Chromoblastomycosis (CBM) is one of the most predominant cutaneous and subcutaneous infections encountered, especially in tropical and subtropical regions of the world (Attapattu, 1997; Bonifaz et al., 2001; Queiroz-Telles et al., 2009; Lu et al., 2013). CBM is a neglected disease by the World Health Organization (WHO, 2013). It is a chronic and granulomatous mycosis caused by a group of melanized and dimorphic fungi belonging to the Herpotrichiellaceae family (order Chaetothyriales). The disease is acquired through the traumatic inoculation of fungal propagules into the human tissue and can affect immunocompetent individuals (Mcginnis, 1938; Rubin et al., 1991; Marques et al., 2006; Guedes et al., 2010; Queiroz-Telles, 2015; Brito and Bittencourt, 2018). The most well-known etiological agent of CBM is Fonsecaea pedrosoi. However, species such as F. monophora, F. nubica, and F. pugnacious are important pathogens to humans (Santos et al., 2007; Najafzadeh et al., 2009; Najafzadeh et al., 2010; de Azevedo et al., 2015; Feng and de Hoog, 2017). All pathogenic species of the Fonsecaea genus are morphologically and genetically similar, but the disease has different clinical aspects and geographic distributions (Najafzadeh et al., 2010; Teixeira et al., 2017; Vicente et al., 2017). Clinically, F. pedrosoi and F. nubica are exclusively related to CBM, whereas F. monophora and F. pugnacious can infect different organs and are considered opportunistic pathogens (Santos et al., 2007; Najafzadeh et al., 2009; Najafzadeh et al., 2010). Except for F. pedrosoi, which is restricted to South and Central America, all other species have also been described in Asia, Africa, and Europe (Xi et al., 2009; Cateau et al., 2014; de Azevedo et al., 2015; Fransisca et al., 2017; Label et al., 2018; Rasamoelina et al., 2020).

An essential characteristic of CBM is the presence of highly melanized thick-walled cells, called sclerotic bodies or muriform cells (MCs), that are crucial for fungal persistence inside host tissues, protecting against environmental stresses (Alviano et al., 1992; López-Martínez and Méndez-Tovar, 2007; Feng and de Hoog, 2017; Dong et al., 2020). These cells are considered the key factor for Fonsecaea pathogenicity and are related to chronic disease. (Hamza et al., 2003; Salgado et al., 2010; Siqueira et al., 2017; Dong et al., 2020).

The capacity of Fonsecaea spp. to undergo the transition between the saprophytic (conidia and hyphae) and the parasitic (MCs) morphotypes is considered the main requirement for fungal persistence on the human tissue (Queiroz-Telles et al., 2017; Siqueira et al., 2017; Dong et al., 2018). The conidial and hyphal forms of Fonsecaea are frequently found in the environment and during the initial stages of CBM, which are marked by fungal inoculation, adhesion to the tissue, and proliferation (Queiroz-Telles, 2015; Siqueira et al., 2017; Dong et al., 2020). Once the fungus has infected the host, MCs become the predominant cells found within the granulomatous and suppurative centers of the lesions observed in CBM (Bonifaz et al., 2001; Minotto et al., 2001; Queiroz-Telles et al., 2017). Nutritional conditions, temperature, and stress factors, such as calcium deprivation and pH, are considered important factors that elicit fungal morphogenesis (Alviano et al., 1992; Mendoza et al., 1993; Silva et al., 2002; Santos et al., 2007). In vitro, highly nutritional medium (e.g., Sabouraud Dextrose Broth and Potato Dextrose Broth) induces mainly conidia and hyphae formation (Alviano et al., 1992; Siqueira et al., 2017). In contrast, in conjunction with propranolol – a beta-adrenergic antagonist – supplementation, as proposed by Alviano et al. (1992), minimum medium conditions can promote MCs production that is morphologically similar to those obtained in vivo (Alviano et al., 1992; Silva et al., 2002; Dong et al., 2018). The molecular mechanisms and virulence factors related to CBM remain not-so-well-known, although it is clear that microenvironmental factors play essential roles on Fonsecaea morphological transition and survival inside the host.

Recently it has been shown that hyphae and MCs of F. pedrosoi are related to the intense pro-inflammatory response found in CBM. At the same time, conidia alone cannot induce the chronic phase of the disease in the animal model of infection nor stimulate the production of important cytokines such as TNF, IL-1β, and IL-6 in vitro (Siqueira et al., 2017). Furthermore, F. pedrosoi hyphae, but not conidia, are capable of promoting IL-1β secretion by macrophages and dendritic cells through NLRP3-caspase-1 inflammasome activation, a crucial mediator of the host immune response during fungal infections (Vanaja et al., 2015; de Castro et al., 2017). Additionally, current research indicates that the early stages of F. pedrosoi infection in a self-healing murine CBM model are mediated mainly by Th17 response through Dectin-2 receptors (Siqueira et al., 2020). In contrast, a pro-inflammatory Th1 response profile becomes predominant in later stages of the disease, which is necessary to combat MCs and is associated with the healing of the lesion (Siqueira et al., 2020). Lastly, it was demonstrated that Treg cells are pivotal for fungal clearance, and deregulation of these cells is involved with improper host immune response (Siqueira et al., 2020).

Extracellular vesicles (EVs) are an embracing term to refer to membranous structures composed of a phospholipid bilayer that may be originated from distinct biological pathways but that have in common the ability to reach the extracellular environment (van Niel et al., 2018; Rizzo et al., 2020b). Bacteria, fungi, plants, and animals can produce vesicles that can be found intra- or extracellularly, which is an evolutionary capacity conserved among all the kingdoms of life (Deatherage and Cookson, 2012; Brown et al., 2015; van Niel et al., 2018). These structures are highly heterogeneous, especially in their biogenesis, size, and the content they carry (Willms et al., 2018; recently revised by Rizzo et al. (2021)).

Pathogenic fungal EVs were first described in 2007 for Cryptococcus neoformans by Rodrigues and co-workers and have gained massive attention since that (Rodrigues et al., 2007). The first characterization of EVs produced by melanized fungi was obtained from Alternaria infectoria (Dematiaceae), an environmental filamentous fungus involved with crop infestations that can also infect immunocompromised individuals (Silva et al., 2014). Only 6 years later, in 2020, human pathogenic black fungi’s EVs were reported for Exophiala dermatitidis (Herpotrichiellaceae), a clinically important extremophilic black yeast (Lavrin et al., 2020). To our knowledge, this present work is the first to collect the data on EVs produced by species of the genus Fonsecaea.

Once fungal EVs are involved with cell-to-cell communication and are associated with fungal pathogenicity mechanisms, those structures are important elements for the host-pathogen interaction during CBM (Rodrigues and Nimrichter, 2022). In the present work, we characterized for the first time the EVs produced by the pathogenic species F. pedrosoi and F. nubica in comparison to the EVs obtained from the environmental species F. erecta. We characterized their size, morphology, and ergosterol/protein content and their ability to modulate pro- and anti-inflammatory cytokine and nitric oxide production by bone marrow-derived macrophages (BMDMs).

In the present work, we described and characterized for the first time EVs produced by F. pedrosoi, F. nubica, and F. erecta. F. pedrosoi and F. nubica species are etiologic agents of chromoblastomycosis (CBM), whereas F. erecta is an environmental fungus (Najafzadeh et al., 2010; Vicente et al., 2014; Queiroz-Telles et al., 2017; You et al., 2019). Fungal EVs have a role not only in fungal metabolism but also in fungal pathogenicity and survival inside the host (Rodrigues et al., 2008; Bielska et al., 2018; Ikeda et al., 2018; Zhao et al., 2019; Rizzo et al., 2020a). Although there is growing evidence about the virulence roles of these vesicles, especially for yeast-like fungi such as C. albicans, C. neoformans, Saccharomyces cerevisiae, and Histoplasma capsulatum, little is known for filamentous and melanized fungi (Albuquerque et al., 2008; Rodrigues et al., 2008; Oliveira et al., 2010a; Kabani and Melki, 2015; Baltazar et al., 2018; Zhao et al., 2019; Cleare et al., 2020).

The influence of factors such as growth conditions, cell morphology, and fungal strains used in experiments appears to be important in the modulation of EVs biogenesis, size, cargo, and function, although how these factors affect fungal EVs production remains poorly understood (Cleare et al., 2020; Marina et al., 2020; Rizzo et al., 2020a). Since morphological transition and MC establishment during CBM is crucial for Fonsecaea survival and pathogenicity, understanding how nutritional conditions and growth factors interfere with EVs characteristics and host-cell communication might grant new insights regarding the roles of extracellular components produced by Fonsecaea spp. (de Castro et al., 2017; Siqueira et al., 2017; Dong et al., 2018; Siqueira et al., 2020). For the comparative characterization of the EVs, three experimental conditions were utilized to isolate the vesicles: non-defined rich medium with mainly conidia and hyphae (mycelium) formation (RM); defined minimum medium with mostly hyphae and few conidia without MCs formation (MM MC-); and defined minimum medium with predominantly MCs and hyphae formation (MM MC+).

The first interesting observation is that the pathogenic species F. pedrosoi and F. nubica induced MC formation, although F. erecta did not display the classical described globular and clump morphology as observed for F. pedrosoi and F. nubica (Mcginnis, 1938; Alviano et al., 1992). The ability of an environmental species to produce MCs, a type of cell strongly related to CBM, might be due to the nature of infection by species of the genus Fonsecaea demonstrated to be accidental and not intentional (de Hoog et al., 2004; Fornari et al., 2018). Vicente et al. (2017) have previously investigated the pathogenicity of F. pedrosoi, F. monophora, and F. erecta. By using G. mellonella’s larvae as a model of infection, they observed that F. monophora (pathogenic) and F. erecta similarly led to a decrease in the life span of the larvae when compared to F. pedrosoi. It was also demonstrated that BALB/c mice infection by F. pedrosoi and F. erecta exhibited similar fungal load levels and kinetics with enhanced production of pro-inflammatory cytokines in the tissue such as TNF, IL-1β, IL-6, and MCP-1 (Vicente et al., 2017). In this sense, non-pathogenic species of Fonsecaea exhibit infecting animal tissues capacity.

Furthermore, it has also been demonstrated that F. erecta presented higher virulent capacity in Tenebrio molitor larvae model than the pathogenic species (Fornari et al., 2018). Initially, the infection by F. erecta did not trigger the host immune defense system which might be explained by the fact that the primary host of F. erecta infections is plants and not animals; hence, the extracellular components produced by this fungus are probably involved with environmental interactions. Thus, the poor capacity of the EVs produced by F. erecta to stimulate cytokine production by macrophages might be related to the fact that these extracellular structures are not originally adapted to the molecular machinery of animal hosts.

Lipids and proteins are fundamental molecular components found in a vast array of fungal EVs that can be used as indicators of the levels of vesicles production (Oliveira et al., 2010b; Baltazar et al., 2018; Zhao et al., 2019; Cleare et al., 2020; Lavrin et al., 2020; Marina et al., 2020). In the present work, it was observed that Fonsecaea EVs isolated from the highly nutritional medium led to enhanced levels of ergosterol and protein content compared to EVs obtained from both MM conditions. When the fungal species were compared, F. erecta displayed the highest amounts of proteins and sterols in nearly all conditions, indicating that this fungus might produce higher numbers of EVs compared to F. pedrosoi and F. nubica ( Figure 2A ). The aptness to generate highest amount of EVs might be because that F. erecta has initially been obtained from the environment and thus, has a predisposition to grow in conditions similar to experimental conditions. Nutritional availability of the culture medium has been shown to modulate the lipid and protein content of EVs produced by C. neoformans and H. capsulatum (Cleare et al., 2020; Marina et al., 2020). EVs produced by C. neoformans in MM (Sabourad Dextrose Broth 50%) presented elevated levels of ergosterol/protein ratio when compared to the vesicles isolated from RM (Sabourad Dextrose Broth 100%) (Marina et al., 2020). For H. capsulatum, three conditions were used to compare EVs features, in which was observed higher ergosterol and protein content in vesicles produced in HAM’s F12 media (Cleare et al., 2020). Also, a comparison between fungi from the same genus, Candida glabrata, C. parapsilosis, and C. tropicalis, showed heterogeneous levels of protein and phospholipid content (Karkowska-Kuleta et al., 2020), reiterating that not only nutritional availability but also fungal species and strain can modulate EVs biogenesis, accordingly to the results presented in this research.

Size analyses of EVs are indicators of their biogenesis’s pathways and cargo (Wolf et al., 2015; de Oliveira et al., 2020; Rizzo et al., 2020b; Rizzo et al., 2021). Overall, for Fonsecaea EVs, three main populations were identified: (a) small EVs ranging between 10-150 nm; (b) intermediate EVs ranging between 200-600 nm; (c) and large EVs ranging between 650-1500 nm. The EVs bigger than 800 nm probably are vesicles’ agglomerates, as observed through TEM ( Figure 2F ) that cannot be read as individual components by DLS. The DLS results for F. pedrosoi, F. nubica, and F. erecta EVs indicate that these structures have similar diameter variations, as encountered for A. fumigatus, A. flavus, C. albicans, and H. capsulatum (Albuquerque et al., 2008; Vargas et al., 2015; Souza et al., 2019; Brauer et al., 2020). However, Nano Tracking Analysis (NTA) assays must be done in the future to confirm with more precision the diameter of the EVs produced by Fonsecaea spp. TEM imaging allowed us to observe the EVs’ morphology and distribution ( Figure 2F ). Most of the EVs observed are similar to previous descriptions, especially for A. infectoria, C. albicans, C. glabrata, C. parapsilosis, E. dermatitis, and Pichia fermentans (Silva et al., 2014; Leone et al., 2017; Karkowska-Kuleta et al., 2020; Lavrin et al, 2020; Vargas et al., 2020). The EVs of F. nubica and F. erecta isolated from MM MC- are morphologically similar to the P. fermentans EVs, exhibiting extremely rounded shape structures, even though Fonsecaea EVs seem more contrasted, probably because of melanin released to the extracellular environment (Alviano et al., 1991; Farbiarz et al., 1992; Leone et al., 2017). An intriguing observation is the presence of electron-dense hexagonal-shaped components associated with the EVs isolated from F. pedrosoi when grown in MM (MC+) ( Figure 2F ). These structures appear to be equivalent in size and shape to electron-dense fragments shed from the cell wall of F. pedrosoi MCs, as reported by Franzen et al. (2006), a type of event that has previously been described (Alviano et al., 1991; Farbiarz et al., 1992). Since melanin, β-1,3-glucan, and α-1,3-glucan are major components of the Fonsecaea cell wall and can be recognized by the host immune cells, more detailed analyses should be done to assess the importance of these structures in the host-cell communication during CBM.

Many studies about fungal EVs have indicated that these structures can modulate cytokine production by macrophages and/or dendritic cells, such as for C. neoformans (Oliveira et al., 2010a; Marina et al., 2020), C. abicans (Vargas et al., 2015; Vargas et al., 2020), Paracoccidioides brasiliensis (Silva et al., 2016), Sporothrix brasiliensis (Ikeda et al., 2018), A. fumigatus (Souza et al., 2019) and A. flavus (Brauer et al., 2020). In the present work, it was demonstrated that only the EVs produced by the pathogenic species of the genus Fonsecaea obtained from RM could stimulate TNF and IL-10 production by BMDMs, whereas the EVs isolated from both MM conditions were not able to promote these patterns ( Figures 3 , 5 ). Moreover, EVs produced in RM and MM (MC-) by F. pedrosoi and F. nubica, and in MM (MC+) by the three species, were able to elicit significant levels of IL-1β when administered with only nigericin, which indicates that, in those conditions, the EVs might act as the first signal in inflammasome activation ( Figure 4 ). For EVs of the filamentous fungus A. fumigatus, it was evidenced that their vesicles carried not only proteins with virulent capacities, like allergens, chaperones, superoxide dismutase, nucleoside diphosphatase kinase, and more, but also could enhance fungal clearance and promote a pro-inflammatory response by macrophages through the production of TNF, IL-1β and CCL2 (Souza et al., 2019). Similar results were found for EVs produced by A. flavus which their vesicles activated TNF, NO, IL-6, and IL-1β, promoted enhanced phagocytosis and stimulated M1 polarization by macrophages in vitro (Brauer et al., 2020). The fungal EVs’ potential to induce M1 polarization has also been shown for P. brasiliensis’ EVs that stimulated TNF, IL-6, and IL-12p production by peritoneal macrophages and promoted M1 activation through the expression of iNOS, a classical marker for M1 polarization (Silva et al., 2016). Altogether, these data show that fungal EVs can act as immunomodulatory components in host-fungi interactions and that growth conditions can affect this ability.

Marina et al. (2020) revealed that C. neoformans EVs not only modulated cytokine production by bone marrow-derived macrophages and dendritic cells but that these modulator capacities were influenced by medium composition. Furthermore, C. neoformans EVs could induce IL-1β only when combined with nigericin, acting as the first signal for inflammasome activation, similar to the results observed in the work here presented. Further analyses of inflammasome signaling pathways, such as the cleavage of pro -IL-1β and production of other cytokines (e.g., IL-18) should aid to comprehend the mechanisms underlying Fonsecaea species’ EVs impact on inflammasome activation.

Besides cytokine production, macrophages have another important molecular mechanism against the infection by Fonsecaea spp. as observed in CBM, releasing oxygen and nitrogen reactive species, such as NO and H₂O₂ (Bocca et al., 2006; Cunha et al., 2010). Fungal EVs have been demonstrated to stimulate NO production by macrophages and/or dendritic cells, which indicates that these structures may enhance fungicidal activity by phagocytes (Oliveira, et al., 2010a; Vargas et al., 2015; Joffe et al., 2016; Silva et al., 2016; Brauer et al., 2020; Marina et al., 2020). The EVs produced by A. flavus, C. albicans, C. neoformans, and P. brasiliensis can promote NO secretion, measured by both iNOS expression and nitrite production (Oliveira, et al., 2010a; Vargas et al., 2015; Silva et al., 2016; Brauer et al., 2020; Marina et al., 2020). Moreover, for A. flavus and P. brasiliensis, the enhanced production of NO after the interaction of macrophages and EVs was related to M1 polarization and increased fungicidal activity (Silva et al., 2016; Brauer et al., 2020). Thus, further investigation about the possible roles for Fonsecaea EVs in M1 polarization should be addressed.

In this research, the EVs isolated from a medium composed mainly of conidia and hyphae (RM), in general, produced EVs with higher immunostimulatory capacities than those obtained from MM mainly composed of hyphae or MCs (MM MC- and MM MC+). Interestingly, it has been shown that hyphae and MCs, but not conidia, can induce IL-1β and TNF production by murine macrophages and a human monocytic cell line in vitro (de Castro et al., 2017). Hyphae and especially MCs are involved with the chronicity of CBM. In contrast, conidia could not reach the chronic phase of the disease, being rapidly eliminated from the injury in a mice model of infection (Siqueira et al., 2017). Moreover, only the cells mentioned above, but not conidia of F. pedrosoi, were able to induce a pro-inflammatory response by macrophages and dendritic cells marked by TNF, IL-1β, IL-6, and MCP-1 production, which corroborates the fact that only the interaction between macrophages and MCs were able to promote the up-regulation of genes related to inflammation, cell migration and fungal recognition (Siqueira et al., 2017). In this sense, the EVs produced by F. pedrosoi exhibit a relatively opposite modulatory capacity in stimulating TNF and NO production when the fungi interact directly with macrophages. EVs from culture containing abundant conidia, but not exclusively hyphae nor MCs, were more capable of inducing a pro-inflammatory response. The host-fungi interactions are extremely dynamic and dependent on interactions between the cells and molecules secreted, which can modulate the host immune response pattern in the inflammatory focus.

In summary, we demonstrated and characterized for the first time EVs produced by pathogenic (F. pedrosoi and F. nubica) and environmental (F. erecta) species of the genus Fonsecaea. These structures can modulate pro- and anti-inflammatory cytokine and nitric oxide production by macrophages in vitro. This ability is influenced by growth conditions, which suggests that environmental factors encountered by Fonsecaea spp. might act as potent regulators of the EVs’ biogenesis and function. Nevertheless, further analysis, especially genomic and proteomic investigations, should be performed to elucidate the possible roles of Fonsecaea EVs in host-cell communication far from inflammatory focus and their influence on the cell morphology transition during CBM.

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

The animal study was reviewed and approved by Animal Ethics Committee of the University of Brasília (UnBDoc n°. 46/2017).

AB formulated the study. LOL-C, AB, and LF wrote the manuscript. LOL-C, CM, and LC performed the experiments. SB, LF, and LOL-C carried out transmission electron microscopy and optical microscopy. LOL-C, CM, RC, LC, LF, and AB analyzed and interpreted the results. CM, RC, SB, VV, LF, and AB contributed with fungal strains, reagents, and analysis tools. All authors reviewed and approved the manuscript.

This work was supported by Fundação de Amparo à Pesquisa do Distrito Federal (193.000.805/2015) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (306515/2019-9). The Doctoral Felloshwip was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (88882.347193/2019-1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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