Immunogold labeling of extracellular vesicles from the fungal pathogen Fusarium graminearum reveals the presence of the protein marker Sur7

Background: Fungal extracellular vesicles (EVs) have been implicated in host-pathogen and pathogen-pathogen communication in some fungal diseases. In depth research into fungal EVs has been hindered by the lack of specific protein markers such as those found in mammalian EVs that have enabled sophisticated isolation and analysis techniques. Despite their role in fungal EV biogenesis, ESCRT proteins such as Vps23 (Tsg101) and Bro1 (ALIX) are not present as fungal EV cargo. Furthermore, tetraspanin homologs are yet to be identified in many fungi including the model yeast S. cerevisiae. Objective: We performed de novo identification of EV protein markers for the major human fungal pathogen Candida albicans with adherence to MISEV2018 guidelines. Materials and methods: EVs were isolated by differential ultracentrifugation from DAY286, ATCC90028 and ATCC10231 yeast cells, as well as DAY286 biofilms. Whole cell lysates (WCL) were also obtained from the EV-releasing cells. Label-free quantitative proteomics was performed to determine the set of proteins consistently enriched in EVs compared to WCL. Results: 47 proteins were consistently enriched in C. albicans EVs. We refined these to 22 putative C. albicans EV protein markers including the claudin-like Sur7 family (Pfam: PF06687) proteins Sur7 and Evp1 (orf19.6741). A complementary set of 62 EV depleted proteins was selected as potential negative markers. Conclusions: The marker proteins for C. albicans EVs identified in this study will be useful tools for studies on EV biogenesis and cargo loading in C. albicans and potentially other fungal species and will also assist in elucidating the role of EVs in C. albicans pathogenesis. Many of the proteins identified as putative markers are fungal specific proteins indicating that the pathways of EV biogenesis and cargo loading may be specific to fungi, and that assumptions made based on studies in mammalian cells could be misleading. Abbreviations: A1 – ATCC10231; A9 – ATCC90028; DAY B – DAY286 biofilm; DAY Y – DAY286 yeast; EV – extracellular vesicle; Evp1 – extracellular vesicle protein 1 (orf19.6741); GO – gene ontology; Log2(FC) – log2(fold change); MCC – membrane compartment of Can1; MDS – multidimensional scaling; MISEV – minimal information for studies of EVs; sEVs – small EVs; SP – signal peptide; TEMs – tetraspanin enriched microdomains; TM – transmembrane; VDM – vesicle-depleted medium; WCL – whole cell lysate

Extracellular vesicle (EV) release in fungi was described for the first time in 2007 in the yeast-like pathogen Cryptococcus neoformans [1]. Since then, the phenomenon of EV production, which is present in all domains of life, has been observed in many different fungal species, including yeast cells and hyphae. Composition of EVs, the impact of their release on fungal pathogenesis, and their potential use as protective immunogens have been explored in a number of original studies and comprehensive reviews (see Fig 1 and [2] for a summary). However, many aspects related to the biological properties of fungal EVs remain obscure. In this manuscript, we will focus our discussion on three fundamental but still unanswered questions about fungal EVs. Fig 1 Overview of the functional aspects of fungal EVs. What Is the Role of Fungal EVs during Infection? It remains unknown whether fungal EVs are produced in vivo, which is likely linked to the lack of protocols and molecular markers for isolation of these membranous compartments from body fluids. Vesicle properties related to their stability in tissues are also obscure. C. neoformans EVs are rapidly disrupted by serum albumin at physiological concentrations [3]. This observation argues against the stability of EVs in vivo, but not against their potential functions. EV disruption might result in the release of internal and potentially immunomodulatory compounds into the extracellular space, possibly impacting the physiology of host cells. Different studies provided indirect evidence supporting the hypothesis that fungal EVs are produced during infection. Sera from patients with cryptococcosis or histoplasmosis reacted with EV components [4,5]. In addition, EVs were isolated from plasma of patients with Malassezia sympodialis-associated atopic eczema [6]. In vivo studies with C. neoformans suggested that EVs are produced during lung infection [1]. The immunobiological activity of fungal EVs and the mechanisms by which they modulate host cell physiology have been first explored in M. sympodialis, where allergen-containing EVs induced IL-4 and TNF-α responses [6]. In further studies with C. neoformans and Candida albicans [7,8], labeling of EVs with DiIC18, a lipophilic and fluorescent stain, allowed observation of vesicle internalization by murine phagocytes and consequent cytokine production. EVs co-localized with the lipid raft marker GM1, suggesting the participation of such domains during vesicle internalization [7,8]. Apparently, fungal EVs are internalized through phagocytosis, since DiIC18 labeling was restricted to the cytoplasm after 15 minutes of incubation with phagocytes. It remains unclear, however, whether fungal EVs also fuse with host cell membranes, as suggested after interaction of C. neoformans EVs with human brain microvascular endothelial cells (HBMEC) [9]. In this situation, vesicle cargo would be directly delivered into the cytoplasm of host cells. This mechanism appears to modulate HBMEC permeability during murine cryptococcosis, facilitating crossing of the blood-brain barrier and brain colonization by the fungus [9]. More recently, Wolf and colleagues investigated a strain of C. albicans lacking expression of a phosphatidylserine synthase [10]. EV cargo lacked characteristic virulence factors, including phospholipase Plb3 and adhesin Sim1. These EVs failed to induce NFκB activation in macrophages [10]. Thus, phospholipid biosynthesis appears to be required for EV cargo and functions. The molecules carried by EVs may impact antigen processing and, consequently, the immune response. C. albicans EVs stimulated dendritic cells (DCs) to produce IL-12p40, IL-10, and TNF-α, and induced upregulation of CD86 and MHC-II [7]. Treatment of murine macrophages with EVs from C. neoformans or C. albicans resulted in production of nitric oxide, IL-12, TGF-β, and IL-10 [7,8]. In addition, EVs from an acapsular strain of C. neoformans induced a high proinflammatory response [8]. The protective effect of EVs on the innate immune system has been suggested using the insect model Galleria mellonella. Treatment of larvae with EVs from C. albicans resulted in significant protection against subsequent challenges with this fungus [7]. These studies suggest that fungal EVs activate the innate immune response and may also promote, in other models, the development of adaptive responses (Table 1). A beneficial contribution of fungal EVs to humoral immunity is also expected. Enolase, HSP60, and GlcCer are examples of immunogens carried by EVs that can induce protective antibodies (reviewed in [11]). Table 1 Functional diversity of fungal EVs. EV Biogenesis: Where Do They Come From? Exosomes and ectosomes are major EVs produced by eukaryotic cells. Exosomes consist of small (40–100 nm) vesicles originated by invagination of the endosomal compartments membrane, which is driven by a protein complex named endosomal sorting complex required for transport (ESCRT) [12]. This complex regulates the release of small vesicles inside the lumen of the endosome, generating the so-called multivesicular bodies (MVBs). Upon fusion with the plasma membrane, MVBs release exosomes as EVs to the outer space [12]. Unlike exosomes, ectosomes are larger (up to 1 μm), ubiquitous vesicles that are assembled at and released from the plasma membrane [13]. In fungi, mechanisms of vesicle biogenesis and extracellular release are still obscure. Therefore, these extracellular membranous compartments are still collectively called EVs. MVB-like structures have been observed in C. neoformans [5]. Saccharomyces cerevisiae mutants lacking expression of ESCRT machinery proteins still produced EVs, but vesicle cargo was modified in the absence of ESCRT regulators [14]. Analysis of fungal EVs by electron microscopy revealed two kinds of populations in the cell wall periphery: large (up to 300 nm), individualized vesicles and small (up to 100 nm), grouped vesicles [15]. Groups of small EVs in the periplasm are consistent with exosome formation, as suggested in early studies with C. neoformans [16]. Observation of individualized and larger vesicles, however, is suggestive of membrane budding, likely resulting in ectosomes [17]. Membrane budding, in fact, has been observed more than a decade ago in C. neoformans [18]. EV formation can also include inverted macropinocytosis, a process by which fractions of the cytoplasm are sequestered by plasma membrane invaginations, resulting in individualized EV-like structures [19]. All the mechanisms described above would be consistent with the diversity in EV composition, which includes a number of cytoplasmic components (reviewed in [11]).

Background: Small vessel cerebral vascular disease (sCVD) is common to older individuals, often asymptomatic, but associated with incident stroke and future mortality. Multiple mechanisms for sCVD have been postulated, all of which include injury to the neurovascular unit. Analysis of extracellular vesicles (EVs), important constituents of the intercellular communication pathway, may offer new ways to evaluate pathological processes of sCVD as well as serve as novel biomarkers or identify new avenues for therapy. This abstract summarizes work by our group to subtype EVs and measure cargo proteins from the various cell types of the neurovascular unit from a group of cognitively normal individuals with a spectrum of sCVD. Method: Study participants consisted of 14 individuals, 50 % female, 76 + 7 years of age, having 17.6 + 1.9 years of education. White matter hyperintensities varied from 0.73 – 38.8 cc. EV isolation used an Exodus ultrafiltration system on platelet depleted plasma. EVs were further fractionated into EV subtypes with resin-conjugated antibodies against cell type-specific markers. Five EV subtypes were isolated by two rounds of immunoprecipitation with the following markers: NCAM1/ATP1A3 (excitatory neuron EV), CD49f/LRP1 (astrocyte EV), CD11b/LCP1 (activated microglia EV), LAMP2/FTH1 (oligodendrocyte EV), CD31/CD146 (activated endothelial EV). Following isolation of EV subtypes, samples were characterized for purity and yield by nanoparticle tracking analysis, imaged for protein expression by super resolution microscopy, and sequenced for biomarker identification by quantitative proteomics. Results: Nanoparticle tracking analysis confirmed high yields of all 5 EV subtypes, > 2E9 EVs/mL, along with identical size and surface charge profiles. Super resolution microscopy showed consistent canonical EV marker distributions on all EVs while EV subtypes expressed unique markers based off their cell type of origin. Quantitative proteomics identified 400 unique and differentially expressed proteins present amongst the various EV subtypes as compared to mean plasma EVs concentrations. Protein abundance varied widely across EV subpopulations, indicating distinct protein profiles. Conclusion: Preliminary results confirm the potential for biomarker discovery from novel EV subpopulations through identification of differentially expressed cargo proteins from the neurovascular unit. Future work will associate these findings with sCVD phenotypes.

Understanding communication mechanisms between unicellular parasites is crucial in the development of novel therapies, as they rely on diverse modes of communication (like extracellular vesicle release, cytoneme, and filopodia formation) to regulate their behavior and survival.

The role of extracellular vesicles (EVs) in interkingdom communication is widely accepted, and their role in intraspecies communication has been strengthened by recent research. Based on the regulation promoted by EV-associated molecules, the interactions between host and pathogens can reveal different pathways that ultimately affect infection outcomes. As a great part of the regulation is ascribable to RNA contained in EVs, many studies have focused on profiling RNAs in fungal and host EVs, tracking their accumulation during infection, and identifying potential target genes. Herein, we overview the main classes of RNA contained in fungal EVs and the biological processes regulated by these molecules, portraying a state-of-the-art picture of RNAs loaded in fungal EVs, while also raising several questions to drive future investigations. Our compiled data show unambiguously that EVs act as key elements in signaling pathways, and play a crucial role in pathosystems. A complete understanding of the processes that govern RNA content loading and trafficking, and its effect on recipient cells, will lead to improved technologies to ward off infectious agents that threaten human health.

Differential fluorescence nanoparticle tracking analysis for enumeration of the extracellular vesicle content in mixed particulate solutions.

Extracellular vesicles (EVs) are lipid bilayered compartments released by virtually all living cells, including fungi. Among the diverse molecules carried by fungal EVs, a number of immunogens, virulence factors and regulators have been characterised. Within EVs, these components could potentially impact disease outcomes by interacting with the host. From this perspective, we previously demonstrated that EVs from Candida albicans could be taken up by and activate macrophages and dendritic cells to produce cytokines and express costimulatory molecules. Moreover, pre-treatment of Galleria mellonella larvae with fungal EVs protected the insects against a subsequent lethal infection with C. albicans yeasts. These data indicate that C. albicans EVs are multi-antigenic compartments that activate the innate immune system and could be exploited as vaccine formulations. Here, we investigated whether immunisation with C. albicans EVs induces a protective effect against murine candidiasis in immunosuppressed mice. Total and fungal antigen-specific serum IgG antibodies increased by 21 days after immunisation, confirming the efficacy of the protocol. Vaccination decreased fungal burden in the liver, spleen and kidney of mice challenged with C. albicans. Splenic levels of cytokines indicated a lower inflammatory response in mice immunised with EVs when compared with EVs + Freund's adjuvant (ADJ). Higher levels of IL-12p70, TNFα and IFNγ were detected in mice vaccinated with EVs + ADJ, while IL-12p70, TGFβ, IL-4 and IL-10 were increased when no adjuvants were added. Full protection of lethally challenged mice was observed when EVs were administered, regardless the presence of adjuvant. Physical properties of the EVs were also investigated and EVs produced by C. albicans were relatively stable after storage at 4, -20 or -80°C, keeping their ability to activate dendritic cells and to protect G. mellonella against a lethal candidiasis. Our data suggest that fungal EVs could be a safe source of antigens to be exploited in vaccine formulations.

3046 Background: Extracellular Vesicles (EV) are of broad interest as carriers of molecular signatures of tumor progression and cancer treatment response. EVs, which contain nucleic acids, lipids, and proteins, are released from cells for waste excretion and communication. Numerous proteins and markers are expressed within and on the surface of EVs, but classification markers for murine EV subsets are lacking. To identify tumor and dendritic cell- derived EV markers for preclinical models of breast cancer, we investigated surface marker repertoires of EVs produced by the murine breast cancer and dendritic cell lines, 4T1 and DC2.4. Methods: Cells were cultured in serum free media for 2 days. EVs were harvested and isolated by ultrafiltration followed by size exclusion chromatography. EV particle size and concentration were estimated by nanoparticle tracking analysis and microBCA. To identify highly expressed EV markers, a mouse EV multiplex flow cytometry assay was performed using detection antibodies, CD9, CD63, and CD81, with sets of >35 barcoded capture beads, representing more than 100 specific capture: detection combinations. EV marker expression was analyzed using the FCMPASS/MPAPASS software (nano.ccr.cancer.gov). > 250 beads were assessed for each capture- and detection- antibody combination for each EV type and dilution tested; mean fluorescent intensity was determined; and pairwise comparisons between test and control sample sets were evaluated by t-tests. Results: Breast cancer (4T1)-derived EVs but not dendritic cell (DC2.4)-derived EVs were strongly detected with CD326 (EpCAM) and CD49b (integrin alpha5, VLA-2) capture beads, using each of the three tetraspanin antibodies. Both types of EVs were detected with anti-CD9 and anti-CD81 when captured by anti-CD44 and anti-CD49e (integrin beta1, VLA-5) beads. DC2.4 EVs were distinctively identified by CD11b capture. CD63 capture and detection antibodies robustly recognized EVs from 4T1 but provided minimal recognition of DC2.4 EVs. Mouse serum EVs from non-tumor bearing mice, showed minimal or no detectable CD326 or CD11b. Conclusions: Multiparametric MPAPASS-processed EV repertoire analysis of EVs from murine breast cancer and dendritic cell lines identified CD9, CD81, CD44, and CD49e as common epitopes among both types of evaluated EVs. CD326, CD49b, and CD63 distinguished 4T1 from DC2.4 EVs, and CD11b distinctively identified the DC2.4 EVs. The absence of detected CD326+ and CD11b+ in the serum of non-tumor bearing mice indicates the potential of these two markers for detection of specific tumor and antigen presenting cell EV subsets in serum from mice bearing CD326+ tumors such as 4T1. These results establish a foundation for further tests of detection and tracking of tumor-specific CD326+ EVs as "liquid biopsies" in blood samples as correlates to tumor progression and/or response to treatment.

Comparing small urinary extracellular vesicle purification methods with a view to RNA sequencing—Enabling robust and non-invasive biomarker research

e17561 Background: Exosomes have been involved in tumor progression and development of therapeutic resistances in several malignancies. This subtype of extracellular vesicles (EVs) promote angiogenesis, premetastatic niches formation and communication between the tumor and its microenvironment. Ovarian cancer (OC) is a highly lethal tumor that typically spreads within the abdominal cavity rather than through blood vessels. Based on this behavior, we aimed to study the presence of EVs in ascitic fluid of OC patients and explore their potential as predictors of tumor volume and chemoresistance. Methods: Observational prospective study in advanced OC patients who underwent a diagnostic laparoscopy or cytoreductive surgery. Abdominal washings of a cohort of non-cancer patients were collected as controls. All cases provided written consent. Clinical data and tumor genomic alterations (NGS, FoundationOne CDx) were recorded. EVs pellets were collected by ultracentrifugation and profiled by NTA and western blot (EVs markers: CD9, TSG101 and ALIX; non EVs related markers: ALB and APOB). EVs proteins were quantified by BCA assay and profiled by mass spectrometry. Results: We obtained 75 peritoneal fluids from 66 OC patients (median age at diagnosis [range] 62 [26-83] years) and 29 peritoneal washings from controls. Our cohort comprises various histological subtypes (high grade serous, n 55; low grade serous, n 2; endometrioid, n 6; clear cell, n 2 and mucinous, n 1) and tumor stages (I-II, n 7; III; n 39; IV, n 20). Samples were collected either from primary (n 36); interval, (n 25); or relapse surgery (n 14). Pathogenic BRCA events: 15 patients. Mean concentration of exosomes (MCE) was 3.0x106 vs. 1.6x106 particles/ml (p/ml) in cases vs. controls (p < 0.0017). No difference in MCE was observed regarding stages I-II vs. III-IV (3.9x106 vs. 3.3x106 p/ml; p = 0.38). A higher MCE was observed in those samples collected after relapse surgeries compared to those collected from primary (7.2x106 vs. 2.8x106 p/ml; p < 0.0001) and interval surgeries (7.2x106 vs. 3.4x106 p/ml; p < 0.0007). Median exosomal protein concentration (MEPC) in EVs of cancer patients vs. controls was 0.99µg/µl and 0.57µg/µl respectively (p = 0.02). Proteomic characterization by mass spectrometry of EVs cargo has identified 1827 proteins, of which 405 proteins were differentially expressed between cases and controls. We also found 38 differentially expressed proteins comparing platinum-sensitive vs. resistant patient samples. Conclusions: Our data show higher concentration of exosomes and exosomal proteins in OC cases vs controls. Differentially expressed proteins have also been found between samples from platinum-sensitive and platinum-resistant patients. These results suggest that exosomes may be involved in the progression and spread of OC and may provide insights into current treatment response.

Fungal Extracellular Vesicles

Characterization of extracellular vesicles derived from two populations of human placenta derived mesenchymal stem/stromal cells

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

Free flow electrophoresis allows preparation of extracellular vesicles with high purity

Extracellular vesicles (EVs) are a broad category of small vesicles released by cells in the central nervous system (CNS) that facilitate intercellular communication and contribute to various diseases. In neuroscience, one of the most important and well-characterized EV subtypes is exosomes. However, current issues with EV nomenclature make it difficult to draw firm conclusions due to variability in usage across different studies. One solution to overcome this challenge is to use established markers for isolating different types of CNS-derived EVs. This article provides an up-to-date list of potential EV markers, focusing on surface markers. We discuss the role of EVs in CNS diseases and explore each candidate's potential to be used as a specific EV surface marker. We also highlight how non-adherence to EV nomenclature leads to confusion and misuse of EV markers. Additional challenges in EV research, such as isolation methods and the lack of comparative studies between plasma and tissue-derived EVs, are discussed. Providing a list of new possible CNS-derived EV marker candidates can lead to more precise isolation and description of EVs, thereby enhancing our understanding of EV signaling and advancing our knowledge of neurological diseases.