminantium-induced pathobiology remains largely unknown. Our work paves the way for understanding this phenomenon by using quantitative proteomic analyses (2D-DIGE-MS/MS, 1DE-nanoLC-MS/MS and biotin-nanoUPLC-MS/MS) of host bovine aorta endothelial cells (BAE) during the in vitro bacterium intracellular replication cycle. We detect 265 bacterial proteins (including virulence factors), at all time-points of the E. ruminantium replication cycle, highlighting a dynamic bacterium–host interaction. We show that E. ruminantium infection modulates the expression of 433 host proteins: 98 being over-expressed, 161 under-expressed, 140 detected only in infected BAE cells and 34 exclusively detected in non-infected cells. Cystoscape integrated data analysis shows that these proteins lead to major changes in host cell immune responses, host cell metabolism and vesicle trafficking, with a clear involvement of inflammation-related proteins in this process. Our findings led to the first model of E. ruminantium infection in host cells in vitro, and we highlight potential biomarkers of E. ruminantium infection in endothelial cells (such as ROCK1, TMEM16K, Albumin and PTPN1), which may be important to further combat Heartwater, namely by developing non-antibiotic-based strategies.

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