Stress Induces Release of Extracellular Vesicles by Trypanosoma cruzi Trypomastigotes

All extracellular forms of Trypanosoma cruzi, the causative agent of Chagas disease, release extracellular vesicles (EVs) containing major surface molecules of the parasite. EV release depends on several mechanisms (internal and external). However, most of the environmental conditions affecting this phenomenon are still unknown. In this work, we evaluated EV release under different stress conditions and their ability to be internalized by the parasites. In addition, we investigated whether the release conditions would affect their immunomodulatory properties in preactivated bone marrow-derived macrophages (BMDM). Sodium azide and methyl-cyclo-β-dextrin (CDB) reduced EV release, indicating that this phenomenon relies on membrane organization. EV release was increased at low temperatures (4°C) and acidic conditions (pH 5.0). Under this pH, trypomastigotes differentiated into amastigotes. EVs are rapidly liberated and reabsorbed by the trypomastigotes in a concentration-dependent manner. Nitrosative stress caused by sodium nitrite in acid medium or S-nitrosoglutathione also stimulated the secretion of EVs. EVs released under all stress conditions also maintained their proinflammatory activity and increased the expression of iNOS, Arg 1, IL-12, and IL-23 genes in IFN-γ and LPS preactivated BMDM. In conclusion, our results suggest a budding mechanism of release, dependent on the membrane structure and parasite integrity. Stress conditions did not affect functional properties of EVs during interaction with host cells. EV release variations under stress conditions may be a physiological response against environmental changes.

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Lipoproteins Are Responsible for the Pro-Inflammatory Property of Staphylococcus aureus Extracellular Vesicles

International Journal of Molecular Sciences ◽

10.3390/ijms22137099 ◽

2021 ◽

Vol 22 (13) ◽

pp. 7099

Author(s):

Pradeep Kumar Kopparapu ◽

Meghshree Deshmukh ◽

Zhicheng Hu ◽

Majd Mohammad ◽

Marco Maugeri ◽

...

Keyword(s):

Extracellular Vesicles ◽

Inflammatory Responses ◽

Surface Proteins ◽

Host Cells ◽

Immune Stimulation ◽

Domains Of Life ◽

Major Surface ◽

Staphylococcal Aureus

Staphylococcal aureus (S. aureus), a Gram-positive bacteria, is known to cause various infections. Extracellular vesicles (EVs) are a heterogeneous array of membranous structures secreted by cells from all three domains of life, i.e., eukaryotes, bacteria, and archaea. Bacterial EVs are implied to be involved in both bacteria–bacteria and bacteria–host interactions during infections. It is still unclear how S. aureus EVs interact with host cells and induce inflammatory responses. In this study, EVs were isolated from S. aureus and mutant strains deficient in either prelipoprotein lipidation (Δlgt) or major surface proteins (ΔsrtAB). Their immunostimulatory capacities were assessed both in vitro and in vivo. We found that S. aureus EVs induced pro-inflammatory responses both in vitro and in vivo. However, this activity was dependent on lipidated lipoproteins (Lpp), since EVs isolated from the Δlgt showed no stimulation. On the other hand, EVs isolated from the ΔsrtAB mutant showed full immune stimulation, indicating the cell wall anchoring of surface proteins did not play a role in immune stimulation. The immune stimulation of S. aureus EVs was mediated mainly by monocytes/macrophages and was TLR2 dependent. In this study, we demonstrated that not only free Lpp but also EV-imbedded Lpp had high pro-inflammatory activity.

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Glucose-restriction increasesTrichomonas vaginaliscellular damage towards HeLa cells and proteolytic activity of cysteine proteinases (CPs), such as TvCP2

Parasitology ◽

10.1017/s0031182019000209 ◽

2019 ◽

Vol 146 (9) ◽

pp. 1156-1166 ◽

Cited By ~ 2

Author(s):

Jesús F. T. Miranda-Ozuna ◽

Luis Alberto Rivera-Rivas ◽

Rosa Elena Cárdenas-Guerra ◽

Mar Sarai Hernández-García ◽

Sarahí Rodríguez-Cruz ◽

...

Keyword(s):

Proteolytic Activity ◽

Hela Cells ◽

Trichomonas Vaginalis ◽

Host Cells ◽

Cellular Damage ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cell Monolayers ◽

Glucose Restriction ◽

Effect Of Glucose

AbstractTrichomonas vaginalisinduces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose onT. vaginaliscytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface ofT. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

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Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2455 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2455-2458 ◽

Cited By ~ 47

Author(s):

J Nakajima-Shimada ◽

Y Hirota ◽

T Aoki

Keyword(s):

Trypanosoma Cruzi ◽

Growth Inhibition ◽

Host Cell ◽

Culture System ◽

Mammalian Cells ◽

Screening Method ◽

Host Cells ◽

Mammalian Host ◽

Dependent Manner ◽

Pyrimidine Analogs

Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.

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Differential regulation of metabolism by nitric oxide andS-nitrosothiols in endothelial cells

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00210.2011 ◽

2011 ◽

Vol 301 (3) ◽

pp. H803-H812 ◽

Cited By ~ 21

Author(s):

Anne R. Diers ◽

Katarzyna A. Broniowska ◽

Victor M. Darley-Usmar ◽

Neil Hogg

Keyword(s):

Nitric Oxide ◽

Endothelial Cells ◽

Oxidative Phosphorylation ◽

Metabolic Pathways ◽

Nitrosative Stress ◽

Surrogate Marker ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

No Donor

S-nitrosation of thiols in key proteins in cell signaling pathways is thought to be an important contributor to nitric oxide (NO)-dependent control of vascular (patho)physiology. Multiple metabolic enzymes are targets of both NO and S-nitrosation, including those involved in glycolysis and oxidative phosphorylation. Thus it is important to understand how these metabolic pathways are integrated by NO-dependent mechanisms. Here, we compared the effects of NO and S-nitrosation on both glycolysis and oxidative phosphorylation in bovine aortic endothelial cells using extracellular flux technology to determine common and unique points of regulation. The compound S-nitroso-l-cysteine (l-CysNO) was used to initiate intracellular S-nitrosation since it is transported into cells and results in stable S-nitrosation in vitro. Its effects were compared with the NO donor DetaNONOate (DetaNO). DetaNO treatment caused only a decrease in the reserve respiratory capacity; however, l-CysNO impaired both this parameter and basal respiration in a concentration-dependent manner. In addition, DetaNO stimulated extracellular acidification rate (ECAR), a surrogate marker of glycolysis, whereas l-CysNO stimulated ECAR at low concentrations and inhibited it at higher concentrations. Moreover, a temporal relationship between NO- and S-nitrosation-mediated effects on metabolism was identified, whereby NO caused a rapid impairment in mitochondrial function, which was eventually overwhelmed by S-nitrosation-dependent processes. Taken together, these results suggest that severe pharmacological nitrosative stress may differentially regulate metabolic pathways through both intracellular S-nitrosation and NO-dependent mechanisms. Moreover, these data provide insight into the role of NO and related compounds in vascular (patho)physiology.

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Identification of the natural product berberine as an antiviral drug

AMB Express ◽

10.1186/s13568-020-01088-2 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Jiping Shao ◽

Debin Zeng ◽

Shuhong Tian ◽

Gezhi Liu ◽

Jian Fu

Keyword(s):

Conformational Changes ◽

Polyacrylamide Gel Electrophoresis ◽

Antiviral Drug ◽

Host Cells ◽

Heptad Repeat ◽

Potential Candidate ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Antiviral Activities ◽

Hiv 1

Abstract Drugs targeting the fusion process of viral entry into host cells have been approved for clinical use in the treatment of AIDS. There remains a great need to improve the use of existing drugs for HIV therapy. Berberine is traditionally used to treat diarrhea, bacillary dysentery, and gastroenteritis in clinics, here our research shows that berberine is effective in inhibiting HIV-1 entry. Native polyacrylamide gel electrophoresis studies reveal that berberine can directly bind to both N36 and C34 to form a novel N36-berberine-C34 complex and effectively block the six-helix bundle formation between the N-terminal heptad repeat peptide N36 and the C-terminal heptad repeat peptide C34. Circular dichroism experiments show that binding of berberine produces conformational changes that damages the secondary structures of 6-HB. Computer-aided molecular docking studies suggest a hydrogen bond with T-639 and two polar bonds with Q-563 and T-639 are established, involving the oxygen atom and the C=O group of the indole ring. Berberine completely inhibits six HIV-1 clade B isolates and exhibits antiviral activities in a concentration-dependent manner with IC50 values varying from 5.5 to 10.25 µg/ml. This compound-peptide interaction may represent a mechanism of action of antiviral activities of berberine. As a summary, these studies successfully identify compound berberine as a potential candidate drug for HIV-1 treatment. As a summary, antiviral activity of berberine in combination with its use in clinical practice, this medicine can be used as a potential clinically anti-HIV drug.

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Infectivity of Trypanosoma cruzi strains is associated with differential expression of surface glycoproteins with differential Ca2+ signalling activity

Biochemical Journal ◽

10.1042/bj3300505 ◽

1998 ◽

Vol 330 (1) ◽

pp. 505-511 ◽

Cited By ~ 107

Author(s):

C. Rita RUIZ ◽

Silvio FAVORETO ◽

L. Miriam DORTA ◽

E. M. Maria OSHIRO ◽

T. Alice FERREIRA ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Hela Cells ◽

Strain Analysis ◽

Inverse Correlation ◽

Host Cells ◽

Target Cells ◽

Metacyclic Trypomastigotes ◽

Cell Components ◽

Major Surface ◽

Surface Glycoproteins

Mammalian cell invasion assays, using metacyclic trypomastigotes of Trypanosoma cruzi G and CL strains, showed that the CL strain enters target cells in several-fold higher numbers as compared with the G strain. Analysis of expression of surface glycoproteins in metacyclic forms of the two strains by iodination, immunoprecipitation and FACS, revealed that gp90, undetectable in the CL strain, is one of the major surface molecules in the G strain, that expression of gp82 is comparable in both strains and that gp35/50 is expressed at lower levels in the CL strain. Purified gp90 and gp35/50 bound more efficiently than gp82 to cultured HeLa cells. However, the intensity of the Ca2+ response triggered in HeLa cells by gp82 was significantly higher than that induced by gp35/50 or gp90. Most of the Ca2+ signalling activity of the metacyclic extract towards HeLa cells was due to gp82 and was inhibitable by gp82-specific monoclonal antibody 3F6. Ca2+ mobilization was also triggered in metacyclic trypomastigotes by host-cell components; it was mainly gp82-mediated and more intense in the CL than in the G strain. We propose that expression of gp90 and gp35/50 at high levels impairs binding of metacyclic forms to host cells through productive gp82-mediated interaction, which leads to the target-cell and parasite Ca2+ mobilization required for invasion. Analysis of metacyclic forms of eight additional T. cruzi strains corroborated the inverse correlation between infectivity and expression of gp90 and gp35/50.

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LuxR Homologue SmcR Is Essential for Vibrio vulnificus Pathogenesis and Biofilm Detachment, and Its Expression is Induced by Host Cells

Infection and Immunity ◽

10.1128/iai.00561-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3721-3730 ◽

Cited By ~ 37

Author(s):

Seung Min Kim ◽

Jin Hwan Park ◽

Hyun Sung Lee ◽

Won Bin Kim ◽

Jung Min Ryu ◽

...

Keyword(s):

Quorum Sensing ◽

Vibrio Vulnificus ◽

Cell Communication ◽

Cellular Level ◽

Host Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Content Type ◽

Biofilm Detachment

ABSTRACTQuorum sensing is a cell-to-cell communication system known to control many bacterial processes. In the present study, the functions of quorum sensing in the pathogenesis ofVibrio vulnificus, a food-borne pathogen, were assessed by evaluating the virulence of a mutant deficient in SmcR, a quorum-sensing regulator and homologue of LuxR. When biofilms were used as an inoculum, thesmcRmutant was impaired in virulence and colonization capacity in the infection of mice. The lack of SmcR also resulted in decreased histopathological damage in mouse jejunum tissue. These results indicated that SmcR is essential forV. vulnificuspathogenesis. Moreover, thesmcRmutant exhibited significantly reduced biofilm detachment. Upon exposure to INT-407 host cells, the wild type, but not thesmcRmutant, revealed accelerated biofilm detachment. The INT-407 cells increasedsmcRexpression by activating the expression of LuxS, an autoinducer-2 synthase, indicating that host cells manipulate the cellular level of SmcR through the quorum-sensing signaling ofV. vulnificus. A whole-genome microarray analysis revealed that the genes primarily involved in biofilm detachment and formation are up- and downregulated by SmcR, respectively. Among the SmcR-regulated genes,vvpEencoding an elastolytic protease was the most upregulated, and the purified VvpE appeared to dissolve established biofilms directly in a concentration-dependent mannerin vitro. These results suggest that the host cell-induced SmcR enhances the detachment ofV. vulnificusbiofilms entering the host intestine and thereby may promote the dispersal of the pathogen to new colonization loci, which is crucial for pathogenesis.

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Pyruvate : ferredoxin oxidoreductase (PFO) is a surface-associated cell-binding protein in Trichomonas vaginalis and is involved in trichomonal adherence to host cells

Microbiology ◽

10.1099/mic.0.053033-0 ◽

2011 ◽

Vol 157 (12) ◽

pp. 3469-3482 ◽

Cited By ~ 25

Author(s):

Patricia Meza-Cervantez ◽

Arturo González-Robles ◽

Rosa Elena Cárdenas-Guerra ◽

Jaime Ortega-López ◽

Emma Saavedra ◽

...

Keyword(s):

Enzyme Activity ◽

Trichomonas Vaginalis ◽

Cellular Localization ◽

Binding Protein ◽

Host Cells ◽

Dependent Manner ◽

Cell Binding ◽

Concentration Dependent Manner ◽

Ferredoxin Oxidoreductase ◽

Pyruvate Ferredoxin Oxidoreductase

The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate : ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.

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Stable tRNA halves can be sorted into extracellular vesicles and delivered to recipient cells in a concentration-dependent manner

RNA Biology ◽

10.1080/15476286.2019.1708548 ◽

2019 ◽

Vol 17 (8) ◽

pp. 1168-1182 ◽

Cited By ~ 9

Author(s):

Fabiana Gámbaro ◽

Marco Li Calzi ◽

Pablo Fagúndez ◽

Bruno Costa ◽

Gonzalo Greif ◽

...

Keyword(s):

Extracellular Vesicles ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Trna Halves

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Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi

Biochemical Journal ◽

10.1042/bj3320695 ◽

1998 ◽

Vol 332 (3) ◽

pp. 695-702 ◽

Cited By ~ 25

Author(s):

Marlene BENCHIMOL ◽

Wanderley de SOUZA ◽

Nicole VANDERHEYDEN ◽

Li ZHONG ◽

Hong-Gang LU ◽

...

Keyword(s):

Plasma Membrane ◽

Trypanosoma Cruzi ◽

Developmental Stages ◽

Functional Expression ◽

Surface Proteins ◽

Dependent Manner ◽

Bafilomycin A1 ◽

Concentration Dependent Manner ◽

Surface Localization ◽

Acidic Compartments

Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4±2.8 (S.D.) nmol of H+/min per 108 amastigotes and 6.37±0.8 nmol of H+/min per 108 trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9±2.2 nmol of H+/min per 108 cells), but not by trypomastigotes (5.1±1.7 nmol of H+/min per 108 cells). The localization of the V-H+-ATPase of T. cruziwas investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.

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