and
(Trypanosomatidae: Kinetoplastida) are parasitic protozoan causing Chagas disease, African Trypanosomiasis and Leishmaniases worldwide. They are vector borne diseases transmitted by triatomine ...bugs, Tsetse fly, and sand flies, respectively. Those diseases cause enormous economic losses and morbidity affecting not only rural and poverty areas but are also spreading to urban areas. During the parasite-host interaction, those organisms release extracellular vesicles (EVs) that are crucial for the immunomodulatory events triggered by the parasites. EVs are involved in cell-cell communication and can act as important pro-inflammatory mediators. Therefore, interface between EVs and host immune responses are crucial for the immunopathological events that those diseases exhibit. Additionally, EVs from these organisms have a role in the invertebrate hosts digestive tracts prior to parasite transmission. This review summarizes the available data on how EVs from those medically important trypanosomatids affect their interaction with vertebrate and invertebrate hosts.
ABSTRACT
The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub‐Saharan ...Africa and Latin America. Control measures have helped reduce the burden of their respective diseases—malaria, leishmaniasis and Chagas disease—in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub‐Saharan Africa and Latin America.
The framework summarises the potential translational utilities of extracellular vesicles in Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi. It highlights three strategic questions to be interrogated to usher in a translational future. TR1, translational agenda 1; TR2, translational agenda 2; TR3, translational agenda 3; CD, Chagas disease.
Chagas disease, caused by the protozoa parasite Trypanosoma cruzi, is a neglected tropical disease and a major public health problem affecting more than 6 million people worldwide. Many challenges ...remain in the quest to control Chagas disease: the diagnosis presents several limitations and the two available treatments cause several side effects, presenting limited efficacy during the chronic phase of the disease. In addition, there are no preventive vaccines or biomarkers of therapeutic response or disease outcome. Trypomastigote form and T. cruzi-infected cells release extracellular vesicles (EVs), which are involved in cell-to-cell communication and can modulate the host immune response. Importantly, EVs have been described as promising tools for the development of new therapeutic strategies, such as vaccines, and for the discovery of new biomarkers. Here, we review and discuss the role of EVs secreted during T. cruzi infection and their immunomodulatory properties. Finally, we briefly describe their potential for biomarker discovery and future perspectives as vaccine development tools for Chagas Disease.
Inflammasomes are large protein complexes that, once activated, initiate inflammatory responses by activating the caspase-1 protease. They play pivotal roles in host defense against pathogens. The ...well-established role of NAIP/NLRC4 inflammasome in bacterial infections involves NAIP proteins functioning as sensors for their ligands. However, recent reports have indicated the involvement of NLRC4 in non-bacterial infections and sterile inflammation, even though the role of NAIP proteins and the exact molecular mechanisms underlying inflammasome activation in these contexts remain to be elucidated. In this study, we investigated the activation of the NAIP/NLRC4 inflammasome in response to
, the protozoan parasite responsible for causing Chagas disease. This parasite has been previously demonstrated to activate NLRP3 inflammasomes. Here we found that NAIP and NLRC4 proteins are also required for IL-1β and Nitric Oxide (NO) release in response to
infection, with their absence rendering macrophages permissive to parasite replication. Moreover,
and
macrophages presented similar impaired responses to
, underscoring the non-redundant roles played by these inflammasomes during infection. Notably, it was the live trypomastigotes rather than soluble antigens or extracellular vesicles (EVs) secreted by them, that activated inflammasomes in a cathepsins-dependent manner. The inhibition of cathepsins effectively abrogated caspase-1 cleavage, IL-1β and NO release, mirroring the phenotype observed in
/
double knockout macrophages. Collectively, our findings shed light on the pivotal role of the NAIP/NLRC4 inflammasome in macrophage responses to
infection, providing new insights into its broader functions that extend beyond bacterial infections.
Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high ...attomole‐low femtomole range) method that uses liquid chromatography‐tandem mass spectrometry (LC‐MSn) for the first large‐scale analysis of GPI‐anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome‐wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI‐anchored proteins. By analyzing the GPIome of T. cruzi insect‐dwelling epimastigote stage using LC‐MSn, we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin‐like gene (TcSMUG S) family are the major GPI‐anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56–85 amino acids) and highly O‐glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.
Synopsis
Glycosylphosphatidylinositol (GPI) anchoring is a common modification of proteins found on the surface of eukaryotic cells. In higher eukaryotes such as mammals, GPI biosynthesis is vital for embryonic development, and GPI‐anchored proteins participate in important biological processes such as cell–cell interactions, signal transduction, endocytosis, complement regulation, and antigenic presentation (Paulick and Bertozzi, 2008). In lower eukaryotes such as protozoan parasites (e.g., Trypanosoma cruzi, Trypanosoma brucei, Leishmania spp., and Plasmodium spp.), which cause major endemic human infectious diseases worldwide (e.g., Chagas disease, sleeping sickness, leishmaniasis, malaria), GPI‐anchored molecules extensively coat the parasite cell surface and actively participate in relevant parasite‐mammalian host interactions (Ferguson, 1999).
T. cruzi is the etiologic agent of Chagas disease, or American trypanosomiasis, a neglected tropical disease that affects over 11 million people and causes an estimated 50 000 annual deaths in Latin America (Dias et al, 2002; Barrett et al, 2003; Moncayo and Ortiz Yanine, 2006). More recently, Chagas disease has become a public health menace for the U.S. and some European countries, where an increasing number of chronically T. cruzi‐infected migrants from endemic countries are residing in (Bern et al, 2007; Piron et al, 2008). There are only two commercial drugs (Benznidazole and Nifurtimox) available for the treatment of Chagas disease, and both are partially effective and highly toxic. In addition, no human vaccine is currently available for treating or preventing Chagas disease (Garg and Bhatia, 2005; Dumonteil, 2007; Hotez et al, 2008). Therefore, there is an urgent need for new therapeutic targets against T. cruzi. In this regard, GPI‐anchored proteins and free GPI anchors seem to be very attractive targets for development of new therapies for preventing or treating Chagas disease. These glycoconjugates play a central role in the parasite infectivity and host immune response against this deadly pathogen (Almeida and Gazzinelli, 2001; Buscaglia et al, 2006; Gazzinelli and Denkers, 2006; Acosta‐Serrano et al, 2007).
T. cruzi has four developmental stages or forms, two (i.e., epimastigote and metacyclic trypomastigote) dwelling in the hematophagous triatomine insect vector (a Reduviidae, popularly known as the kissing bug), and two (i.e., amastigote and trypomastigote) in the mammalian host. The parasite can be transmitted by contaminated excrement of the insect vector, blood transfusion, organ transplantation, or congenitally. Each developmental stage of T. cruzi has been proposed to express a different subset of GPI‐anchored proteins on the cell surface. These proteins are encoded by thousands of members of multigene families, such as trans‐sialidase (TS)/gp85 glycoprotein, mucin, mucin‐associated surface protein (MASP), and metalloproteinase gp63 (Buscaglia et al, 2006; Acosta‐Serrano et al, 2007). Although some of the expressed members (proteins) of these multigene families have been shown to be modified by GPI‐anchor addition, it has not been known how many of these gene products could possibly be GPI anchored. To answer this question, we performed a genome‐wise GPI‐anchoring prediction analysis. Here we show that approximately 12% of the annotated protein sequences of T. cruzi possibly code for GPI‐anchored proteins. This number is much higher compared with other lower and higher eukaryotes that have in average from 0.5 to 2% of proteins predicted to be GPI anchored.
Despite the overall importance of GPI anchors, there is no universal methodology for the systematic analysis of these molecules. One of the major hurdles to develop a method for the large scale analysis of GPIs is their complex structure. The general structure of a GPI anchor comprises a hydrophobic lipid tail and a hydrophilic carbohydrate (glycan) core, which together provide a highly amphiphilic character for these molecules (McConville and Ferguson, 1993; Ferguson, 1999). The complex structure and amphiphilic nature of GPIs make their extraction and purification more difficult, and multiple techniques are required to determine their fine structure.
To overcome this problem, here we have developed a fast, simple, and highly sensitive approach that uses liquid chromatography‐tandem mass spectrometry (LC‐MSn) for the first large‐scale analysis of GPI‐anchored molecules (i.e., the GPIome) of a eukaryote, T. cruzi. In our study, we analyzed GPI‐anchored molecules purified from the noninfective epimastigote forms of the parasite (Figure 3). Our analyses resulted in the identification 78 species of free GPIs (or GIPLs), of which 70 were novel species. Also, we identified 11 (8 novel) GPI species derived from GPI‐anchored proteins (Supplementary Table I). Finally, we determined that mucins encoded by the T. cruzi small mucin‐like gene (TcSMUG S) family are the major GPI‐anchored proteins expressed on the epimastigote cell surface. Taken together, our results and others from the literature suggest that the T. cruzi epimastigote cell surface is covered by a variety of GIPLs, and to a less extent by short and highly glycosylated GPI‐anchored mucins (Buscaglia et al, 2006). This thick layer of surface glycoconjugates may participate in the interaction of the parasite with the vector midgut and also may protect T. cruzi against the insect digestive enzymes. We also propose the use of this LC‐MSn method for the global analysis of the GPIome of other pathogenic eukaryotes and mammalian cells, including healthy and modified (cancer) cells.
The genome‐wise prediction analysis of the human pathogen T. cruzi reveals that approximately 12% of its annotated sequences have potential glycosylphosphatidylinositol (GPI)‐anchoring sites. This number is much higher compared to other lower eukaryotes, or even, higher eukaryotes.
Currently available methods for the analysis of GPI anchors have low resolution and require large amounts of sample. Here, we show that a polystyrene/divinylbenzene‐based resin linked to C4 groups achieves high performance in the purification of GPI‐anchored proteins and protein‐free GPIs (glycoinositolphospholipids, GIPLs).
We have developed a liquid chromatography‐mass spectrometry‐based method for the large‐scale analysis of the GPI‐anchored molecules (GPIome) of T. cruzi epimastigotes. This analysis led to the characterization of 90 GPI species, of which 79 were novel. Also, we identified mucins of the TcSMUG S family as the major GPI‐anchored glycoproteins expressed on T. cruzi epimastigote cell surface.
The T. cruzi epimastigote GPIome is rich in short, heavily O‐glycosylated GPI‐anchored polypeptides and free GPIs (GIPLs). These glycoconjugates may protect the parasite against the insect digestive enzymes and promote the parasite interaction with the insect's midgut.
Extracellular vesicles (EVs) are present in numerous peripheral bodily fluids and function in critical biological processes, including cell-to-cell communication. Most relevant to the present study, ...EVs contain microRNAs (miRNAs), and initial evidence from the field indicates that miRNAs detected in circulating EVs have been previously associated with mental health disorders. Here, we conducted an exploratory longitudinal and cross-sectional analysis of miRNA expression in serum EVs from adolescent participants. We analyzed data from a larger ongoing cohort study, evaluating 116 adolescent participants at two time points (wave 1 and wave 2) separated by three years. Two separate data analyses were employed: A cross-sectional analysis compared individuals diagnosed with Major Depressive Disorder (MDD), Anxiety disorders (ANX) and Attention deficit/Hyperactivity disorder (ADHD) with individuals without psychiatric diagnosis at each time point. A longitudinal analysis assessed changes in miRNA expression over time between four groups showing different diagnostic trajectories (persistent diagnosis, first incidence, remitted and typically developing/control). Total EVs were isolated, characterized by size distribution and membrane proteins, and miRNAs were isolated and sequenced. We then selected differentially expressed miRNAs for target prediction and pathway enrichment analysis. In the longitudinal analysis, we did not observe any statistically significant results. In the cross-sectional analysis: in the ADHD group, we observed an upregulation of miR-328-3p at wave 1 only; in the MDD group, we observed a downregulation of miR-4433b-5p, miR-584-5p, miR-625-3p, miR-432-5p and miR-409-3p at wave 2 only; and in the ANX group, we observed a downregulation of miR-432-5p, miR-151a-5p and miR-584-5p in ANX cases at wave 2 only. Our results identified previously observed and novel differentially expressed miRNAs and their relationship with three mental health disorders. These data are consistent with the notion that these miRNAs might regulate the expression of genes associated with these traits in genome-wide association studies. The findings support the promise of continued identification of miRNAs contained within peripheral EVs as biomarkers for mental health disorders.
We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B(2) receptor (B(2)R), thereby stimulating adaptive immunity. In this study, we show ...that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B(2)R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c(+) dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B(2)R-dependent up-regulation of IFN-gamma production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2(-/-) mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2(-/-) mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin "danger" signals is intensified through cooperative activation of TLR2 and B(2)R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B(2)R, and ACE.
Extracellular vesicles (EVs) shed by trypomastigote forms of
have the ability to interact with host tissues, increase invasion, and modulate the host innate response. In this study, EVs shed from
...-infected macrophages were investigated as immunomodulatory agents during the initial steps of infection. Initially, by scanning electron microscopy and nanoparticle tracking analysis, we determined that
-infected macrophages release higher numbers of EVs (50-300 nm) as compared to non-infected cells. Using Toll-like-receptor 2 (TLR2)-transfected CHO cells, we observed that pre-incubation of these host cells with parasite-derived EVs led to an increase in the percentage of infected cells. In addition, EVs from parasite or
-infected macrophages or not were able to elicit translocation of NF-κB by interacting with TLR2, and as a consequence, to alter the EVs the gene expression of proinflammatory cytokines (TNF-α, IL-6, and IL-1β), and STAT-1 and STAT-3 signaling pathways. By proteomic analysis, we observed highly significant changes in the protein composition between non-infected and infected host cell-derived EVs. Thus, we observed the potential of EVs derived from
during infection to maintain the inflammatory response in the host.
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