Extracellular vesicles are small membrane structures containing proteins and nucleic acids that are gaining a lot of attention lately. They are produced by most cells and can be detected in several ...body fluids, having a huge potential in therapeutic and diagnostic approaches. EVs produced by infected cells usually have a molecular signature that is very distinct from healthy cells. For intracellular pathogens like viruses, EVs can have an even more complex function, since the viral biogenesis pathway can overlap with EV pathways in several ways, generating a continuum of particles, like naked virions, EVs containing infective viral genomes and quasi-enveloped viruses, besides the classical complete viral particles that are secreted to the extracellular space. Those particles can act in recipient cells in different ways. Besides being directly infective, they also can prime neighbor cells rendering them more susceptible to infection, block antiviral responses and deliver isolated viral molecules. On the other hand, they can trigger antiviral responses and cytokine secretion even in uninfected cells near the infection site, helping to fight the infection and protect other cells from the virus. This protective response can also backfire, when a massive inflammation facilitated by those EVs can be responsible for bad clinical outcomes. EVs can help or harm the antiviral response, and sometimes both mechanisms are observed in infections by the same virus. Since those pathways are intrinsically interlinked, understand the role of EVs during viral infections is crucial to comprehend viral mechanisms and respond better to emerging viral diseases.
RNA-binding proteins (RBPs) have important functions in the regulation of gene expression. RBPs play key roles in post-transcriptional processes in all eukaryotes, such as splicing regulation, mRNA ...transport and modulation of mRNA translation and decay. RBPs assemble into different mRNA-protein complexes, which form messenger ribonucleoprotein complexes (mRNPs). Gene expression regulation in trypanosomatids occurs mainly at the post-transcriptional level and RBPs play a key role in all processes. However, the functional characterization of RBPs in Trypanosoma cruzi has been impaired due to the lack of reliable reverse genetic manipulation tools. The comparison of RBPs from Saccharomyces cerevisiae and T. cruzi might allow inferring on the function of these proteins based on the information available for the orthologous RNA-binding proteins from the S. cerevisiae model organism. In this review, we discuss the role of some RBPs from T. cruzi and their homologues in regulating gene expression in yeast.
The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. Despite the importance of this type of control in Trypanosoma cruzi, few RNA binding proteins ...have been characterized. The RRM domain (RNA Recognition Motif) is one of the most abundant domains found in RNA-binding proteins in higher eukaryotes. Proteins containing the RRM domain are involved in the majority of post-transcriptional processes regulating gene expression. In this work, we aimed to characterize the protein TcNRBD1 from T. cruzi. TcNRBD1 is an RNA-binding protein that contains 2 RRM domains and is the ortholog of the P34 and P37 proteins from Trypanosoma brucei. The TcNRBD1 protein is expressed in all developmental stages of T. cruzi, and its localization pattern is concentrated at the perinuclear region. TcNRBD1 is associated with polysomes and with the 80S monosomes. Furthermore, sequencing of the mRNAs bound to TcNRBD1 allowed the identification of several transcripts that encode ribosomal proteins. Immunoprecipitation assays followed by mass spectrometry showed that the protein complexes with several ribosomal proteins from both the 40S and 60S subunits. In summary, the results indicate that TcNRBD1 is associated with different parts of the translation process, either by regulating mRNAs that encode ribosomal proteins or by acting in some step of ribosome assembly in T. cruzi.
The extracellular vesicle (EVs) traffic has been highlighted as a very important pathway of cellular communication. EVs are produced by prokaryotes and eukaryotes organisms and can carry molecules to ...help maintain homeostasis, responding to general disbalance, infections, and allowing rapid modulation of the immune system. In the context of infection, EVs from both the host and the pathogen have been identified as playing roles in the recruitment of immunological molecules that can lead to the resolution of the infection or the host's defeat. Bacterial vesicles RNA cargo play roles in the host cell by regulating gene expression and modulating immune response. In fungi the RNA molecules present in EVs are diverse and participate in communication between the host and pathogenic fungi. Little is known about how cross-kingdom sRNA trafficking occurs, although in recent years, there has been an increase in studies that relate EV participation in sRNA delivery. This review aims to elucidate and update the reader concerning the role of extracellular vesicles, with emphasis in the RNA content. We describe the EVs during infection from the host point-of-view, as well as the bacteria and fungi pathogens producing EVs that help the establishment of the disease.
Endothelial-like cells may be obtained from CD133+ mononuclear cells isolated from human umbilical cord blood (hUCB) and expanded using endothelial-inducing medium (E-CD133 cells). Their use in ...regenerative medicine has been explored by the potential not only to form vessels but also by the secretion of bioactive elements. Extracellular vesicles (EVs) are prominent messengers of this paracrine activity, transporting bioactive molecules that may guide cellular response under different conditions. Using RNA-Seq, we characterized the miRNA content of EVs derived from E-CD133 cells cultivated under normoxia (N-EVs) and hypoxia (H-EVs) and observed that changing the O2 status led to variations in the selective loading of miRNAs in the EVs. In silico analysis showed that among the targets of differentially loaded miRNAs, there are transcripts involved in pathways related to cell growth and survival, such as FoxO and HIF-1 pathways. The data obtained reinforce the pro-regenerative potential of EVs obtained from E-CD133 cells and shows that fine tuning of their properties may be regulated by culture conditions.
In this study, we investigated the influence of fungal extracellular vesicles (EVs) during biofilm formation and morphogenesis in Candida albicans. Using crystal violet staining and scanning electron ...microscopy (SEM), we demonstrated that C. albicans EVs inhibited biofilm formation
. By time-lapse microscopy and SEM, we showed that C. albicans EV treatment stopped filamentation and promoted pseudohyphae formation with multiple budding sites. The ability of C. albicans EVs to regulate dimorphism was further compared to EVs isolated from different C. albicans strains, Saccharomyces cerevisiae, and Histoplasma capsulatum. C. albicans EVs from distinct strains inhibited yeast-to-hyphae differentiation with morphological changes occurring in less than 4 h. EVs from S. cerevisiae and H. capsulatum modestly reduced morphogenesis, and the effect was evident after 24 h of incubation. The inhibitory activity of C. albicans EVs on phase transition was promoted by a combination of lipid compounds, which were identified by gas chromatography-tandem mass spectrometry analysis as sesquiterpenes, diterpenes, and fatty acids. Remarkably, C. albicans EVs were also able to reverse filamentation. Finally, C. albicans cells treated with C. albicans EVs for 24 h lost their capacity to penetrate agar and were avirulent when inoculated into Galleria mellonella. Our results indicate that fungal EVs can regulate yeast-to-hypha differentiation, thereby inhibiting biofilm formation and attenuating virulence.
The ability to undergo morphological changes during adaptation to distinct environments is exploited by Candida albicans and has a direct impact on biofilm formation and virulence. Morphogenesis is controlled by a diversity of stimuli, including osmotic stress, pH, starvation, presence of serum, and microbial components, among others. Apart from external inducers, C. albicans also produces autoregulatory substances. Farnesol and tyrosol are examples of quorum-sensing molecules (QSM) released by C. albicans to regulate yeast-to-hypha conversion. Here, we demonstrate that fungal EVs are messengers impacting biofilm formation, morphogenesis, and virulence in C. albicans. The major players exported in C. albicans EVs included sesquiterpenes, diterpenes, and fatty acids. The understanding of how C. albicans cells communicate to regulate physiology and pathogenesis can lead to novel therapeutic tools to combat candidiasis.
Immunoprecipitation is a helpful tool to assess interactions between proteins and proteins or nucleic acids (DNA or RNA). Its principle consists in capturing and enriching one or multiple target ...proteins from a complex sample with a specific antibody conjugated to a solid matrix and isolating the RNA and/or protein molecules associated to those target(s) group of proteins that can be further identified by advanced techniques such as RNA-seq and/or mass spectrometry. Since this technique allows for identifying, mapping, and checking new protein-protein and protein-RNA interactions, its use is very convenient in situations where many proteins remain with their functions uncharacterized, as is the case of the protozoan Trypanosoma cruzi. Here we describe a protocol that is based on the cryogrinding method for cell lysis and the use of antibodies conjugated to magnetic beads to capture and purify protein complexes in a robust and efficient way.
RNA-binding proteins (RBPs) are well known as key factors in gene expression regulation in eukaryotes. These proteins associate with mRNAs and other proteins to form mRNP complexes that ultimately ...determine the fate of target transcripts in the cell. This association is usually mediated by an RNA-recognition motif (RRM). In the case of trypanosomatids, these proteins play a paramount role, as gene expression regulation is mostly posttranscriptional. Despite their relevance in the life cycle of Trypanosoma cruzi, the causative agent of Chagas' disease, to date, few RBPs have been characterized in this parasite.
We investigated the role of DRBD2 in T. cruzi, an RBP with two RRM domains that is associated with cytoplasmic translational complexes. We show that DRBD2 is an ortholog of the Gbp2 in yeast, an SR-rich protein involved in mRNA quality control and export. We used an immunoprecipitation assay followed by shotgun proteomics and RNA-seq to assess the interaction partners of the DRBD2-mRNP complex in epimastigotes. The analysis identified mostly proteins involved in RNA metabolism and regulation, such as ALBA1, ALBA3, ALBA4, UBP1, UBP2, DRBD3, and PABP2. The RNA-seq results showed that most of the transcripts regulated by the DRBD2 complex mapped to hypothetical proteins related to multiple processes, such as to biosynthetic process, DNA metabolic process, protein modification, and response to stress.
The identification of regulatory proteins in the DRBD2-mRNP complex corroborates the important role of DRBD2 in gene expression regulation in T. cruzi. We consider these results an important contribution to future studies regarding gene expression regulation in T. cruzi, especially in the field of RNA-binding proteins.
Chagas disease is a neglected tropical disease caused by
. This protozoan developed several mechanisms to infect, propagate, and survive in different hosts. The specific expression of proteins is ...responsible for morphological and metabolic changes in different parasite stages along the parasite life cycle. The virulence strategies at the cellular and molecular levels consist of molecules responsible for mediating resistance mechanisms to oxidative damage, cellular invasion, and immune evasion, performed mainly by surface proteins. Since parasite surface coat remodeling is crucial to invasion and infectivity, surface proteins are essential virulence elements. Understanding the factors involved in these processes improves the knowledge of parasite pathogenesis. Genome sequencing has opened the door to high-throughput technologies, allowing us to obtain a deeper understanding of gene reprogramming along the parasite life cycle and identify critical molecules for survival. This review therefore focuses on proteins regulated during differentiation into infective forms considered virulence factors and addresses the current known mechanisms acting in the modulation of gene expression, emphasizing mRNA signals, regulatory factors, and protein complexes.
Post-transcriptional regulation of gene expression is a critical process for adapting to and surviving
, a parasite with a complex life cycle. RNA-binding proteins (RBPs) are key players in this ...regulation, forming ribonucleoprotein complexes (messenger ribonucleoproteins) and RNA granules that control transcript stability, localization, degradation, and translation modulation. Understanding the specific roles of individual RBPs is crucial for unraveling the details of this regulatory network. In this study, we generated null mutants of the TcZC3HTTP gene, a specific RBP in the
family characterized by a C3H zinc finger and a DNAJ domain associated with RNA and protein binding, respectively. Through cell growth assays, we demonstrated that the absence of TcZC3HTTP or the expression of an additional tagged version impacted epimastigote growth, indicating its contribution to cell proliferation. TcZC3HTTP was found to associate with mRNAs involved in cell cycle and division in epimastigotes, while in nutritionally stressed parasites it exhibited associations with mRNAs coding for other RBPs and rRNA. Furthermore, our analysis identified that TcZC3HTTP protein partners were different during normal growth conditions compared to starvation conditions, with the latter showing enrichment of ribosomal proteins and other RBPs. Therefore, this study provides insights into TcZC3HTTP's role in the post-transcriptional regulation of gene expression during normal growth and nutritional stress in
, uncovering its versatile functions in different cellular contexts.IMPORTANCEUnderstanding how
, the causative agent of Chagas disease, regulates gene expression is crucial for developing targeted interventions. In this study, we investigated the role of TcZC3HTTP, an RNA-binding protein, in post-transcriptional regulation. Our findings demonstrate that TcZC3HTTP is relevant for the growth and proliferation of epimastigotes, a stage of the parasite's life cycle. We identified its associations with specific mRNAs involved in cell cycle and division and its interactions with enzymes and other RNA-binding proteins (RBPs) under normal and starvation conditions. These insights shed light on the regulatory network underlying gene expression in
and reveal the multifaceted functions of RBPs in this parasite. Such knowledge enhances our understanding of the parasite's biology and opens avenues for developing novel therapeutic strategies targeting post-transcriptional gene regulation in
.
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