Parasitic diseases remain a major global health problem for humans. Parasites employ a variety of strategies to invade and survive within their hosts and to manipulate host defense mechanisms, always ...in the pathogen's favor. Extracellular vesicles (EVs), membrane-bound nanospheres carrying a variety of bioactive compounds, were shown to be released by the parasites during all stages of the infection, enabling growth and expansion within the host and adaptation to frequently changing environmental stressors. In this review, we discuss how the use of existing nanotechnologies and high-resolution imaging tools assisted in revealing the role of EVs during parasitic infections, enabling the quantitation, visualization, and detailed characterization of EVs. We discuss here the cases of malaria, Chagas disease and leishmaniasis as examples of parasitic neglected tropical diseases (NTDs). Unraveling the EVs' role in the NTD pathogenesis may enormously contribute to their early and reliable diagnostic, effective treatment, and prevention.
Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to ...digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature.
Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied.
Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.
Mesenchymal stromal cells (MSCs) can generate immunological tolerance due to their regulatory activity in many immune cells. Extracellular vesicles (EVs) release is a pivotal mechanism by which MSCs ...exert their actions. In this study, we evaluate whether mesenchymal stromal cell extracellular vesicles (MSC-EVs) can modulate T cell response. MSCs were expanded and EVs were obtained by differential ultracentrifugation of the supernatant. The incorporation of MSC-EVs by T cells was detected by confocal microscopy. Expression of surface markers was detected by flow cytometry or CytoFLEX and cytokines were detected by RT-PCR, FACS and confocal microscopy and a miRNA PCR array was performed. We demonstrated that MSC-EVs were incorporated by lymphocytes in vitro and decreased T cell proliferation and Th1 differentiation. Interestingly, in Th1 polarization, MSC-EVs increased Foxp3 expression and generated a subpopulation of IFN-γ
/Foxp3
T cells with suppressive capacity. A differential expression profile of miRNAs in MSC-EVs-treated Th1 cells was seen, and also a modulation of one of their target genes,
. MSC-EVs altered the metabolism of Th1-differentiated T cells, suggesting the involvement of the TGF-β pathway in this metabolic modulation. The addition of MSC-EVs in vivo, in an OVA immunization model, generated cells Foxp3
. Thus, our findings suggest that MSC-EVs are able to specifically modulate activated T cells at an alternative regulatory profile by miRNAs and metabolism shifting.
Abstract
Cell culture‐conditioned medium (CCM) is a valuable source of extracellular vesicles (EVs) for basic scientific, therapeutic and diagnostic applications. Cell culturing parameters affect the ...biochemical composition, release and possibly the function of CCM‐derived EVs (CCM‐EV). The CCM‐EV task force of the Rigor and Standardization Subcommittee of the International Society for Extracellular Vesicles aims to identify relevant cell culturing parameters, describe their effects based on current knowledge, recommend reporting parameters and identify outstanding questions. While some recommendations are valid for all cell types, cell‐specific recommendations may need to be established for non‐mammalian sources, such as bacteria, yeast and plant cells. Current progress towards these goals is summarized in this perspective paper, along with a checklist to facilitate transparent reporting of cell culturing parameters to improve the reproducibility of CCM‐EV research.
Ergosterol biosynthesis inhibitors, such as posaconazole and ravuconazole, have been proposed as drug candidates for Chagas disease, a neglected infectious tropical disease caused by the protozoan ...parasite Trypanosoma cruzi. To understand better the mechanism of action and resistance to these inhibitors, a clone of the T. cruzi Y strain was cultured under intermittent and increasing concentrations of ravuconazole until phenotypic stability was achieved. The ravuconazole-selected clone exhibited loss in fitness in vitro when compared to the wild-type parental clone, as observed in reduced invasion capacity and slowed population growth in both mammalian and insect stages of the parasite. In drug activity assays, the resistant clone was above 300-fold more tolerant to ravuconazole than the sensitive parental clone, when the half-maximum effective concentration (EC50) was considered. The resistant clones also showed reduced virulence in vivo, when compared to parental sensitive clones. Cross-resistance to posaconazole and other CYP51 inhibitors, but not to other antichagasic drugs that act independently of CYP51, such as benznidazole and nifurtimox, was also observed. A novel amino acid residue change, T297M, was found in the TcCYP51 gene in the resistant but not in the sensitive clones. The structural effects of the T297M, and of the previously described P355S residue changes, were modelled to understand their impact on interaction with CYP51 inhibitors.
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•A ravuconazole-resistant T. cruzi clone presented reduced in vitro and in vivo fitness.•The ravuconazole-resistant clone presented cross-resistance to other CYP51 inhibitors.•There was no cross-resistance to benznidazole and nifurtimox.•Resistance is associated with a novel structural mutation in the TcCYP51 protein.
It has been proposed that self and protozoan-derived GPI anchors are natural ligands of CD1d. In this study, we investigated the ability of GPI anchors from Trypanosoma cruzi to bind to CD1d and ...mediate activation of NKT cells. We observed that GPI-anchored mucin-like glycoproteins (GPI mucins), glycoinositolphospholipids (GIPLs), and their phosphatidylinositol moieties bind to rCD1d and inhibit the stimulation of a NKT hybridoma by the alpha-galactosylceramide-CD1 complex. However, these GPI anchors and related structures were unable to activate NKT cells in vitro or in vivo. We found that high titers of Ab anti-GPI mucins, but not anti-GIPLs, were detected in sera from wild-type as well as in TAP1(-/-), CD1d(-/-), and MHC class II(-/-) mice after immunization. However, T-dependent anti-GPI mucin Ab isotypes, such as IgG1, IgG2a, IgG2b, and IgG3, were absent on MHC class II(-/-), but were conserved in CD1d(-/-) and TAP1(-/-) mice. Furthermore, we found that CD1d(-/-) mice presented a robust cytokine as well as anti-GPI mucins and anti-GIPL Ab responses, upon infection with T. cruzi parasites. These results indicate that, despite binding to CD1d, GPI mucins and related structures expressed by T. cruzi appear not to evoke dominant CD1d-restricted immune responses in vivo. In contrast, MHC class II is critical for the production of the major Ig G isotypes against GPI mucins from T. cruzi parasites.
•This review summarizes the current literature of themes focusing on toxoplasmosis.•The themes are focusing on human toxoplasmosis and animal toxoplasmosis.•Studies in experimental models and genetic ...characterization of T. gondii strains.•T. gondii outbreaks caused by food and water and toxoplasmosis and One Health.•Clinical forms and diagnosis of toxoplasmosis.
Toxoplasmosis is a unique health disease that significantly affects the health of humans, domestic animals, wildlife and is present in ecosystems, including water, soil and food. Toxoplasma gondii is one of the best-adapted parasites in the word. This parasite is able to persist for long periods in its hosts, in different geographic regions of the word. This review summarizes the current literature of these themes, focusing on: (1) toxoplasmosis, a zoonotic infection; (2) One health approach and toxoplasmosis; (3) human toxoplasmosis; (4) animal toxoplasmosis; (5) toxoplasmosis diagnosis, as immunological, parasitological and molecular diagnosis; (6) T. gondii outbreaks caused by infected meat, milk and dairy products, as well as, vegetables and water consume; (7) studies in experimental models; (8) genetic characterization of T. gondii strains; (9) extracellular vesicles and miRNA; and (10) future perspectives on T. gondii and toxoplasmosis. The vast prevalence of toxoplasmosis in both humans and animals and the dispersion and resistence of T. gondii parasites in environment highlight the importance of the one health approach in diagnostic and control of the disease. Here the different aspects of the one health approach are presented and discussed.
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BACKGROUND: Leishmania enriettii is a species non-infectious to man, whose reservoir is the guinea pig Cavia porcellus. Many aspects of the parasite-host interaction in this model are unknown, ...especially those involving parasite surface molecules. While lipophosphoglycans (LPGs) and glycoinositolphospholipids (GIPLs) of Leishmania species from the Old and New World have already been described, glycoconjugates of L. enriettii and their importance are still unknown. METHODS: Mice peritoneal macrophages from C57BL/6 and knock-out (TLR2 −/−, TLR4 −/−) were primed with IFN-γ and stimulated with purified LPG and GIPLs from both species. Nitric oxide and cytokine production were performed. MAPKs (p38 and JNK) and NF-kB activation were evaluated in J774.1 macrophages and CHO cells, respectively. RESULTS: LPGs were extracted, purified and analysed by western-blot, showing that LPG from L88 strain was longer than that of Cobaia strain. LPGs and GIPLs were depolymerised and their sugar content was determined. LPGs from both strains did not present side chains, having the common disaccharide Gal(β1,4)Man(α1)-PO₄. The GIPL from L88 strain presented galactose in its structure, suggestive of type II GIPL. On the other hand, the GIPL of Cobaia strain presented an abundance of glucose, a characteristic not previously observed. Mice peritoneal macrophages from C57BL/6 and knock-outs (TLR2 -/- and TLR4 -/-) were primed with IFN-γ and stimulated with glycoconjugates and live parasites. No activation of NO or cytokines was observed with live parasites. On the other hand, LPGs and GIPLs were able to activate the production of NO, IL-6, IL-12 and TNF–α preferably via TRL2. However, in CHO cells, only GIPLs were able to activate TRL2 and TRL4. In vivo studies using male guinea pigs (Cavia porcellus) showed that only strain L88 was able to develop more severe ulcerated lesions especially in the presence of salivary gland extract (SGE). CONCLUSION: The two L. enriettii strains exhibited polymorphisms in their LPGs and GIPLs and those features may be related to a more pro-inflammatory profile in the L88 strain.
Malaria is the most serious mosquito‐borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum. The parasite invades human red blood cells and releases extracellular ...vesicles (EVs) to alter its host responses. It becomes clear that EVs are generally composed of sub‐populations. Seeking to identify EV subpopulations, we subject malaria‐derived EVs to size‐separation analysis, using asymmetric flow field‐flow fractionation. Multi‐technique analysis reveals surprising characteristics: we identify two distinct EV subpopulations differing in size and protein content. Small EVs are enriched in complement‐system proteins and large EVs in proteasome subunits. We then measure the membrane fusion abilities of each subpopulation with three types of host cellular membranes: plasma, late and early endosome. Remarkably, small EVs fuse to early endosome liposomes at significantly greater levels than large EVs. Atomic force microscope imaging combined with machine‐learning methods further emphasizes the difference in biophysical properties between the two subpopulations. These results shed light on the sophisticated mechanism by which malaria parasites utilize EV subpopulations as a communication tool to target different cellular destinations or host systems.
Synopsis
Plasmodium falciparum invades human red blood cells and releases two extracellular vesicle subsets secreted by infected cells. These EV subpopulations harbor different protein cargo and have specific mechanical membrane properties, suggesting distinct host cell targets.
Two distinct subsets of malaria‐derived EVs with different sizes are identified using asymmetric flow field‐flow fractionation.
Small EVs are rich in complement system proteins, whereas large EVs contain 20S proteasome subunits.
Small EVs are more efficient in fusing under endosomal conditions as compared to the large subset.
The EV subpopulations possess distinct membrane mechanical properties, suggesting different lipid compositions.
Plasmodium falciparum invades human red blood cells and releases two extracellular vesicle subsets secreted by infected cells. These EV subpopulations harbor different protein cargo and have specific mechanical membrane properties, suggesting distinct host cell targets.
B‐1 cells are a B‐lymphocyte subtype whose roles in immunity are not completely defined. These cells can produce cytokines (mainly IL‐10) and natural and specific antibodies. Currently, extracellular ...vesicles (EVs) released by immune cells have emerged as new important entities in cell‐cell communication. Immune cells release EVs that can activate and/or modulate other immune cells. Here, we characterized the EVs released by peritoneal B‐1 cells infected or not with Leishmania (Leishmania) amazonensis. This Leishmania species causes cutaneous leishmaniasis and can infect macrophages and B‐1 cells. Our results showed that peritoneal B‐1 cells spontaneously release EVs, but the parasite stimulated an increase in EVs production by peritoneal B‐1 cells. The treatment of BALB/c and C57BL/6 bone marrow‐derived macrophages (BMDM) with EVs from infected peritoneal B‐1 cells led to differential expression of iNOS, IL‐6, IL‐10, and TNF‐α. Additionally, BALB/c mice previous treated with EVs released by peritoneal B‐1 cells showed a significant lower lesion size and parasite burden. Thus, this study demonstrated that peritoneal B‐1 cells could release EVs that can alter the functions of macrophages in vitro and in vivo these EVs altered the course of L. amazonensis infection. These findings represent the first evidence that EVs from peritoneal B‐1 cells can act as a new mechanism of cellular communication between macrophages and B‐1 cells, contributing to immunity against experimental leishmaniasis.
Graphical
EVs from peritoneal B‐1 cells contribute to immunity against experimental leishmaniasis
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