Figure - available from: Frontiers in Immunology
This content is subject to copyright.
Nanoparticle-tracking analysis of extracellular vesicles (EVs) produced by T. interdigitale. EVs were purified from T. interdigitale culture supernatants and quantified using a NanoSight NS300. (A) Histogram showing the EV particle-size distribution (EVs × 10⁹/mL vs size in nanometers). (B) Screenshot from video recorded using the NanoSight NS300, showing the distribution of EVs from T. interdigitale.
Source publication
The release of biomolecules critically affects all pathogens and their establishment of diseases. For the export of several biomolecules in diverse species, the use of extracellular vesicles (EVs) is considered to represent an alternative transport mechanism, but no study to date has investigated EVs from dermatophytes. Here, we describe biological...
Citations
... In relation to filamentous fungi of medical relevance, the data available so far are limited [19]. EVs were isolated and characterized from Trichophyton interdigitale [32], Rhizopus delemar [33], Aspergillus flavus [34] and A. fumigatus [35]. More recently, the first characterization of EVs in protoplasts obtained from germinating conidia of A. fumigatus was described, showing that EV production was a common feature of different morphologi-cal stages of this opportunistic fungal pathogen. ...
... The electron density of the EVs varied considerably, suggesting distinct contents. The micrographs also showed that the majority of EVs presented spherical or ovoid shape with lipid bilayer, sometimes resembling multivesicular compartments ( Figure 3B) similar to those described for several fungi pathogens: C. neoformans, H. capsulatum, Candida parapsilosis, C. albicans, Sporothrix schenckii; Saccharomyces cerevisiae, P. brasiliensis, Malassezia sympodialis, A. infectoria, Paracoccidioides lutzii, S. brasiliensis, Cryptococcus gattii, R. delemar, T. interdigitale, A. fumigatus, A. flavus and Candida auris [21,23,[25][26][27][28][29][30][31][32][33][34][35]37,50,[53][54][55][56]. ...
... The electron density of the EVs varied considerably, suggesting distinct contents. The micrographs also showed that the majority of EVs presented spherical or ovoid shape with lipid bilayer, sometimes resembling multivesicular compartments ( Figure 3B) [21,23,[25][26][27][28][29][30][31][32][33][34][35]37,50,[53][54][55][56]. It is well-known that sterols are structural components of fungal EVs [37]; for this reason, these molecules were used as molecular markers of indirect identification of vesicular secretion ( Figure 4A). ...
The release of extracellular vesicles (EVs) has been implicated as an alternative transport mechanism for the passage of macromolecules through the fungal cell wall, a phenomenon widely reported in yeasts but poorly explored in mycelial cells. In the present work, we have purified and characterized the EVs released by mycelia of the emerging, opportunistic, widespread and multidrug-resistant filamentous fungus Scedosporium apiospermum. Transmission electron microscopy images and light scattering measurements revealed the fungal EVs, which were observed individually or grouped with heterogeneous morphology, size and electron density. The mean diameter of the EVs, evaluated by the light scattering technique, was 179.7 nm. Overall, the structural stability of S. apiospermum EVs was preserved during incubation under various storage conditions. The lipid, carbohydrate and protein contents were quantified, and the EVs’ protein profile was evidenced by SDS-PAGE, revealing proteins with molecular masses ranging from 20 to 118 kDa. Through immunoblotting, ELISA and immunocytochemistry assays, antigenic molecules were evidenced in EVs using a polyclonal serum (called anti-secreted molecules) from a rabbit inoculated with conditioned cell-free supernatant obtained from S. apiospermum mycelial cells. By Western blotting, several antigenic proteins were identified. The ELISA assay confirmed that the anti-secreted molecules exhibited a positive reaction up to a serum dilution of 1:3200. Despite transporting immunogenic molecules, S. apiospermum EVs slightly induced an in vitro cytotoxicity effect after 48 h of contact with either macrophages or lung epithelial cells. Interestingly, the pretreatment of both mammalian cells with purified EVs significantly increased the association index with S. apiospermum conidia. Furthermore, EVs were highly toxic to Galleria mellonella, leading to larval death in a typically dose- and time-dependent manner. Collectively, the results represent the first report of detecting EVs in the S. apiospermum filamentous form, highlighting a possible implication in fungal pathogenesis.
... Subsequently, the coverslips were removed and stained with Panotico Rapido dye (Laborclin, Pinhais, PR, Brazil). Approximately 100 macrophages were counted to determine the average number of conidia inside macrophages (conidia/100 macrophages) and the percentage of macrophages that had internalized at least one conidium (% macrophage with conidia) 25 . ...
... To investigate the macrophages' ability to control fungal proliferation after phagocytosis, a killing assay was performed 25 . Cells were treated and challenged under the same conditions as those described above for the phagocytosis assay. ...
... Similarly, IL-23 secreted by macrophages promotes Th17 differentiation (108). On the other hand, macrophages can engulf abnormally proliferating KCs, alleviate psoriatic lesions (130), and play multiple roles in the immunopathological process of psoriasis. Further studies would contribute to a deep understanding of their regulatory mechanisms, offering new strategies and targets for treating psoriasis. ...
Psoriasis is a chronic autoimmune inflammatory disease characterized by erroneous metabolism of keratinocytes. The development of psoriasis is closely related to abnormal activation and disorders of the immune system. Dysregulated skin protective mechanisms can activate inflammatory pathways within the epithelial immune microenvironment (EIME), leading to the development of autoimmune-related and inflammatory skin diseases. In this review, we initially emphasized the pathogenesis of psoriasis, paying particular attention to the interactions between the abnormal activation of immune cells and the production of cytokines in psoriasis. Subsequently, we delved into the significance of the interactions between EIME and immune cells in the emergence of psoriasis. A thorough understanding of these immune processes is crucial to the development of targeted therapies for psoriasis. Finally, we discussed the potential novel targeted therapies aimed at modulating the EIME in psoriasis. This comprehensive examination sheds light on the intricate underlying immune mechanisms and provides insights into potential therapeutic avenues of immune-mediated inflammatory diseases.
... Although the cell wall was initially thought to prevent vesicular transit due to its rigidity and absence of large pores, we now know that cell walls are flexible structures that can be easily rearranged for cellular division or during EV generation. In the last decades, it has been shown that F-EVs are fundamental for key biological functions, such as cell wall remodeling [16], biofilm matrix formation [17], and host-pathogen interactions [18]; and that these particles are clearly able to modify the behavior of recipient cells [19]. To date, F-EV production has been confirmed in several fungal species, including yeasts such as Saccharomyces cerevisiae [16], known for its role in beer production, or the opportunistic pathogen Candida albicans [20], which is also a common member of the human microbiota. ...
Extracellular vesicles (EVs) represent a complex mechanism of molecular exchange that has garnered significant attention in recent times. Nonetheless, identifying sustainable sources of biologically safe EVs remains challenging. This chapter delves into the utilization of fermented food industry by-products as a circular and secure reservoir of biocompatible EVs, dubbed as BP-EVs. BP-EVs demonstrate excellent oral bioavailability and biodistribution, with negligible cytotoxicity, and a preferential targeting capacity toward the central nervous system, liver, and skeletal tissues. The ease of editing BP-EVs is also depicted using the most common EV editing methods in this chapter. Globally, these groundbreaking findings are poised to unlock significant avenues for leveraging BP-EVs as an optimal source of biocompatible nanovesicles across a wide array of applications within the bioeconomy and biomedical fields. These applications primarily target molecule delivery into the central nervous system and skeletal tissue but are not limited to these two organism systems.
... Both conidia and hyphae can induce the formation of Neutrophil extracellular traps (NETs) but it is more involved in the killing and elimination of hyphae indicating that phagocytosis is the main mechanism of conidial inactivation by neutrophils (Reis et al., 2022). Extracellular vesicles are also produced by dermatophytes and were identified in T. interdigitale (Bitencourt et al., 2018). ...
Dermatophytosis is a common superficial mycosis among humans and
animals. Dermatophytes are keratinophilic organisms and can utilize
keratin as a sole source of nutrients. Dermatophytes are classified into 7
genera namely Microsporum, Trichophyton, Epidermophyton, Nannizzia,
Lophophyton, Paraphyton and Arthroderma. Based on the ecology and
host preference, they are classified into anthropophiles, zoophiles and
geophiles. The characteristic lesion produced in dermatophytosis is called
as ringworm and in humans the disease is known as Tinea. Dermatophytosis is considered as a zoonotic disease and can be transmitted
from pet and farm animals. The disease is transmitted by the infectious
propagules called arthroconidia. Dermatophytes secrete various endo- and
exo-proteases which assist in degrading the keratin tissue. Dermatophytes
can induce both immediate hypersensitivity (IH) reactions and delayed
type hypersensitivity (DTH) reactions. The disease is diagnosed by the
clinical signs, microscopic examination of clinical samples, isolation of the
organism, identification of etiological agent by growth characteristics,
microscopic examination, laboratory test and molecular techniques. The
dermatophytes produce colonies with characteristic morphology and
reverse pigmentation. Most of the dermatophytes produce macroconidia of
peculiar shape and size. The commonly employed laboratory tests include
urease production test, hair perforation test and growth pattern in
Trichophyton agar. The molecular techniques such as PCR, sequencing,
RFLP, AFLP and MALDI-TOF are utilized for species and strain
differentiation. Allylamines such as terbinafine and azoles such as
ketoconazole, itraconazole are the most commonly used drugs for
dermatophytosis.Emergence of dug resistant strains of
dermatophytes are creating challenges in the therapy and necessitates the
development of newer formulations of drugs. Vaccines are not commonly
used for the control of dermatophytosis but a low virulent strain of T.
verrucosum named LTF-130 is being used for immunization against
dermatophytosis in cattle.
... Furthermore, applying an innovative protocol for the new isolation of Cryptococcus gattii EVs, Reis et al. studied Cryptococcus gattii and revealed the involvement of scrablase a phospholipid translocase, as a virulence factor in Cryptococcus gattii secretion (Reis et al., 2019). EVs have also been observed in fungal culture supernatants and body fluids of various human pathogenic fungi, including Histoplasma capsulatum (Albuquerque et al., 2008), Sporothrix brasiliensis (Ikeda et al., 2018), Sporothrix schenckii (Albuquerque et al., 2008), Malassezia sympodialis (Gehrmann et al., 2011), and Trichophyton interdigitale (Bitencourt et al., 2018). Zhao et al. demonstrated that EVs enriched with Saccharomyces cerevisiae Fks1 and Chs3 protected yeast cells from cell wall disruption, suggesting that EVs prevent the host immune system from killing fungal organisms through a potential role in intraspecific fungal communication (Zhao et al., 2019). ...
... Bitencourt et al. were the first to report that Trichophyton interdigitale secretes EVs, which stimulate the transcription of the M1-polarization marker inducible nitric oxide synthase (iNOS) and inhibit the expression of the M2 markers arginase-1 and Ym-1. These EVs also induce the production of pro-inflammatory mediators by bone marrow-derived macrophages (BMDMs) and keratinocytes in a dose-dependent manner (Bitencourt et al., 2018). Gehrmann et al. discovered that EVs derived from DCs co-cultured with Malassezia sympodialis can carry Malassezia sympodialis antigens and stimulate cytokine release in autologous CD14 and CD34-depleted PBMCs from patients. ...
Invasive fungal disease (IFD) poses a significant threat to immunocompromised patients and remains a global challenge due to limited treatment options, high mortality and morbidity rates, and the emergence of drug-resistant strains. Despite advancements in antifungal agents and diagnostic techniques, the lack of effective vaccines, standardized diagnostic tools, and efficient antifungal drugs contributes to the ongoing impact of invasive fungal infections (IFI). Recent studies have highlighted the presence of extracellular vesicles (EVs) released by fungi carrying various components such as enzymes, lipids, nucleic acids, and virulence proteins, which play roles in both physiological and pathological processes. These fungal EVs have been shown to interact with the host immune system during the development of fungal infections whereas their functional role and potential application in patients are not yet fully understood. This review summarizes the current understanding of the biologically relevant findings regarding EV in host-pathogen interaction, and aim to describe our knowledge of the roles of EV as diagnostic tools and vaccine vehicles, offering promising prospects for the treatment of IFI patients.
... Whilst a shortfall of protocols and specific biomarkers for EV isolation continues to be a challenge in the field of EV biology, particularly for filamentous fungi, numerous studies on fungal EV production remain biased towards human fungal yeast pathogens (Albuquerque et al., 2008;Gehrmann et al., 2011;Vallejo et al., 2011;Vallejo et al., 2012;Vargas et al., 2015;Leone et al., 2017;Bielska et al., 2018;Ikeda et al., 2018;Peres da Silva et al., 2019;Lavrin et al., 2020). Consequently, very little is known about the release of EVs in plant pathogenic fungi, although a large number of studies have confirmed their presence in these organisms (Silva et al., 2014;Bitencourt et al., 2018;Liu et al., 2018;de Paula et al., 2019;Souza et al., 2019;Bleackley et al., 2020;Brauer et al., 2020;Rizzo et al., 2020;Costa et al., 2021;Garcia-Ceron et al., 2021). However, the production of EVs in fungi is considered unconventional because they mostly transport proteins lacking signal peptides (Samuel et al., 2015). ...
Organisms from all kingdoms of life release membrane vesicles, which are tiny and spherical structures made of a lipid bilayer. Membrane vesicles carry out a number of functions, such as forming new cell membranes, removing waste products from the cell, and transporting lipids and other substances from parent to recipient cells. The payloads often contained in the vesicles are sorted via the endosomal sorting complex required for transport (ESCRT) pathway in a stepwise manner. Alterations to this endomembrane system reduces formation of vesicles and aberrant endosomal compartments. Furthermore, in pathogenic fungi, the deletion of ESCRT genes negatively effects virulence and growth, suggesting the ESCRT pathway has links to disease. However, only a few fungal species have to date been evaluated for the ESCRT pathway. In this review, we evaluate recent developments in the ESCRT pathway of fungi that infect plant hosts and its role in pathogenesis. This will provide an overview of EV-mediated cell-cell communication during host-pathogen interactions.
... Such protocol granted a reliable and cost-effective method to isolate EVs from many other fungi until today. In a few hours, with only one ultracentrifuge and a flask of TBE or PBS, a pellet of 1x10 8 to 10 11 particles/mL could be easily obtained (Bitencourt et al., 2018;Ikeda et al., 2018;De Paula et al., 2019). However, the main disadvantages regarding centrifugation are working with large volumes and the fact that other molecules, such as proteins, lipoproteins, and nonexosomal particles, will also be isolated given their similar size and density (Gardiner et al., 2016;Mathieu et al., 2019). ...
... marneffei (Yang et al., 2021) and T. interdigitale (Bitencourt et al, 2018). ...
... Macrophages are another cell that plays a vital role in controlling fungi infection (Heung, 2020). Fungi EVs were able to modulate these cells, increasing the fungicidal capacity and/or production of inflammatory mediators as observed in A. flavus (Brauer et al., 2020), A. fumigatus (Souza et al., 2019;Freitas et al. 2023), C. albicans , C. neoformans (Oliveira et al., 2010), P. brasiliensis (da Silva et al., 2016), S. brasiliensis (Campos et al., 2021) and Trichophyton interdigitale (Bitencourt et al., 2018). Otherwise, in H. capsulatum (Baltazar et al., 2018) and one strain of C. auris 6 (Zamith-Miranda et al., 2021), EVs reduced the fungicidal rate of macrophages, revealing different effects of EVs on host cells. ...
Like other organisms, fungi produce extracellular vesicles (EVs) that are involved in various biological processes, including intercellular communication and the transport of molecules between cells. These EVs can be applied in fungal pathogenesis, virulence, and interactions with other organisms, including host cells, in the case of fungal infections. While some types of mycoses are relatively common and easily treatable, certain neglected mycoses pose significant public health challenges, such as sporotrichosis, chromoblastomycosis, and paracoccidioidomycosis. These infectious diseases can cause significant morbidity and disability, leading to a reduced quality of life for the patients. So, research about the virulence factor is essential to understand how fungi escape the immune system. In this context, this manuscript reviews the study of fungi EVs, their cargo, their obtaining, and their role during the infectious process, which is extremely important for understanding this neglected mycosis.
... Extracellular vesicles (EVs) are spherical bilayered compartments secreted by all live cells [12,14]. EV production was reported in several fungal species, such as Aspergillus flavus [15], Aspergillus fumigatus [16], C. albicans [12], Candida parapsilosis [12], Cryptococcus gattii [17], Histoplasma capsulatum [12], Malassezia sympodialis [18], Sporothrix brasiliensis [19], Sporothrix schenckii [12], Saccharomyces cerevisiae [20], Paracoccidioides brasiliensis [21], Trichophyton interdigitale [22], and several other fungi. EVs are composed of proteins, polysaccharides, lipids, RNA, and pigments, and these structures may be associated with pathogenesis during fungal infection [6,12,13,[23][24][25]. ...
... The pellet (only EVs) was resuspended in 1 mL sterile nuclease-free water (Sigma Aldrich). All steps were conducted at 4 • C. Characterization and quantification of EV preparation was performed using nanoparticle tracking analysis (NTA) in NanoSight NS300 (Malvern Instruments, Malvern, UK), as previously described [22]. The experiments were performed in triplicate. ...
Candida albicans is a commensal fungus in healthy humans that causes infection in immunocompromised individuals through the secretion of several virulence factors. The successful establishment of infection is owing to elaborate strategies to cope with defensive molecules secreted by the host, including responses toward oxidative stress. Extracellular vesicle (EV) release is considered an alternative to the biomolecule secretory mechanism that favors fungal interactions with the host cells. During candidiasis establishment, the host environment becomes oxidative, and it impacts EV release and cargo. To simulate the host oxidative environment, we added menadione (an oxidative stress inducer) to the culture medium, and we explored C. albicans EV metabolites by metabolomics analysis. This study characterized lipidic molecules transported to an extracellular milieu by C. albicans after menadione exposure. Through Liquid Chromatography coupled with Mass Spectrometry (LC-MS) analyses, we identified biomolecules transported by EVs and supernatant. The identified molecules are related to several biological processes, such as glycerophospholipid and sphingolipid pathways, which may act at different levels by tuning compound production in accordance with cell requirements that favor a myriad of adaptive responses. Taken together, our results provide new insights into the role of EVs in fungal biology and host–pathogen interactions.
... EVs from Cryptococcus neoformans are phagocytosed by macrophages, inducing cell activation and NO and cytokine production [36], whereas C. albicans EVs activate RAW 264.7 cells resulting in NO production and release of IL-12, IL-10, TGF-β, and TNF-α [69]. Another example of EV interaction with the immune host system is EVs released from Trichophyton interdigitale, which stimulate the release of NO, TNF-α, IL-6, and IL-1β, but not IL-10, from murine macrophages and human keratinocytes in a dose-dependent manner [37]. ...
Members of the Candida haemulonii species complex are multidrug-resistant emergent yeast pathogens able to cause superficial and invasive infections in risk populations. Fungal extracellular vesicles (EVs) play a critical role in the pathogenicity and virulence of several species and may perform essential functions during infections, such as carrying virulence factors that behave in two-way communications with the host, affecting survival and fungal resistance. Our study aimed to describe EV production from Candida haemulonii var. vulnera and evaluate whether murine macrophage RAW 264.7 cells respond to their stimuli by generating an oxidative response after 24 h. For this purpose, reactive oxygen species detection assays demonstrated that high concentrations of yeast and EVs (1010 particles/mL) of Candida haemulonii did not change macrophage viability. However, the macrophages recognized these EVs and triggered an oxidative response through the classical NOX-2 pathway, increasing O2•− and H2O2 levels. However, this stress did not cause lipid peroxidation in the RAW 264.7 cells and neither lead to the activation of the COX-2–PGE2 pathway. Thus, our data suggest that low concentrations of C. haemulonii EVs are not recognized by the classical pathway of the oxidative burst generated by macrophages, which might be an advantage allowing the transport of virulence factors via EVs, not identified by the host immune system that could work as fine tube regulators during infections caused by C. haemulonii. In contrast, C. haemulonii var. vulnera and high EV concentrations activated microbicidal actions in macrophages. Therefore, we propose that EVs could participate in the virulence of the species and that these particles could be a source of antigens to be exploited as new therapeutic targets.
