Search Results (300)

Botrytis cinerea is a highly versatile pathogenic fungus, causing significant damage across a wide range of plant species. A central focus of this review is the recent advances made through proteomics, an advanced molecular tool, in understanding the mechanisms of B. cinerea infection. Recent advances in mass spectrometry-based proteomics—including LC-MS/MS, iTRAQ, MALDI-TOF, and surface shaving—have enabled the in-depth characterization of B. cinerea subproteomes such as the secretome, surfactome, phosphoproteome, and extracellular vesicles, revealing condition-specific pathogenic mechanisms. Notably, in under a decade, the proportion of predicted proteins experimentally identified has increased from 10% to 52%, reflecting the rapid progress in proteomic capabilities. We explore how proteomic studies have significantly enhanced our knowledge of the fungus secretome and the role of extracellular vesicles (EVs), which play key roles in pathogenesis, by identifying secreted proteins—such as pH-responsive elements—that may serve as biomarkers and therapeutic targets. These technologies have also uncovered fine regulatory mechanisms across multiple levels of the fungal proteome, including post-translational modifications (PTMs), the phosphomembranome, and the surfactome, providing a more integrated view of its infection strategy. Moreover, proteomic approaches have contributed to a better understanding of host–pathogen interactions, including aspects of the plant’s defensive responses. Furthermore, this review discusses how proteomic data have helped to identify metabolic pathways affected by novel, more environmentally friendly antifungal compounds. A further update on the advances achieved in the field of proteomics discovery for the organism under consideration is provided in this paper, along with a perspective on emerging tools and future developments expected to accelerate research and improve targeted intervention strategies. Full article

Human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis is a complex parasitic disease marked by dynamic host–parasite interactions and immunomodulation. Extracellular vesicles (EV) derived from immune cells have emerged as key mediators of intercellular communication and potential biomarkers in infectious diseases. In this study, we combined a modified lymphocyte proliferation assay with nano-flow cytometry to quantify and phenotype EV released by CD4⁺, CD8⁺, and CD14⁺ cells in PBMC cultures from CL patients at different clinical stages: before treatment (PBT), during treatment (PDT), and post-treatment (PET) with antimonial. Healthy individuals (HI) were included as physiological controls. Upon stimulation with L. (V.) braziliensis antigens, we observed a distinct modulation of EV subsets. In the PBT group, CD4⁺ and CD14⁺ EV were significantly reduced, while CD8⁺ EV remained elevated. During PDT and PET, EV concentrations were restored across all subsets. These findings suggest that L. (V.) braziliensis selectively modulates the release of immune cell–derived EV, possibly as an immune evasion mechanism. The restoration of EV release following antimonial therapy highlights their potential as sensitive biomarkers for disease activity and treatment monitoring. This study offers novel insights into the immunoregulatory roles of EV in CL and underscores their relevance in host–parasite interactions. Full article

The expanding distribution and geographic range of mosquitoes have potentially contributed to increased flaviviral dissemination and transmission. Despite the growing burden of flaviviral infections, there are no effective antiviral treatments or vaccines, highlighting the need for novel therapeutic targets. Tetraspanins, a superfamily of transmembrane domain glycoproteins involved in cellular organization, signaling, and protein–protein interactions have been recognized as potential mediators of flaviviral infection and transmission. While their roles in vertebrate hosts have been explored, their involvement in flaviviral replication and dissemination within medically important vectors remains poorly understood. In this study, we investigated the role of arthropod tetraspanins in mosquito cells and extracellular vesicles (EVs) derived from cells infected with Zika virus (ZIKV) and dengue virus (serotype 2; DENV2). Among several of the tetraspanins analyzed, only CD151 was significantly upregulated in both mosquito cells and in EVs derived from ZIKV/DENV2-infected cells. RNAi-mediated silencing of CD151 led to a marked reduction in viral burden, suggesting its crucial role in flavivirus replication. Inhibition of EV biogenesis using GW4869 further demonstrated that EV-mediated viral transmission contributes to flavivirus propagation. Additionally, co-immunoprecipitation and immunofluorescence analyses revealed direct interactions between CD151 and ZIKV NS2B and DENV2 capsid proteins. Overall, our findings highlight the functional importance of mosquito CD151 in the replication and transmission of ZIKV and DENV2. This study provides new insights into the molecular mechanisms of flaviviral infection in mosquitoes and suggests that targeting vector tetraspanins may offer a potential approach to controlling mosquito-borne flaviviruses. Full article

GW4869, a cell-permeable, selective inhibitor of neutral sphingomyelinase is a pharmacological agent that blocks the production and release of extracellular vesicles (EVs). Our previous studies have shown that GW4869 inhibits flaviviral loads in tick, mosquito and mammalian cells, including murine cortical neurons. Yet the mechanism(s) of GW4869 inhibitor upon viral infections were not addressed. In the current study, we focused on how GW4869 interferes with Langat Virus (LGTV, a tick-borne flavivirus) replication in ISE6 tick cells. First, we found that GW4869 is neither cytotoxic at tested doses of 50, 100, and 150 µM in tick cells, nor does it directly bind to the free LGTV present in cell culture supernatants. When tick cells were treated with GW4869, followed by infection with viral stock at dilutions of 10⁻², 10⁻³, 10⁻⁴ (the infectious dose determination by viral dilution assay), it affected LGTV replication in tick cells. A reduction in viral burden was noted in GW4869-treated tick cells, which constituted more than half the amount of decrease when compared to the mock control. Next, GW4869 treatment not only resulted in decreased LGTV transcript levels in tick cells and EVs derived from these infected cells, but also revealed diminished EVs concentrations. Enhanced IsSMase transcripts in the LGTV-infected group was noted upon GW4869 treatment, thus suggesting a host response to perhaps inhibit virus replication. In addition, GW4869 treatment reduced LGTV loads in density gradient EVs fractions, which correlated with decreased EVs concentration in those fractions. These data not only indicate that GW4869 affects LGTV replication, but that it also interferes with EV secretion and release from tick cells. Lastly, we found that GW4869 inhibits LGTV replication in tick cells but does not directly affect the infectivity of LGTV viral particles. Overall, our study suggests that GW4869 is a potential therapeutic inhibitor in controlling tick-borne diseases. Full article

A key feature that differentiates Gram-positive and Gram-negative bacteria is the outer membrane, an asymmetric membrane composed of lipopolysaccharides, phospholipids, lipoproteins and integral proteins, including the outer-membrane proteins (OMPs). By being in direct contact with the extracellular milieu, the outer membrane and OMPs participate in multiple functions in Gram-negative bacteria, including controlling nutrient and molecule access to the cytoplasm, membrane vesicle formation and resistance to environmental stresses. OMPs have a characteristic barrel shape formed by antiparallel β-strands, with or without channels that allow diffusion of substrates through the outer membrane. The marine bacterium Vibrio cholerae is responsible for non-invasive gastroenteritis and cholera disease by consumption of contaminated water or food. Its OMPs, besides having a porin function, contribute to resistance to osmotic pressure and antimicrobial agents, intracellular signaling, adhesion to host cells and biofilm formation, amongst other functions. In this review, in addition to quickly reviewing the general structure of the outer membrane, the OMPs and how they reach the outer membrane, the functions attributed to these proteins are compiled. The mechanisms used by each of the described OMP to accomplish these functions in the marine pathogenic bacterium V. cholerae are discussed. Potential clinical and bioengineering applications of OMPs, such as diagnostic tools, vaccine development, and targeted antimicrobial or anti-virulence strategies are presented. What is known about the OMPs of V. cholerae is presented below. Full article

Severe dengue is a global health threat, affecting 4 billion people, with nearly 1 million hospitalizations during epidemics and around 25,000 annual deaths. Severe dengue presentations are characterized by vascular leakage, hemorrhagic manifestations, and shock, which can lead to multiorgan failure. Recent studies highlight the crucial role of extracellular vesicles (EVs) in the pathogenesis of dengue, influencing immune response and disease progression. EVs, nanometric structures secreted by cells, mediate viral dissemination, immune modulation, and endothelial dysfunction by transporting biomolecules such as microRNAs (miRNAs) and viral proteins. Infected cell-derived EVs carry viral components, including NS protein and miRNAs like miR-21 and miR-126-5p, which compromise endothelial integrity and activate immune pathways such as Toll-like receptor, NF-κB, and JAK-STAT signaling. This, together with the immune response, leads to the release of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IFN-γ. EVs also facilitate viral immune evasion by suppressing antiviral responses. Recent analyses of miRNAs within EVs suggest their potential as biomarkers for disease progression. Differentially expressed miRNAs in circulating EVs correlate with severe outcomes, providing tools for risk stratification and therapeutic monitoring. Advanced techniques, such as nanoparticle tracking analysis and flow cytometry, allow precise EV characterization, supporting their integration into clinical applications. Full article

Epidermolysis bullosa (EB) is a debilitating genetic skin disorder characterized by extreme fragility, chronic wounds, and severe complications, particularly in its most severe form, recessive dystrophic EB (RDEB). Current treatments focus on symptomatic relief through wound care and pain management, with recent FDA approvals of Vyjuvek and Filsuvez providing new but limited therapeutic options. However, emerging research highlights the potential of extracellular vesicles (EVs) derived from mesenchymal stem cells as a promising approach to address both the symptoms and underlying pathology of EB. EVs function as carriers of bioactive molecules, modulating inflammation, promoting tissue regeneration, and even delivering functional type VII collagen to RDEB patient cells. Unlike whole-cell therapies, EVs are non-immunogenic, have greater stability, and avoid risks such as graft-versus-host disease or tumorigenic transformation. Additionally, EVs offer diverse administration routes, including topical application, local injection, and intravenous delivery, which could extend their therapeutic reach beyond skin lesions to systemic manifestations of EB. However, challenges remain, including standardization of EV production, scalability, and ensuring consistent therapeutic potency. Despite these hurdles, EV-based therapies represent a transformative step toward addressing the complex pathology of EB, with the potential to improve wound healing, reduce fibrosis, and enhance patient quality of life. Full article

Bacterial extracellular vesicles (BEVs) have emerged as pivotal mediators of host–microbe interactions, profoundly influencing cancer biology. These nanoscale vesicles, produced by both Gram-positive and Gram-negative bacteria, carry diverse biomolecular cargo such as proteins, lipids, nucleic acids, and metabolites. BEVs play dualistic roles in tumor promotion and suppression by modulating the tumor microenvironment, immune responses, and genetic regulation. This review synthesizes the current understanding of BEVs in various cancers, including gastrointestinal, ovarian, breast, lung, brain, and renal malignancies. BEVs are highlighted for their potential as diagnostic biomarkers, prognostic indicators, and therapeutic agents, including their applications in immunotherapy and advanced engineering for precision medicine. Challenges such as heterogeneity, standardization, and clinical scalability are critically analyzed, with case examples providing actionable insights. Future directions emphasize interdisciplinary collaborations, emerging technologies, and the integration of BEV-based tools into clinical workflows. This review underscores the transformative potential of BEVs in advancing cancer diagnostics and therapeutics, paving the way for innovations in precision oncology. Full article

Helicobacter pylori (H. pylori) is a Gram-negative bacterium that colonizes the human stomach and is associated with several gastric diseases, including gastritis, peptic ulcer disease, and gastric cancer. The bacterium’s ability to thrive in the harsh gastric environment is due, to some extent, to its stress response mechanisms, with its heat shock proteins (HSPs) playing a putative, yet not fully understood, role in these adaptive processes. HSPs are a family of molecules, highly conserved throughout phylogenesis, that assist in protein folding, prevent aggregation, and ensure cellular homeostasis under stressful conditions. In H. pylori, HSPs contribute to survival in the stomach’s acidic environment and oxidative stress. Furthermore, they aid in the bacterium’s ability to adhere to gastric epithelial cells, modulate the host immune response, and form biofilms, all contributing to chronic infection and pathogenicity. The role of microbial HSPs in antibiotic resistance has also emerged as a critical area of research, as these proteins help stabilize efflux pumps, protect essential proteins targeted by antibiotics, and promote biofilm formation, thereby reducing the efficacy of antimicrobial treatments. Among bacterial HSPs, GroEL and DnaK are probably the major proteins that control most of the H. pylori’s functioning. Indeed, both proteins possess remarkable acid resistance, high substrate affinity, and dual roles in protein homeostasis and host interaction. These features make them critical for H. pylori’s adaptation, persistence, and pathogenicity in the gastric niche. In addition, recent findings have also highlighted the involvement of HSPs in the crosstalk between H. pylori and gastric epithelial cells mediated by the release of bacterial outer membrane vesicles and host-derived exosomes, both of these extracellular vesicles being part of the muco-microbiotic layer of the stomach and influencing cellular signalling and immune modulation. Considering their critical role in the survival and persistence of bacteria, microbial HSPs also represent potential therapeutic targets. Strategies aimed at inhibiting microbial HSP function, combined with conventional antibiotics or developing vaccines targeting microbial HSPs, could provide new avenues for the treatment of H. pylori infections and combat antibiotic resistance. This review explores the multifaceted roles of microbial HSPs in the pathogenesis of H. pylori, highlighting their contributions to bacterial adhesion, immune evasion, stress response, and antibiotic resistance. Full article

Extracellular vesicles (EVs) are nanometer-sized lipid structures actively secreted by Gram-negative and Gram-positive bacteria, representing a sophisticated microbial adaptation and communication strategy. These structures are involved in biomolecular transport, the regulation of biological processes, the modulation of host–pathogen interactions, and adaptation to hostile environmental conditions. EVs also play a crucial role in virulence, antibiotic resistance, and biofilm formation. This review will explore the biogenesis, composition, and biological mechanisms of outer membrane vesicles (OMVs) secreted by Gram-negative bacteria and membrane vesicles (MVs) generated by Gram-positive bacteria. In detail, the modulation of EVs in response to antibiotic exposure will be addressed. The role of EV morpho-functional adaptations will be studied in antimicrobial resistance, the gene determinant spread, and survival in adverse environments. This study aims to provide a comprehensive overview of the EV role in bacterial physiology, highlighting their ecological, evolutionary, and biotechnological implications. An overview of the enzymes and proteins mainly involved in OMV-mediated resistance mechanisms will also be provided. These insights could open new perspectives for developing therapeutic strategies that counteract EV secretion and biotechnological applications, such as vaccines and drug delivery systems. Full article

Background: Heterorhabditis bacteriophora entomopathogenic nematodes are commonly used in agricultural practices for the biological control of insect pests. These parasites are also used in basic research for unveiling the molecular basis of nematode parasitism in relation to the insect anti-nematode response. We have recently shown that H. bacteriophora excreted–secreted products reduce the expression of the antimicrobial peptide gene Diptericin in Drosophila melanogaster, which increases fly mortality due to enhanced propagation of the mutualistic bacteria Photorhabdus luminescens. However, the effect of entomopathogenic nematode extracellular vesicles (EVs) on the insect host defense remains unknown. Methods: Here, we injected adult flies with H. bacteriophora EVs and used quantitative RT-PCR together with gene-specific primers to analyze the activity of immune-related signaling pathways. Results: We found that H. bacteriophora EVs are lethal to Drosophila melanogaster, and they downregulate the expression of Attacin, Cecropin, and Prophenoloxidase 3 in adult flies. Conclusions: These findings build on previous knowledge and strengthen the notion that H. bacteriophora entomopathogenic nematodes release a variety of effector molecules to modify the insect’s innate immune signaling. This information is important because it contributes toward clarifying the molecular interplay between entomopathogenic nematode components and the host’s innate immune system. Full article

Introduction: Coronavirus (CoV) is an extremely contagious, enveloped positive-single-stranded RNA virus, which has become a global pandemic that causes several illnesses in humans and animals. Hence, it is necessary to investigate viral-induced reactions across diverse hosts. Herein, we propose utilizing naturally secreted extracellular vesicles (EVs), mainly focusing on exosomes to examine virus–host responses following CoV infection. Exosomes are small membrane-bound vesicles originating from the endosomal pathway, which play a pivotal role in intracellular communication and physiological and pathological processes. We suggested that CoV could impact EV formation, content, and diverse immune responses in vitro. Methods: In this study, we infected A-72, which is a canine fibroblast cell line, with a feline coronavirus (FCoV) and canine coronavirus (CCoV) independently in an exosome-free media at 0.001 multiplicity of infection (MOI), with incubation periods of 48 and 72 h. The cell viability was significantly downregulated with increased incubation time following FCoV and CCoV infection, which was identified by performing the 3-(4,5-dimethylthiazo-1-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After the infection, EVs were isolated through ultracentrifugation, and the subsequent analysis involved quantifying and characterizing the purified EVs using various techniques. Results: NanoSight particle tracking analysis (NTA) verified that EV dimensions fell between 100 and 200 nm at both incubation periods. At both periods, total protein and RNA levels were significantly upregulated in A-72-derived EVs following FCoV and CCoV infections. However, total DNA levels were gradually upregulated with increased incubation time. Dot blot analysis indicated that the expression levels of ACE2, IL-1β, Flotillin-1, CD63, caspase-8, and Hsp90 were modified in A-72-derived EVs following both CoV infections. Conclusions: Our results indicated that FCoV and CCoV infections could modulate the EV production and content, which could play a role in the development of viral diseases. Investigating diverse animal CoV will provide in-depth insight into host exosome biology during CoV infection. Hence, our findings contribute to the comprehension and characterization of EVs in virus–host interactions during CoV infection. Full article

Bacterial outer membrane vesicles (OMVs) are nanoscale extracellular structures produced by Gram-negative bacteria that are critical for microbial biology and host-pathogen interactions and have great potential in biotechnological applications. Despite the availability of fluorescent dyes for OMV studies, many are repurposed from eukaryotic extracellular vesicle research and are not explicitly optimized for OMVs, leading to challenges in achieving consistent labeling, minimizing background noise, and preserving vesicle integrity during analyses. This study evaluates B2, a benzimidazole-derived fluorophore, for OMV labeling in advanced techniques like flow cytometry and confocal microscopy. OMVs were isolated from Escherichia coli strains BL21 and O157, and their integrity was confirmed using transmission electron microscopy (TEM). B2 staining protocols were optimized for OMVs, and fluorescence analyses revealed specific interactions with the vesicle membranes, reducing aggregation and enhancing signal uniformity. Flow cytometry indicated near-complete labeling efficiency (98–100%) with minimal background interference. Confocal microscopy further validated B2’s effectiveness, showing evident OMV internalization into epithelial HT-29 cells and compatibility with other fluorophores. Density functional theory (DFT) calculations, including Fukui function analysis, identified key electrophilic and nucleophilic regions in B2 that facilitate specific hydrogen bonding and polar interactions with membrane components. Non-covalent interaction (NCI) analysis revealed pronounced intramolecular hydrogen bonding along with discrete regions of weak van der Waals interactions. Molecular dynamics simulations suggest that B2 exhibits an affinity for both the hydrophobic core of the lipid bilayer and the core oligosaccharide region of the LPS layer, which collectively ensures sustained retention of the dye. The findings presented in this study position B2 as a valuable fluorophore for OMV research. Full article

Trypanosomatids constitute a family of parasitic protozoa that cause significant human and veterinary diseases that are classified as neglected zoonotic diseases (NZDs). In a rapidly evolving world, these diseases have the potential to become a world health problem no longer solely associated with low-income countries. Therefore, the development of new strategies to control and restrain the dissemination of trypanosomatids is imperative. Extracellular vesicles (EVs) are a heterogeneous group of membrane-enclosed vesicles released by prokaryotic and eukaryotic cells. They can be found in diverse body fluids that carry biologically active molecules, including proteins, nucleic acids, lipids, and carbohydrates. EVs participate in cell-to-cell communication by delivering their cargo content to recipient cells. Thus, EVs play a role in regulating normal physiological processes, including immune surveillance and tissue repair, as well as being involved in pathological conditions, like cancer. In recent years, EVs have attracted significant attention from the scientific community, mainly due to their immune regulatory properties. Therefore, this review examines the role played by trypanosomatid-derived EVs in leishmaniases and trypanosomiasis, highlighting their biological role in host–parasite communication and exploring their potential future applications in controlling NZDs, especially those caused by trypanosomatids. Full article

Bacterial extracellular vesicles (BEVs) produced by members of the intestinal microbiota can not only contribute to digestion but also mediate microbe–host cell communication via the transfer of functional biomolecules to mammalian host cells. An unresolved question is which host factors and conditions influence BEV cargo and how they impact host cell function. To address this question, we analysed and compared the proteomes of BEVs released by the major human gastrointestinal tract (GIT) symbiont Bacteroides thetaiotaomicron (Bt) in vivo in fed versus fasted animals using nano-liquid chromatography with tandem mass spectrometry (LC-MSMS). Among the proteins whose abundance was negatively affected by fasting, nine of ten proteins of the serine protease family, including the regulatory protein dipeptidyl peptidase-4 (DPP-4), were significantly decreased in BEVs produced in the GITs of fasted animals. Strikingly, in extracellular vesicles produced by the intestinal epithelia of the same fasted mice, the proteins with the most increased abundance were serine protease inhibitors (serpins). Together, these findings suggest a dynamic interaction between GI bacteria and the host. Additionally, they indicate a regulatory role for the host in determining the balance between bacterial serine proteases and host serpins exported in bacterial and host extracellular vesicles. Full article