Bacteria-host Interactions Research Articles

Targeted Proteomics for Studying Pathogenic Bacteria.

Mass spectrometry-based proteomics has been extensively used to map bacterial proteomes, which has led to a better understanding of the molecular mechanisms underlying bacterial infection and bacteria-host interactions. Quantitative proteomics using selected or parallel reaction monitoring is considered one of the most sensitive and specific quantitative MS-based approaches and has significantly advanced proteome studies of pathogenic bacteria. Here, recent applications of targeted proteomics for bacteria identification, biomarker discovery, and the characterization of bacterial virulence and antimicrobial resistance are reviewed among others. Results of such studies are expected to further contribute to improve the fight against the most common human pathogenic bacteria.

Current Understanding of Human Metaproteome Association and Modulation.

During the last decade, metaproteomics has provided a better understanding and functional characterization of the microbiome. A large body of evidence now reveals interspecies, species of bacteria-host interactions, via the secreted modulatory microbial protein "metaproteome". Although high-throughput state-of-art mass spectrometry has recently empowered metaproteomics, its profile remains unclear, and, most importantly, the exact consequences and underlying mechanism of these protein molecules on the host are insufficiently understood. Here we address the current progress in the study of the human metaproteome, suggesting possible modulation, a metaproteome dysbiotic signature, challenges, and future perspectives.

Confirmed and Potential Roles of Bacterial T6SSs in the Intestinal Ecosystem.

The contact-dependent type VI secretion system (T6SS) in diverse microbes plays crucial roles in both inter-bacterial and bacteria-host interactions. As numerous microorganisms inhabit the intestinal ecosystem at a high density, it is necessary to consider the functions of T6SS in intestinal bacteria. In this mini-review, we discuss T6SS-dependent functions in intestinal microbes, including commensal microbes and enteric pathogens, and list experimentally verified species of intestinal bacteria containing T6SS clusters. Several seminal studies have shown that T6SS plays crucial antibacterial roles in colonization resistance, niche occupancy, activation of host innate immune responses, and modulation of host intestinal mechanics. Some potential roles of T6SS in the intestinal ecosystem, such as targeting of single cell eukaryotic competitors, competition for micronutrients, and stress resistance are also discussed. Considering the distinct activities of T6SS in diverse bacteria residing in the intestine, we suggest that T6SS research in intestinal microbes may be beneficial for the future development of new medicines and clinical treatments.

Histological and Comparative Transcriptome Analyses Provide Insights into Small Intestine Health in Diarrheal Piglets after Infection with Clostridium Perfringens Type C.

Simple SummaryClostridium perfringens (C. perfringens), formerly called Clostridium welchii, is a spore-forming pathogenic bacterium. C. perfringens type C can produce fatal toxins, which are absorbed by the small intestine into the body causing diarrhea in humans and animals, especially piglets. Each year, diarrhea induced by this pathogen causes significant economic loss to the pig industry worldwide. Nevertheless, the regulatory mechanisms of the duodenum, jejunum, and ileum in piglets challenged by C. perfringens type C are poorly understood. This study aimed to identify pathological changes and genes associated with the small intestine in piglets after infection with C. perfringens type C. RNA-Sequencing (RNA-Seq), enzyme-linked immunosorbent assay (ELISA), and hematoxylin & eosin (H&E) staining were used to analyze duodenal, jejunal, and ileal tissues. Our results showed that treated piglets were successfully infected with C. perfringens type C. These findings will help to elucidate the pathogenicity of piglets infected with this pathogen.C. perfringens type C can induce enteritis accompanied by diarrhea and annually causes significant economic losses to the global pig industry. The pathogenic mechanisms of C. perfringens type C in pigs are still largely unknown. To investigate this, we challenged seven-day-old piglets with C. perfringens type C to cause diarrhea. We performed hematoxylin & eosin (H&E) staining of the small intestine (including duodenum, jejunum, and ileum) and assessed gene expression in the ileal tissue. H&E staining of the duodenum, jejunum, and ileum demonstrated inflammation and edema of the lamina propria and submucosa. A total of 2181 differentially expressed genes (DEGs) were obtained in ileal tissues. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of DEGs indicated that the main pathways were enriched in the T cell receptor signaling pathway, NF-kappa B signaling pathway, and (tumor necrosis factor) TNF signaling pathway. These results provide insights into the pathogenicity of C. perfringens type C and improve our understanding of host–bacteria interactions.

Differential Helicobacter pylori Plasticity in the Gastric Niche of Subjects at Increased Gastric Cancer Risk.

Helicobacter pylori (H. pylori) represents an independent risk factor for Gastric Cancer (GC). First Degree Relatives (FDR) of GC subjects and Autoimmune Gastritis (AG) patients are both at increased risk for GC. H. pylori genetic heterogeneity within the gastric niche of FDR and AG individuals has been little explored. To understand whether they exploit an increased H. pylori stability and virulence, 14 AG, 25 FDR, 39 GC and 13 dyspeptic patients (D) were investigated by a cultural PCR-based approach characterizing single colonies-forming-units. We chose three loci within the Cytotoxin-associated gene-A Pathogenicity Island (CagPAI) (cagA,cagE,virB11), vacA, homA and homB as markers of virulence with reported association to GC. Inflammatory/precancerous lesions were staged according to Sydney System. When compared to D, FDR, similarly to GC patients, were associated to higher atrophy (OR = 6.29; 95% CI:1.23–31.96 in FDR; OR = 7.50; 95% CI:1.67–33.72 in GC) and a lower frequency of mixed infections (OR = 0.16; 95% CI:0.03–0.81 in FDR; OR = 0.10; 95% CI:0.02–0.48 in GC). FDR presented also an increased neutrophil infiltration (OR = 7.19; 95% CI:1.16–44.65). Both FDR and GC carried a higher proportion of CagPAI+vacAs1i1mx+homB+ profiles (OR = 2.71; 95% CI: 1.66–4.41 and OR = 3.43; 95% CI: 2.16–5.44, respectively). Conversely, AG patients presented a lower frequency of subtypes carrying a stable CagPAI and vacAs1i1mx. These results underline different H. pylori plasticity in FDR and AG individuals, and thus, a different host-bacterium interaction capacity that should be considered in the context of eradication therapies.

The Immunomodulatory Properties of Extracellular Vesicles Derived from Probiotics: A Novel Approach for the Management of Gastrointestinal Diseases.

Probiotics, included in functional foods, nutritional supplements, or nutraceuticals, exhibit different beneficial effects on gut function. They are extensively used to improve the digestive processes as well as reduce the symptoms and progression of different diseases. Probiotics have shown to improve dysbiosis and modulate the immune response of the host by interacting with different cell types. Probiotics and the host can interact in a direct way, but it is becoming apparent that communication occurs also through extracellular vesicles (EVs) derived from probiotics. EVs are key for bacteria–bacteria and bacteria–host interactions, since they carry a wide variety of components that can modulate different signaling pathways, including those involved in the immune response. Interestingly, EVs are recently starting to be considered as an alternative to probiotics in those cases for which the use of live bacteria could be dangerous, such as immunocompromised individuals or situations where the intestinal barrier is impaired. EVs can spread through the mucus layer and interact with the host, avoiding the risk of sepsis. This review summarizes the existing knowledge about EVs from different probiotic strains, their properties, and their potential use for the prevention or treatment of different gastrointestinal diseases.

Characterization of AIM2 expression in human macrophages during M. tuberculosis infection

Abstract Tuberculosis (TB) is a leading contributor to global mortality and is a well-known threat to global health. The disease-causing bacterium, Mycobacterium tuberculosis (M.tb), has demonstrated resistance to current treatments, thus necessitating novel methods to fight the disease. The investigation of host-bacterium interactions may provide insight for development of effective host-directed therapies, thus circumventing antibiotic resistance. The body’s defense system against airborne M.tb includes alveolar macrophages (AMs) responsible for elimination of foreign agents. However, they do not effectively eliminate M.tb, leading to bacterial propagation. It was previously found that the NOD-like receptor, absent in melanoma 2 (AIM2), plays key roles in M.tb infection in murine macrophages. However, the expression of AIM2 in primary human cells during M.tb infection remains unknown. To this end, we characterized AIM2 expression in human monocyte-derived macrophages (MDMs) and AMs during M.tb infection via quantitative real-time PCR (qRT-PCR). We found that M.tb induces AIM2 transcripts in human MDMs and AMs, in a time-dependent manner. We also investigated the contribution of peroxisome proliferator-activated receptor (PPAR)γ to AIM2 expression due to its known regulation of several genes involved in the human macrophage response to M.tb. We found that PPARγ mediates AIM2 expression. In conclusion, the increased AIM2 gene expression observed in MDMs and AMs during M.tb infection necessitates further investigation into its function and its potential use as a target for M.tb host-directed therapy.

Indoles: metabolites produced by intestinal bacteria capable of controlling liver disease manifestation.

Alterations in the bacteria that reside in our gastrointestinal tract play a role in the pathogenesis and progression of many disorders including liver and gastrointestinal diseases. Both qualitative (composition) and quantitative (amount) changes in gut microbes are associated with increased susceptibility to liver disease. Importantly, the intestinal microbiota is involved in the regulation of many host signalling pathways via the generation of different metabolites. Hence, dysbiosis influences disease development and progression by directly affecting the host-bacteria metabolic interaction. Microbe-derived harmful metabolites can translocate to distant organs due to increased intestinal permeability as observed during dysbiosis. Contrary, certain bacterial metabolites such as tryptophan metabolites contribute to intestinal and systemic homeostasis. Here, we provide an overview of current evidence describing to what extent microbial metabolites modulate the development of chronic liver diseases such as alcoholic steatohepatitis and nonalcoholic fatty liver disease with a special emphasis on indoles.

The Role of Staphylococcus aureus in Patients with Chronic Sinusitis and Nasal Polyposis.

Staphylococcus aureus (S. aureus) is correlated with the development of persistent severe inflammatory disease of the upper airway including chronic rhinosinusitis with nasal polyps (CRSwNP). The presence of S. aureus is associated with atopic disease including allergic rhinitis and atopic dermatitis and is associated with poor outcomes. Several different strains of S. aureus generate different toxins and gene products that can account for organism pathogenicity. S. aureus bacteria and its antigens shape the bacterial and fungal microbiome and the mucosal niche which generates host responses that can account for inflammation. The multiple disease phenotypes and molecular endotypes seen in CRSwNP can be characterized by T-helper cell environment within the inflammatory milieu, the presence of epithelial barrier dysfunction, aberrant eicosanoid metabolism, poor wound healing, and dysfunctional host-bacteria interactions which lead to recalcitrant disease and worse surgical outcomes. Understanding the pathomechanisms that S. aureus utilizes to promote nasal polyp formation, prolonged tissue inflammation, and bacterial dysbiosis are essential in our efforts to identify new therapeutic approaches to resolve this chronic inflammatory process.

Acid Sphingomyelinase-Ceramide System in Bacterial Infections.

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.

Whole-Genome Comparisons Among the Genus Shewanella Reveal the Enrichment of Genes Encoding Ankyrin-Repeats Containing Proteins in Sponge-Associated Bacteria.

The bacterial members of the genus Shewanella are widely distributed and inhabit both freshwater and marine environments. Some members of Shewanella have gained considerable attention due to its ability to survive in redox-stratified environments. However, a gap of knowledge exists on the key genomic features of the sponge-associated Shewanella sp. involving the successful host-bacteria interaction, as sponge-symbiotic Shewanella are largely underrepresented in the public repositories. With the aim of identifying the genomic signatures of sponge-Shewanella association, we generated a high-quality genome data of a sponge-associated, Shewanella sp. OPT22, isolated from the intertidal marine sponge Ophlitaspongia papilla and performed comprehensive comparative analyses of 68 genome strains of the genus Shewanella including two previously reported genomes of sponge-associated bacteria, Shewanella spongiae KCTC 22492 and Shewanella sp. Alg231_23. The 16S rRNA-based phylogenetic reconstruction showed the well-supported affiliation of OPT22 and KCTC 22492 with previously reported sponge-associated bacteria, affirming the “sponge-specific” nature of these two bacterial strains isolated from different marine sponge species from the Atlantic and Pacific (East Sea) Oceans, respectively. The genome comparison of the 68 strains of Shewanella inhabiting different habitats revealed the unusual/previously unreported abundance of genes encoding for ankyrin-repeat containing proteins (ANKs) in the genomes of the two sponge-associated strains, OPT22 (ANKs; n = 45) and KCTC 22492 (ANKs; n = 52), which might be involved in sponge-Shewanella interactions. Focused analyses detected the syntenic organization of the gene cluster encoding major secretion system (type III/IV/VI) components and the presence of effector homologs in OPT22 and KCTC 22492 that seem to play a role in the virulence of the sponge bacteria. The genomic island (GI) of Shewanella sp. OPT22 was identified to localize a gene cluster encoding T4SS components and ANK (n = 1), whereas S. spongiae KCTC 22492 harbored a total of seven ANKs within multiple GIs. GIs may play a pivotal role in the dissemination of symbioses-related genes (ANKs) through the horizontal gene transfer, contributing to the diversification and adaptation of sponge-associated Shewanella. Overall, the genome analyses of Shewanella isolates from marine sponges revealed genomic repertoires that might be involved in establishing successful symbiotic relationships with the sponge hosts.

Microbiological Aspects of Acute and Chronic Pediatric Rhinosinusitis.

The microbiology of acute and chronic rhinosinusitis has been extensively studied, but there are still some differences of view concerning the etiology of the former, and many disagreements regarding the microbiology of the latter. Establishing the concomitant distribution of the causative micro-organisms in cases that involve multiple sinuses is scientifically and practically important. The main problems are the variety of aerobes and anaerobes that may be involved, and the fact that different tracts of the sinuses of the same patient may be simultaneously affected. Rhinosinusitis may also involve the formation of biofilm, which may play a significant role in its pathogenesis and persistence. Biofilms have a number of advantages in terms of bacterial survival, and their perpetuation can create a certain degree of instability in host-bacteria interactions. Sinonasal microflora may further complicate pathogenesis and the identification of the pathogen(s) involved. Furthermore, the concentration, uniformity, and type/number of strains of nasal microbiota may vary from one site to another. The relative and total micro-organism counts can also be affected by various factors, and microbiota can modulate the course of both acute and chronic rhinosinusitis.

Probiotics: How Effective Are They in the Fight against Obesity?

Obesity has been associated with structural and functional changes in the gut microbiota. The abundance in, and diversity of, certain bacteria may favor energy harvest and metabolic pathways leading to obesity. Therefore, gut microbiota has become a potential target that can be manipulated to obtain optimal health. Probiotics have been shown to influence the composition of the gut microbiota, improve gut integrity, and restore the microbial shifts characteristic of obesity. Based on physical and biochemical parameters, metabolic and inflammatory markers, and alterations in gut microbe diversity, animal studies revealed beneficial results in obese models whereas the results in humans are sparse and inconsistent. Thus, the purpose of this review is to present evidence from animal studies and human clinical trials demonstrating the effects of various probiotic strains and their potential efficacy in improving obesity and associated metabolic dysfunctions. Furthermore, the review discusses current gaps in our understanding of how probiotics modulate gut microflora to protect against obesity. Finally, we propose future studies and methodological approaches that may shed light on the challenges facing the scientific community in deciphering the host–bacteria interaction in obesity.

Lable-free based comparative proteomic analysis of secretory proteins of rough Brucella mutants

Brucella rough mutants are reported to induce infected macrophage death, which is type IV secretion system (T4SS) dependent. T4SS and its secretory proteins play a major role in host-bacteria interactions, but the crucial secretory proteins to promote macrophage death during Brucella rough mutant infection have not been characterized. In this study, we found that T4SS components played no role for macrophage death induced by Brucella rough mutant infection, but some T4SS effectors did. Proteomics of secretory proteins from Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123 was analyzed by liquid chromatography/tandem mass spectrometry and 861 unique proteins were identified, among which 37 were differential secretory proteins. Gene ontology and pathway analysis showed that differential secretory proteins involved in cellular process and metabolic process, distributed in the cell and membrane, possessed molecular function of catalytic activity and binding, and were associated with ribosome, NOD-like receptor signaling pathway, two-component system and bacterial secretion system. Cell death analysis showed that T4SS effector VceC, and two differential secretory proteins OmpW family protein (BAB1_1579) and protein BAB1_1185 were associated with Brucella cytotoxicity. This study provides new insights into the molecular mechanisms associated with Brucella cytotoxicity and valuable information for screening vaccine candidates for Brucella. SignificanceBrucella rough mutants induce infected macrophage death, which is T4SS dependent. In the present report, a comparative proteomics analysis revealed 37 differential secretory proteins between Brucella rough mutants ΔrfbE and ΔrfbEΔvirB123. Further study demonstrated OmpW family protein (BAB1_1579) and uncharacterized protein BAB1_1185, two differential secretory proteins, were associated with Brucella cytotoxicity. This study provides novel information of the secretory proteins from the Brucella rough mutants and their effects on the Brucella cytotoxicity.

Distinct gene expression characteristics in epithelial cell-Porphyromonas gingivalis interactions by integrating transcriptome analyses.

Porphyromonas gingivalis is a pivotal periodontal pathogen, and the epithelial cells serve as the first physical barrier to defend the host from bacterial attack. Within this host-bacteria interaction, P. gingivalis can modify the host immune reaction and adjust the gene expression, which is associated with periodontitis pathogenesis and developing strategies. Herein, a meta-analysis was made to get the differential gene expression profiles in epithelial cells with or without P. gingivalis infection. The network-based meta-analysis program for gene expression profiling was used. Both the gene ontology analysis and the pathway enrichment analysis of the differentially expressed genes were conducted. Our results determined that 290 genes were consistently up-regulated in P. gingivalis infected epithelial cells. 229 gene ontology biological process terms of up-regulated genes were discovered, including “negative regulation of apoptotic process” and “positive regulation of cell proliferation/migration/angiogenesis”. In addition to the well-known inflammatory signaling pathways, the pathway associated with a transcriptional misregulation in cancer has also been increased. Our findings indicated that P. gingivalis benefited from the survival of epithelial cells, and got its success as a colonizer in oral epithelium. The results also suggested that infection of P. gingivalis might contribute to oral cancer through chronic inflammation. Negative regulation of the apoptotic process and transcriptional misregulation in cancer pathway are important contributors to the cellular physiology changes during infection development, which have particular relevance to the pathogenesis and progressions of periodontitis, even to the occurrence of oral cancer.

Adherence of Proteus mirabilis to Uroepithelial Cells.

Bacterial adherence to eukaryotic cells is mediated by different adhesins that can act at different stages in bacteria-host interaction. Abundant evidence has suggested that adherence is critical for infection by bacterial pathogens. Proteus mirabilis is an opportunistic pathogen which frequently infects the human urinary tract, particularly in patients with indwelling urinary catheters. Sequencing of the genome of this pathogen has revealed the existence of a remarkable amount of complete fimbrial operons. In this chapter, we describe in vitro adherence assays of P. mirabilis to uroepithelial cells, which can provide relevant results to assess virulence of uropathogenic strains.

Subgingival lipid A profile and endotoxin activity in periodontal health and disease

Regulation of lipopolysaccharide (LPS) chemical composition, particularly its lipid A domain, is an important, naturally occurring mechanism that drives bacteria-host immune system interactions into either a symbiotic or pathogenic relationship. Members of the subgingival oral microbiota can critically modulate host immuno-inflammatory responses by synthesizing different LPS isoforms. The objectives of this study were to analyze subgingival lipid A profiles and endotoxin activities in periodontal health and disease and to evaluate the use of the recombinant factor C assay as a new, lipid A-based biosensor for personalized, point-of-care periodontal therapy. Subgingival plaque samples were collected from healthy individuals and chronic periodontitis patients before and after periodontal therapy. Chemical composition of subgingival lipid A moieties was determined by ESI-Mass Spectrometry. Endotoxin activity of subgingival LPS extracts was assessed using the recombinant factor C assay, and their inflammatory potential was examined in THP-1-derived macrophages by measuring TNF-α and IL-8 production. Characteristic lipid A molecular signatures, corresponding to over-acylated, bi-phosphorylated lipid A isoforms, were observed in diseased samples. Healthy and post-treatment samples were characterized by lower m/z peaks, related to under-acylated, hypo-phosphorylated lipid A structures. Endotoxin activity levels and inflammatory potentials of subgingival LPS extracts from periodontitis patients were significantly higher compared to healthy and post-treatment samples. This is the first study to consider structure-function-clinical implications of different lipid A isoforms present in the subgingival niche and sheds new light on molecular pathogenic mechanisms of subgingival biofilm communities. Subgingival endotoxin activity (determined by lipid A chemical composition) could be a reliable, bacterially derived biomarker and a risk assessment tool for personalized periodontal care.

Bacteria-Host Interactions in Multiple Sclerosis.

Multiple sclerosis (MS) is caused by a complex interaction of genetic and environmental factors. Numerous causative factors have been identified that play a role in MS, including exposure to bacteria. Mycobacteria, Chlamydia pneumoniae, Helicobacter pylori, and other bacteria have been proposed as risk factors for MS with different mechanisms of action. Conversely, some pathogens may have a protective effect on its etiology. In terms of acquired immunity, molecular mimicry has been hypothesized as the mechanism by which bacterial structures such as DNA, the cell wall, and intracytoplasmic components can activate autoreactive T cells or produce autoantibodies in certain host genetic backgrounds of susceptible individuals. In innate immunity, Toll-like receptors play an essential role in combating invading bacteria, and their activation leads to the release of cytokines or chemokines that mediate effective adaptive immune responses. These receptors may also be involved in central nervous system autoimmunity, and their contribution depends on the infection site and on the pathogen. We have reviewed the current knowledge of the influence of bacteria on MS development, emphasizing the potential mechanisms of action by which bacteria affect MS initiation and/or progression.

Tug of war between Acinetobacter baumannii and host immune responses.

Acinetobacter baumannii is an emerging nosocomial, opportunistic pathogen with growing clinical significance. Acinetobacter baumannii has an exceptional ability to rapidly develop drug resistance and to adhere to abiotic surfaces, including medical equipment, significantly promoting bacterial spread and also limiting our ability to control A. baumannii infections. Consequently, A. baumannii is frequently responsible for ventilator-associated pneumonia in clinical settings. In order to develop an effective treatment strategy, understanding host-pathogen interactions during A. baumannii infection is crucial. Various A. baumannii virulence factors have been identified as targets of host innate pattern-recognition receptors, which leads to activation of downstream inflammasomes to develop inflammatory responses, and the recruitment of innate immune effectors against A. baumannii infection. To counteract host immune attack, A. baumannii regulates its expression of different virulence factors. This review summarizes the significance of mechanisms of host-bacteria interaction, as well as different bacteria and host defense mechanisms during A. baumannii infection.

Mycoplasmas are no exception to extracellular vesicles release: Revisiting old concepts.

Release of extracellular vesicles (EV) by Gram-negative and positive bacteria is being frequently reported. EV are nano-sized, membrane-derived, non-self-replicating, spherical structures shed into the extracellular environment that could play a role in bacteria-host interactions. Evidence of EV production in bacteria belonging to the class Mollicutes, which are wall-less, is mainly restricted to the genus Acholeplasma and is scanty for the Mycoplasma genus that comprises major human and animal pathogens. Here EV release by six Mycoplasma (sub)species of clinical importance was investigated. EV were obtained under nutritional stress conditions, purified by ultracentrifugation and observed by electron microscopy. The membrane proteins of EV from three different species were further identified by mass spectrometry as a preliminary approach to determining their potential role in host-pathogen interactions. EV were shown to be released by all six (sub)species although their quantities and sizes (30–220 nm) were very variable. EV purification was complicated by the minute size of viable mycoplasmal cells. The proteins of EV-membranes from three (sub)species included major components of host-pathogen interactions, suggesting that EV could contribute to make the host-pathogen interplay more complex. The process behind EV release has yet to be deciphered, although several observations demonstrated their active release from the plasma membrane of living cells. This work shed new light on old concepts of “elementary bodies” and “not-cell bound antigens”.