Elucidating the role of exosomes in Candida – macrophage interaction

Escola de Ciências Cidália Manuela Lopes Pereira Elucidating the role of exosomes in Candida - macrophage interaction setembro de 2022 Universidade do Minho Escola de Ciências Cidália Manuela Lopes Pereira Elucidating the role of exosomes in Candida – macrophage interaction Dissertação de Mestrado Mestrado em Genética Molecular Trabalho efetuado sob a orientação de Doutora Ana Paula Fernandes Monteiro Sampaio Carvalho e de Doutora Andreia Ferreira Castro Gomes setembro de 2022 i DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS Este é um trabalho académico que pode ser utilizado por terceiros desde que respeitadas as regras e boas práticas internacionalmente aceites, no que concerne aos direitos de autor e direitos conexos. Assim, o presente trabalho pode ser utilizado nos termos previstos na licença abaixo indicada. Caso o utilizador necessite de permissão para poder fazer um uso do trabalho em condições não previstas no licenciamento indicado, deverá contactar o autor, através do RepositóriUM da Universidade do Minho. Licença concedida aos utilizadores deste trabalho Atribuição-NãoComercial-SemDerivações CC BY-NC-ND https://creativecommons.org/licenses/by-nc-nd/4.0/ ii STATEMENT OF INTEGRITY I hereby declare having conducted this academic work with integrity. I confirm that I have not used plagiarism or any form of undue use of information or falsification of results along the process leading to its elaboration. I further declare that I have fully acknowledged the Code of Ethical Conduct of the University of Minho. iii AGRADECIMENTOS Great people are those who make others feel that they, too, can become great. - Mark Twain Em primeiro lugar gostaria de agradecer às minhas orientadoras, à Professora Andreia Gomes e à Professora Paula Sampaio, por me terem permitido realizar este projeto que tanto acresceu ao meu crescimento pessoal e profissional. Pela orientação e partilha de conhecimento ao longo deste caminho. Um enorme agradecimento ao Augusto, por me ter ensinado grande parte de tudo o que sei sobre investigação e trabalho laboratorial, como também por ser uma pessoa dentro e fora do contexto laboratorial cujo exemplo gostaria de seguir. Por toda a paciência que teve comigo ao longo deste percurso, pela motivação e pela disponibilidade que sempre me deu mesmo quando o seu tempo era escasso. À Inês e ao Félix, meus colegas de mestrado e acima de tudo amigos, e à Natacha, à Sónia, à Adília, à Carmo e à Cristiana, minhas amigas, por todo o apoio, preocupação e conselhos ao longo desta fase, mas principalmente pelas jantaradas, gargalhadas e cartadas que tão felizes me fazem. A todos os meus colegas e amigos de laboratório da Micro II e do LBA, duas famílias que tive a felicidade de fazer parte e que me acolheram com todo o carinho. Um agradecimento especial pelos conselhos, esclarecimento de dúvidas e ajuda que sempre se propuseram a dar. Aos amigos do LGM, companheiros de almoços e conversas que nos alegram sempre o dia. Obrigada pelos chocolates, pelos conselhos e pela amizade ao longo deste percurso. Ao Luís, o meu companheiro de todas as horas, por todo o carinho e apoio que sempre me dá. Por ser o meu alicerce e estar sempre do meu lado, mesmo nos momentos mais difíceis. Por me ouvir a falar da mesma coisa mil vezes, por me acalmar, por me fazer rir, por me dar um ombro onde chorar. Mas principalmente, por sempre acreditar em mim mesmo quando eu não acredito. Por último, agradeço à minha família pelo constante apoio e carinho durante esta fase que tantos sobressaltos revelou. Aos meus pais que me possibilitaram estar onde estou hoje, mesmo quando a situação não era a ideal, ajudaram-me a atingir os meus objetivos. Ao meu avô que sempre quis o melhor para mim. Não me conseguiu acompanhar nesta fase, mas esteve e estará para sempre comigo no pensamento, e sei que onde quer que esteja estará orgulhoso de mim. iv RESUMO Os fungos têm sido identificados como causadores de graves doenças humanas, sendo as espécies do género Candida e Aspergillus as principais causas de infeções fúngicas oportunistas em todo o mundo. Ao longo dos anos, houve um aumento na incidência destas infeções e no número de espécies resistentes aos medicamentos, o que contribuiu para maiores percentagens de morbilidade e mortalidade, principalmente entre indivíduos imunocomprometidos. Uma vez que as infeções por Candida têm um efeito significativo na saúde pública, a necessidade de melhor compreender a sua patogénese de forma a guiar o desenvolvimento de medicamentos antifúngicos otimizados é premente. No decorrer da última década, tem sido dado mais destaque aos tratamentos cujo alvo é específico à interação hospedeiro-patógeno, o que em teoria pode possibilitar um impacto mínimo dos medicamentos na sobrevivência dos organismos comensais, tornandoos menos suscetíveis a conduzir uma resistência universal no microbioma. Assim sendo, o melhor conhecimento sobre as interações hospedeiro-espécies de Candida pode levar a novos alvos para os medicamentos antifúngicos e consequentemente auxiliar no controlo destas infeções. Recentemente, as vesículas extracelulares ganharam muito interesse na investigação como um mecanismo omnipresente na transferência de informação entre células e organismos em todos os três domínios da vida. Para além das funções na fisiologia normal, estas partículas têm sido expostas como mediadores de um mecanismo anteriormente não reconhecido para empacotar e proteger carga para absorção e integração noutras células, nomeadamente produtos secretados por patógenos, emergindo assim como um tipo de interação hospedeiro-patógeno funcionalmente diverso. Considerando as potenciais funções relatadas para estas vesículas nas interações hospedeiroCandida , há motivação significativa para aumentar a nossa compreensão sobre a biologia e funções das mesmas, o que pode resultar num melhor entendimento da patogénese de Candida e ajudar a elucidar possíveis alvos terapêuticos para o desenvolvimento de vacinas ou imunoterapias contra este tipo de infeções. O objetivo desta dissertação foi estudar a biologia dos exossomas secretados por C. albicans SC5314 e C. albicans 124A e as suas funções num ambiente de infeção, através do seu isolamento para posterior caraterização, avaliação de citotoxicidade, análise de fagocitose e produção de citocinas quando em cultura com uma linha celular de macrófagos de murinos. Adicionalmente, este trabalho teve como objetivo estabelecer um modelo de embrião de peixe-zebra para estudar as interações hospedeiroCandida , permitindo estudos adicionais sobre o papel de exossomas na patogénese de Candida na escala de um organismo inteiro. v ABSTRACT Since the 1980s, fungi have been revealed as major causes of human disease, where Candida species represent the most common cause of opportunistic fungal infections worldwide. Through the years, there has been an increase in the occurrence of these infections, and in the number of drug-resistant species which contributed to greater percentages of morbidity and mortality, particularly amongst the immunocompromised. Accordingly, Candida infections have become a significant influence in public health which leads to a necessity of better understanding their pathogenesis to improve therapeutic options and facilitate the development of enhanced antifungal drugs. Over the past decade, more prominence has been placed on treatments aiming to target specifically the host-pathogen interaction which, in theory, could enable a minimal impact from the drugs on the survival of commensal organisms, being less susceptible to eliciting resistance in the microbiome. Therefore, improved knowledge about hostCandida species interactions may identify new targets for the antifungal drugs and consequently assist in controlling these infections. More recently, extracellular vesicles (EVs) have gained much interest in research as a ubiquitous mechanism for transferring information between cells and organisms across all three domains of life. In addition to their influence on normal physiology, these particles have also been pinpointed as mediators of a previously unrecognized mechanism to package and protect cargo for uptake and integration into other cells, namely pathogen secretion products, emerging as a functionally diverse type of host-pathogen interaction. Considering the potential roles reported for EVs in hostCandida interactions, improving existing comprehension on the biology and functions of Candida EVs could open new views for understanding Candida pathogenesis and help elucidate possible therapeutic targets and develop potential vaccines or immunotherapy against this infection. The aim of this thesis was to study the biology of C. albicans SC5314 and C. albicans 124A exosomes, and their roles in an infection environment, by isolating them for further characterization, cytotoxicity evaluation, phagocytosis assessment and cytokine production analysis when in culture with a murine macrophage-like cell line. Furthermore, this work aimed to establish an effective zebrafish embryo model to study hostCandida interactions, to allow further studies on the role of exosomes in Candida pathogenesis in the scale of a whole organism. vi TABLE OF CONTENTS DIREITOS DE AUTOR E CONDIÇÕES DE UTILIZAÇÃO DO TRABALHO POR TERCEIROS ............ i STATEMENT OF INTEGRITY ................................................................................................ ii AGRADECIMENTOS ........................................................................................................... iii RESUMO ........................................................................................................................... iv ABSTRACT ......................................................................................................................... v LIST OF FIGURES ............................................................................................................. xii LIST OF TABLES .............................................................................................................. xiv Chapter 1. Introduction ..................................................................................................... 1 1. Candida and candidiasis ................................................................................................ 2 1.1 Clinical features of candidiasis .................................................................................... 2 1.2 Epidemiology of candidiasis ......................................................................................... 4 1.3 Virulence factors of Candida species............................................................................ 7 1.3.1 Adhesion .............................................................................................................. 8 1.3.2 Hyphal morphogenesis .......................................................................................... 9 1.3.3 Hydrolytic enzymes ............................................................................................. 10 1.3.4 Phenotypic switching .......................................................................................... 11 1.3.5 Biofilm formation ................................................................................................ 12 1.4 Challenges in the treatment of candidiasis ................................................................. 13 1.5 Extracellular vesicles and infectious diseases ............................................................. 14 1.5.1 Fungal extracellular vesicles ............................................................................... 16 1.6 Models to assay Candida virulence ............................................................................ 18 1.7 Zebrafish .................................................................................................................. 19 1.7.1 Species characteristics ....................................................................................... 19 1.7.1.1 Life cycle...................................................................................................... 21 1.7.1.2 Reproduction ................................................................................................ 22 xiii Figure 19. Western blot analysis of C. albicans -free supernatants concentrated samples alongside with their residues using anti-Hsp70 exosome antibody. ......................................................................................... 50 Figure 20. Viability of J774A.1 cells after 24 h incubation with C. albicans SC5314 and C. albicans 124A - free supernatants. .................................................................................................................................. 52 Figure 21. Quantification of macrophage phagocytosis of yeast cells evaluated by fluorescence microscopy, where macrophages from test conditions were previously incubated with C. albicans SC5314 and C. albicans 124A -free supernatants for 24 h. ............................................................................................................ 53 Figure 22. Illustration of macrophage phagocytosis of yeast cells by fluorescence microscopy in the control and the test condition where macrophages were previously incubated with C. albicans SC5314 -free supernatant for 24 h. .............................................................................................................................. 53 Figure 23. Production of TNF-α by J774A.1 cells after 24 h incubation with C. albicans SC5314 and C. albicans 124A -free supernatants, and after 45 min incubation with C. albicans SC5314 yeast cells. ........ 54 Figure 24. The survival rates of zebrafish embryo infected with different doses of C. albicans SC5314 along 80 hpf in three independent replicates .................................................................................................... 56 xiv LIST OF TABLES Table 1. Agents of opportunistic mycosis. ................................................................................................ 5 Table 2. Species distribution of Candida bloodstream isolates.. ............................................................... 6 Table 3. Mortality rates of Candida bloodstream infections. ..................................................................... 7 Table 4. Strains and media used in this study. ...................................................................................... 30 Table 5. Concentrations achieved in the two different concentration systems. ........................................ 42 Table 6. Concentration achieved in the Amicon® Ultra-15 Centrifugal Filter Unit. ................................... 45 Table 7. Count rates and attenuators of samples obtained from the two concentration systems. ............ 75 Table 8. Count rates and attenuators of samples obtained from the Amicon® Ultra-15 Centrifugal Filter Unit which growth was performed in YEPD medium. ....................................................................................... 75 Table 9. Count rates and attenuators of samples obtained from the Amicon® Ultra-15 Centrifugal Filter Unit which growth was performed in minimal medium. ................................................................................... 75 1 Chapter 1. Introduction 2 1. Candida and candidiasis Since the 1980s, fungi have been revealed as major causes of human disease, particularly in immunocompromised individuals and with critical diseases. These infections are associated with considerable percentages of morbidity and mortality, making it clear that fungal diseases have a significant influence on public health [1]. The main concerns about this situation are related to the growing number of fungal infections, as well as the diversity and complexity associated to the etiological agents involved [2]. This increased incidence is associated with several factors, such as a greater use of antibiotic drugs, frequent invasive medical procedures, and an increased number of immunocompromised individuals due to mucosal or cutaneous barrier disruption, neutropenia, qualitative neutrophil dysfunction, defective cellular immunity, metabolic dysfunction, and extremes of age [3], [4]. These immunodeficiencies are commonly attributed to cytotoxic and mucotoxic chemotherapy, radiation therapy, transplantations, and steroids administration, that further increase the risk for different fungal pathogens infections [3]. The most common fungi causing life-threatening infections are Candida , Cryptococcus and Aspergillus species. In addition to these, there are many other agents that can cause human disease, such as Saccharomyces , Coccidioides , and Histoplasma species [5]. Despite the vast list of pathogenic agents, Candida spp. remain the most regular cause of human fungal infections [1]. This genus consists of approximately 200 species, of which more than 30 have been identified as causes of human infection such as C. albicans , C. parapsilosis , C. auris , C. dubliniensis , C. glabrata , C. tropicalis , C. krusei , and the list continues to extend [6]. These are opportunistic fungal pathogens that commonly live as commensals in several locations of the human body, such as the oral cavity, the gastrointestinal tract, and the genitourinary system. In the commensal state, these species live as benign members of the microflora of healthy individuals, causing no disease. However, in response to disorders of the normal flora balance or changes in host physiology that compromise the immune defenses that normally suppress the growth of these commensals, they become able to invade tissues and cause opportunistic infections, denominated candidiasis [7]. 1.1 Clinical features of candidiasis Most infections due to Candida species are endogenous, meaning that are caused by prior colonization of locations of the human body, such as the mouth, skin, gastrointestinal tract, and vagina. The infection is asymptomatic until some disorder in the normal flora balance or change in host physiology occurs and 3 compromises the immune system, which allows the infection to become symptomatic [7]. Additionally, candidiasis can also be acquired by exogenous sources, mainly in healthcare environments. Several Candida species have been isolated from different sources in healthcare facilities, such as the floor, countertops, portable equipment, doorknobs, and many other surfaces [8], [9]. Candida species can cause a wide spectrum of infections, from superficial lesions in mucosa and skin that are frequently community acquired and responsible for substantial morbidity to deep-seated, invasive, and systemic infections that are typically nosocomial in origin [10]. Superficial candidiasis is the most common form of candidiasis. It is frequent in individuals with prior colonization, emerging when the immune defenses become compromised and primarily affecting the mucosa of the human oral cavity and the vagina [11]. Some of the most common clinical manifestations of this type of candidiasis include oral candidiasis, vulvovaginal candidiasis and candiduria [12], [13], [14]. Candida species are commonly present in the mouth of healthy individuals as commensals, with carriage rates ranging from 20% to 75% [15]. However, changes in the oral cavity environment can induce the overgrowth of Candida , which can lead to local discomfort, alteration of the taste sensation, and dysphagia. Oral candidiasis is the most common human fungal infection especially in early and later life. Other predisposing factors include diabetes mellitus, immunocompromised individuals such as patients diagnosed with HIV/AIDS or leukemia, those using broad spectrum antibiotics, and individuals in critical conditions [12]. Vulvovaginal candidiasis is the second most common vaginal infection, which affects 75% of the female population at least once in their life span [14]. In addition, approximately 50% of these women will experience a single recurrence, and 5% – 8% will experience recurrent vulvovaginal candidiasis (RVVC), which is defined as four episodes, at least, of infection per year and requires extensive treatments [16]. Possible risk factors for RVVC include treatment-resistant Candida species, use of antibiotics, immunodeficiencies, use of contraceptives, sexual activity, and inadequate treatment for the vulvovaginal candidiasis [17]. Candiduria, the presence of Candida species in urine, is a common clinical finding, particularly in hospitalized patients. Multiple studies indicate that more than 10% -- 15% of hospital acquired urinary tract infections (UTIs) are caused by Candida species, being that all common Candida species can cause this type of infection [13]. Invasive candidiasis only manifests itself in severe cases of debilitation or when the immune system is critically compromised. In this type of infection, Candida species may enter the bloodstream, which is defined as candidaemia, and penetrate multiple organs of the human body like the lung, liver, kidneys, and bone. The mortality rate among individuals with invasive candidiasis is around 40%, even when antifungal therapy is prescribed. Particularly, candidaemia is associated with the highest crude mortality of all bloodstream infections, being that in several studies, the crude mortality of candidaemia exceeds 50% even in non-neutropaenic individuals [18], [19]. 4 Natural: Penetration of mucosal surfaces Iatrogenic: Damage of barriers Iatrogenic: Medical devices colonized with biofilms Figure 1. Main routes of entry into the bloodstream by Candida . Adapted from [19]. Candida cells can enter the bloodstream through three main routes. The entry can occur by the natural way, where yeast cells penetrate epithelial cells from mucosal surfaces into deeper tissues and blood capillaries or vessels, or by the iatrogenic way, through medical devices (catheters, artificial joints, and prosthetic heart valves) in which biofilms play an important role, or through the damage of defense barriers due to surgeries, polytraumas or drug treatment (Figure 1) [19]. Although Candida species can infect several locations in the human body, it is known that the immune protection against them is site-specific. In animal models of candidiasis, the protection against systemic infections does not show results against superficial infections. T-cell immune responses are key in protection against superficial candidiasis but resistance to systemic candidiasis is regularly associated with a functional phagocytic response involving neutrophils or mononuclear phagocytes [20]. 1.2 Epidemiology of candidiasis Fungal infections are major causes of human disease, particularly among critically ill or immunocompromised individuals, with substantial rates of morbidity and mortality associated. Additionally, Candida species are the most common cause of opportunistic fungal infections worldwide (Table 1) [1]. 5 Table 1. Agents of opportunistic mycosis. Adapted from [1]. Over the last decades, several epidemiologic studies have been conducted to assess the incidence of superficial and invasive candidiasis. Regarding UTIs, studies indicate that at least 10% - 15% of hospitalacquired UTIs are caused by Candida species. Candiduria occurs most commonly in adult patients present in intensive care units (ICUs) who often have multiple predisposing factors such as diabetes mellitus, indwelling urinary catheters, and exposure to antimicrobials, as well as in neonates and children present in ICUs, particularly in premature infants [13]. Moreover, Candida species are considered the secondary cause for onychomycosis, being responsible for 2% - 11% of the cases [21], can cause at least one episode of vulvovaginal candidiasis in the life span of 75% of female population [16], and are also responsible for 13.2% of all intra-abdominal infections, which are considered the most common type of deep-seated candidiasis [22], [23]. The incidence of invasive Candida infections has been studied by several multi-institutional surveys, such as the European Confederation of Medical Mycology (ECMM) survey [24], and the National Epidemiology of Mycosis Survey [25]. These studies demonstrated that candidaemia frequency among hospitalized patients has doubled over the past two decades, being now considered the fourth most common nosocomial bloodstream infection in the United States and the fourth most common catheter-related bloodstream infection in Europe [19]. Data from ECMM indicate that C. albicans is the species most frequently isolated from the blood of patients diagnosed with invasive fungal infection [24]. Accordingly, it is considered the most prevalent and the most pathogenic of all Candida species, being responsible for most systemic and oral candidiasis manifestations along with community-onset and nosocomial candidaemia [26]. However, over the years there has been a significant increase in the incidence of infections caused by other Candida species, Organism (s) No. of cases/million/yr Case/fatality ratio (%) Yeasts Candida species 72.8 33.9 Cryptococcus species 65.5 12.7 Other yeasts Hyaline molds Aspergillus species 12.4 23.3 Zygomycetes 1.7 30.0 Other hyaline molds 1.2 14.3 Dematiaceous molds 1.0 0 Pneumocystis jiroveci 6 which may be explained by the introduction of fluconazole in 1990 [27]. This agent had good activity against C. albicans with reduced toxicity when compared to the other anti-fungal that existed, so it became the agent of choice for many fungal infections as well as for prophylactic purposes. Although there is no conclusive evidence, many specialists in the field consider that it was the selective pressure applied by this therapeutic concept that caused changes in the epidemiology [28]. The most common non-albicans Candida species are C. parapsilosis , C. glabrata , C. tropicalis , and C. krusei , although the incidence rates can slightly vary according to the population in analysis (Table 2) [19]. Table 2. Species distribution of Candida bloodstream isolates. Adapted from [19]. Candida species Candidaemia, Europe C. albicans 49 – 53% C. parapsilosis 11 – 21% C. glabrata 10 – 12% C. tropicalis 6 – 11% C. krusei 1 – 9% Others 1 – 10% C. parapsilosis incidence has dramatically increased over time, being frequently reported as the second Candida species most frequently isolated from blood cultures [29]. It is generally a commensal of human skin whose pathogenicity is limited by an intact integument. This species is well known for its ability to grow in total parenteral nutrition, the formation of biofilms on catheters and other implanted devices, for nosocomial spread by hand transferences, and for its persistence in the hospital environment [30]. This fungal pathogen is particularly important since its incidence is much higher among young children, specifically those aged <2 years, when compared with adults [31]. C. glabrata is often described as the second or third most common cause of candidiasis, being commonly isolated from immunocompromised patients such as intensive-care, neutropenic, postsurgical, and bone marrow transplant patients [32]. This species is of special importance due to its innately reduced susceptibility to antifungal agents, specifically the azoles, which results in harder to treat infections [33]. C. tropicalis is considered the third most frequently isolated non-albicans Candida species from blood and urine cultures, being commonly associated with patients with malignancy and neutropenia [34]. Lastly, C. krusei is described as the fifth most commonly isolated Candida species from bloodstream isolates. Nonetheless, this species is usually intrinsically resistant to fluconazole and can cause serious infections in susceptible patients, which results in a significant challenge to clinicians [35]. 7 Range reflects data from all participating countries. Includes 142 other less common Candida species not reported in detail. a b The mortality rate from candidaemia ranges approximately from 30% to 60% in European surveys, depending on the species and geographic location in analysis (Table 3) [36]. Similar studies have been conducted in the United States, which revealed slight differences in the incidence of candidiasis when compared to Europe. This variability is likely due to differences in patient demographics and comorbidities, as well as distinct medical practices [1]. In addition to high mortality and morbidity rates, candidaemia also extends hospitalization times of those surviving these infections, which consequently increases medical care costs. Systemic Candida infections have been associated with an attributable intensive care unit cost of US $21,590 [24]. Table 3. Mortality rates of Candida bloodstream infections. Adapted from [36]. 1.3 Virulence factors of Candida species To develop an infection, opportunistic pathogens must be able to colonize a host, permeate its surface, evade the immune system, endure and divide in the specific host environment, and disseminate to other tissues [37]. As common commensals of the normal flora, Candida species are adapted to and specialized for survival on host surfaces. Throughout the transition to superficial and invasive infections, these pathogens need to adapt to drastically changing environments with extreme changes in several parameters, such as oxygen and carbohydrate levels, pH, osmolarity, availability of nutrients and temperature. The capacity of causing infections at such a vast range of body sites demonstrates the need of specific factors that are adapted and essential to each stage of infection to ensure survival and proliferation. The mechanisms required Candida species ECMM survey No. of episodes Mortality rate, % (range) C. albicans 1090 38.5 (29 -- 45) C. glabrata 269 45.0 (25 – 64) C. parapsilosis 263 25.9 (0 – 34) C. tropicalis 140 41.4 (20 – 44) C. krusei 38 55.3 (20 – 67) Total 1942 37.9 (27 – 40) 8 to surpass all these adaptations are designated as virulence factors [19]. The virulence factors required by Candida species to cause infections may differ depending on the type of infection, the site and phase of infection, and the nature of the host response. Many factors have been proposed to be virulence attributes for Candida , yet the most studied are hyphal formation, adhesion capacity, phenotypic switching, hydrolytic enzyme production, and biofilm production [38]. 1.3.1 Adhesion An effective Candida infection begins with the adherence of the pathogen to specific host cells and/or to the surface of medical devices. Therefore, adhesion is an essential step in the infection process, and its extension is dependent on microbial, host and abiotic surface properties, such as cell-surface hydrophobicity and cell-wall composition [39]. The presence of specific cell-wall proteins, commonly referred to as adhesins, is considered a significant factor in the adhesion ability of Candida species. Adhesins are defined as biomolecules that promote the adherence of Candida species to host cells or host-cell ligands, and various have already been identified and characterized [20], [39]. Mutants deficient in genes encoding these adhesins not only manifested attenuated adherence to host substrates in vitro but also a corresponding reduction in virulence in multiple experimental models of Candida infection. The scope of these reductions has been rather variable, which is thought to be caused by other adhesins actions with redundant or overlapping functions [40]. The majority of the Candida adhesins are glycoproteins, being the most studied from the agglutinin-like sequence (Als) family. The Als protein family is encoded by eight genes, ALS1 – 7 and ALS9, and expresses three common domains amongst the belonging proteins [37]. The central domain contains variable numbers of a tandem repeat sequence which is highly glycosylated, and the N-terminal domain consists of highly variable sequences which is a feature thought to be responsible for differences in Als proteins’ functions and interactions with the host [19]. For example, Als1p has an adhesin function to human buccal epithelial cells important during the early stage of infection, while Als5p provides adherence to fibronectin, collagen, bovine serum albumin, and laminin [37], [40]. Furthermore, the ALS genes are differentially expressed among Candida species and depend on the growth conditions, for instance ALS3 is hypha specific while ALS4 expression is associated with growth phase [19]. Multiple strains of C. albicans express all eight ALS genes, nonetheless only five ALS genes were detected in C. parapsilosis and three in C. tropicalis and C. dubliniensis [39], [41]. Additional adhesins have been identified, including the epithelial adhesin family in C. glabrata , the glycophosphatidylinositol-anchored protein 30 in C. parapsilosis , and the Ala1 and Hwp1 in C. albicans [19], [39]. 15 factors such as hormones, cytokines, and inflammatory mediators that are able to communicate with several cells either locally or at dispersed sites [67]. Research on this topic has been largely focused on soluble proteins secreted by pathogens, which have been proposed to down-modulate the host immune response, and to be involved in metabolic adaptation to the host environment and tissue invasion [68], [69]. More recently, extracellular vesicles (EVs) have gained much interest in cell biology and biomedicine as a ubiquitous mechanism for transferring information between cells and organisms across all three domains of life, including fifteen different fungal genera [66]. The term EVs is used to describe cell-derived double-layer phospholipid membrane particles that reach the extracellular environment, and these are highly heterogeneous, differing in several aspects such as biogenesis pathways, size, cargo, membrane composition, and biological functions. The cargo of EVs consists of a diverse suite of molecules including proteins, lipids and nucleic acids, some of which are reported to have immunomodulatory properties. EVs control various biological roles through the delivery of effectors to target cells, from the regulation of physiological events to the response to specific environmental conditions [70]. In addition to their influence on normal physiology, these particles have also been pinpointed as mediators of a previously unrecognized mechanism to package and protect cargo for uptake and integration into other cells, namely pathogen secretion products, thus emerging as a functionally diverse type of host-pathogen interaction. Pathogenderived EVs are known to mediate highly complex interkingdom communications by transporting molecules from pathogens to hosts, spreading antigens, as well as infectious agents, and transferring genetic material to host cells or other pathogens [66]. Accordingly, these have been reported to have various functions in immune cell activation and suppression, and to play a role in tissue homeostasis and disease development, such as cancer and various infectious diseases [71], [72], [73], [74]. Based on their biosynthetic pathways, EVs are typically classified into two broad classes: microvesicles (also entitled ectosomes or microparticles) and exosomes [70]. Microvesicles are generally larger vesicles, ranging in size from 50 nm to 1000 nm in diameter but can have up to 10 µm in the case of oncosomes. These particles are generated by the direct outward budding and fission of the plasma membrane and the subsequent release of vesicles into the extracellular space. Exosomes are smaller vesicles derived from the endocytic pathway, ranging in size from 30 to 150 nm in diameter. Their biogenesis initiates by inward budding when endosomes maturate to form multivesicular bodies [75]. These structures can fuse with the hydrolytic lysosome, where the vesicle cargo is subsequently degraded or can fuse directly with the plasma membrane, releasing their intraluminal vesicles, now known as exosomes, into the extracellular space. Consequently, exosomes express markers of their parent cells, but are also enriched in other molecules associated with their biogenesis or that are selectively packaged into them, while microvesicles incorporate certain lipids, surface proteins, and other molecules before fission [66]. Nowadays, it is widely accepted that 16 cells from almost every type of organism are capable of secreting these nanoto micrometer-scale vesicles [76], however EVs are dynamic and strongly dependent on the source cell, cell topography, state, and environmental conditions. These factors determine their functionality and target specificity, meaning that the same cell type may secrete distinct types of vesicles depending on the specific factors it is exposed to [77]. 1.5.1 Fungal extracellular vesicles Fungal diseases result from a combination of fungal virulence factors and the defective host’s capacity to control the fungal growth successfully, in which EVs are known to play a role in both of these factors [78]. The first fungal EVs to be isolated and described were those of Cryptococcus neoformans in 2007, and ever since they have been indicated as a crucial mechanism for molecular export in a variety of fungal species [79]. Nowadays, several fungal species’ EVs have been characterized including those of C. albicans , C. glabrata , Saccharomyces cerevisiae , and Aspergillus fumigatus . In C. albicans , the existence of vesicle-like compartments was demonstrated for the first time in cell wall pimples from opaque cultures of C. albicans cells in 1990 [80]. C. albicans EVs were first isolated and observed by transmission electron microscopy in 2008, an analysis that demonstrated the presence of bilayered compartments similar to those initially described for C. neoformans and H. capsulatum [81]. These were later further analyzed to unravel their characterization, composition and implications for human immune responses in wild-type and mutant strains [82]. C. albicans EVs were defined as being EVs typical of fungi, containing proteins, lipids, nucleic acids and carbohydrates. Microscopical observations reveal spherical structures with heterogeneous sizes, with the majority of the vesicles having sizes between 50 and 100 nm, and a smaller percentage having larger sizes reaching more than 200 nm, being that the distribution of EV particle size differs slightly for different strains [82] [83]. Several studies on C. albicans EVs attributed different roles to these in an infection situation. EVs from C. albicans were capable of activating murine macrophages and dendritic cells which indicated a potential role in modulating the innate immune response to the fungus [84], [85]. Contrarily, it was reported that the cargo of EVs produced by C. albicans biofilms includes key proteins and carbohydrates for biofilm biogenesis, making these vesicles crucial contributors to the establishment of biofilms, which are a critical component of C. albicans pathogenesis. Moreover, adding back EVs from wildtype biofilms to biofilms formed by strains with deletions in genes encoding important enzymes for the generation of biofilm matrix restored the loss of azole resistance in these biofilms. This goes accordingly with the fact that the biofilm matrix provides a protective layer against antifungal drugs and reveal a role in antifungal drug resistance for these vesicles [86]. 17 EVs produced by fungi are multi-antigenic compartments that carry a number of biologically active molecules, including virulence factors, regulators and highly immunogenic components which indicates that these could activate the innate immune system and directly influence disease development [87]. Some of these elements are conserved among the different species, but others are species-specific [88]. In fact, recent studies propose that fungal EVs may represent a new alternative for the development of multivalent vaccine formulations. Various in vitro experiments demonstrate that fungal EVs are capable of stimulating phagocyte activity, promoting an increase in cytokine levels, modulating phagocytosis and regulating macrophage polarization. Altogether, this data indicates that fungal EVs activate the immune response during disease development, inducing pathogen killing [85], [89], [90]. Furthermore, there are several reports revealing a protective role from fungal EVs when these are exposed to the host models. For example, pretreatment of Galleria mellonella larvae with fungal EVs stimulates a protective response against a lethal challenge with C. albicans , C. neoformans and Aspergillus flavus , and more recently it was demonstrated that immunization of mice with EVs from C. albicans confers full protection against systemic candidiasis [87], [85], [91], [92]. However, in some models the outcome of the interaction between the host cells and the fungal EVs have also been associated with virulence and disease development, as demonstrated for C. neoformans and S. brasiliensis in murine models, respectively [93], [94]. Recently, a study revealed significant differences in the composition and roles of EVs released from two distinct strains of C. auris and C. albicans ATCC 90028 . Results revealed that EVs from only one of the C. auris isolates potentiated the adhesion of the yeasts to epithelial cells and promoted the survival of the fungal cells modulating the host cell defense mechanisms when in culture with macrophages cells, which suggests that EVs from the same species can promote distinct changes in host cells. EVs from C. albicans had no effect on the adhesion factor but it had the capacity to prime the macrophages for enhanced intracellular yeast killing, following the pattern exhibited by most fungal EVs [78]. In addition, fungal EVs have been suggested to be involved in the development of antifungal resistance and a messenger compartment for transferring virulent factors [95], [86]. The multiple activities attributed to these vesicles indicate that the outcome of the interaction between fungal EVs and the host is indeed dependent on the model, species and strain that it is being investigated, which could be, more specifically, dependent on EVs characterization and composition [78]. Given the potential roles for EVs in host-pathogen interactions, specifically between hostCandida interactions, there is significant motivation to better understand the biology and functions of Candida EVs, which could open new views for understanding Candida pathogenesis. 18 1.6 Models to assay Candida virulence A great amount of research on virulence can be performed with laboratory experimentation using tissue cultures. This method allows direct observation of the interaction between human cells and the pathogenic organisms and has already contributed with considerable knowledge in this field. It is intrinsically challenging to use this system to investigate the interaction of multiple cells and tissue types. Moreover, aside from the experiments performed with freshly harvested primary cells, tissue culture remains rather artificial by using standardized cell line preparations, which results in several cells that may or may not accurately reflect the biology of naturally developing cells in vivo [65]. Therefore, since these do not take into account the responses on the scale of an organism as a whole, cell culture systems are suitable for an initial data screening with the objective to assess fundamental data and use it to define future tests on other systems [96], [97]. With these limitations, it was concluded that infection modelling requires a whole organism where the complex hostfungus interactions can be investigated, and the disease development can be monitored right from the moment that fungal cells are introduced into the host. In order to be a good model, the specimen should be reproducible, relatively easy to set up, cost effective, and it should reproduce the major clinical symptoms that are known to occur in the human disease. With these conditions fulfilled, in-depth investigation of Candida pathogenesis can be performed and, consequently, inferences about Candida ’s virulence in humans can be made [98]. Thus, to obtain a more comprehensive understanding of Candida species pathogenesis, it is of greatest interest to establish an effective animal model system that allows the study of Candida -host cells interaction. Several large animals have been used to study Candida infections, such as macaques [99], piglets [100], rabbits [101] and guinea pigs [102], but nowadays the majority of Candida virulence research is performed with rodent models, due to advantages like ease of handling, economic factors, organism complexity, and the availability of genetically modified mouse strains that allows mimicking of human genetic conditions which offers proper predictivity [98]. In order to study Candida oral and vaginal infections, mucosal infection models have been performed mostly in rats and mice. Using these models, a lot of information about Candida virulence has been obtained through several mutant strains and clinical isolates, such as the fact that C. albicans pathogenesis requires yeast to hypha switching in oral infection [103]. For invasive infection models the mouse has been the most popular host of choice. There are two crucial models of Candida invasive infection: the intravenous infection model and the gastrointestinal colonization and dissemination model [104], being through the last one that it was demonstrated that C. parapsilosis had lower virulence compared to C. albicans but could still successfully establish persistent colonization of the gastrointestinal tract [105]. 19 Nonetheless, several disadvantages limit the use of this model in large-scale studies, such as limited number of offspring produced, high experimental costs when comparing with other models, being timeconsuming and the ethical constraints attached to it [97]. Various non-vertebrate model organisms have been developed to overcome these limitations, for instance, the fruit fly ( Drosophila melanogaster ), the wax moth ( Galleria mellonella ) and a nematode ( Caenorhabditis elegans ). These hosts have several advantages when compared with rodent models, like the simplicity, ethical expedient, inexpensive care systems, small size which enables high throughput studies designed to scan pathogen genomes for virulence-related genes or chemical libraries for antimicrobial compounds, well-developed molecular tools, and a conserved innate immunity [106]. Several studies have utilized these invertebrate models to improve the understanding of the virulence mechanism of Candida infection. For example, C. elegans model was used to compare the in vivo virulence of seven different Candida species with the in vitro production of proteinases and phospholipases, biofilm, and hemolytic activity [107]. In a wild-type Drosophila melanogaster model, a model with simple innate immunity, it was demonstrated that Pho84 gene is required for virulence of C. albicans , and in the Galleria mellonella model, it was demonstrated that a combination of molecular mechanisms, such as the presence of point mutation in the ERG11 gene, the overexpression of ERG11, and genes encoding efflux pumps, are involved in fluconazole resistance in C. parapsilosis [108] [109]. Despite the advantages of these invertebrate models, they still lack many structures, organ systems, and adaptive immunity that are involved in human disease pathogenesis, which limits their role in modelling human disease [110]. In this context, zebrafish has been used as a model organism alternative in numerous studies in the last years. 1.7 Zebrafish Zebrafish represent a very interesting alternative model of increasing popularity, for studying C. albicans - related pathogenesis and other types of infections [111], [112], [113]. 1.7.1 Species characteristics Zebrafish, Danio rerio (Hamilton, 1822), is a small teleost representative of the Cyprinidae family, native to freshwater habitats in South Asia [114]. This vertebrate, illustrated in Figure 3, reaches 3 – 4 cm of body length as an adult and it is broadly distributed through India, Bhutan, Nepal, Bangladesh, and Pakistan, being 20 A B C B A Figure 3. Zebrafish adults. (A) female and (B) male. Adapted from [147]. Figure 4. Zebrafish embryonic development. (A) Embryo at 8 h post-fertilization; (B) Embryo at 32 h post-fertilization; (C) Hatched larvae at 56 h post-fertilization. typically found in stagnant clear water or with a slow flow of shallow depth, approximately 30 cm, such as in streams, canals, ponds, and rice paddies [115], [116]. Zebrafish is omnivorous, and it has a varied diet that is mainly composed by zooplankton and insects but can also include phytoplankton, algae, fish scales, invertebrate’s eggs and more [117], [118]. It can also tolerate and adapt to a wide range of environmental conditions, such as temperature and pH [114], [119]. In the embryonic and larval phases, zebrafish are optically transparent, which allows for an easier analysis of the development during these phases (Figure 4). Reaching the juvenile and adult phases, the formation of blue colored bands along the trunk occurs, thus losing the transparency. Males usually appear to be more gold in color on the ventral side of their body and present a flatter abdomen than females which exhibit a more silver coloration along the trunk and a protruding belly (Figure 3) [120], [121]. Females have the capability to lay around 200 – 300 eggs every 2 to 3 days, which rapidly develop into larvae, ex-utero , within 120 h post-fertilization (hpf) [96]. 21 Figure 5. Life cycle of zebrafish. Retrieved from [162]. 1.7.1.1 Life cycle Zebrafish has a short generation time, typically 3 to 4 months from fertilization to adult stage, with considerably large eggs, 0.7 mm in diameter at fertilization, when compared to other fish [114]. Zebrafish life cycle is illustrated in Figure 5. It features a rapid embryonic development being that at 30 hpf the embryos present their first spontaneous movements demonstrating muscle activity, at 42 hpf the main organs become fully developed, and through 48 and 72 hpf hatching occurs. After hatching, the process of inflating the swim bladder occurs, which allows the larvae to start demonstrating a free-swimming behavior. Up to 7 days post fertilization (dpf), the larvae has completed most of its morphogenesis, with developments such as the protrusion of the mouth, the gut tube dropping more ventrally and the yolk extension being nearly empty, and it continues to grow rapidly. Whereas during the hatching period the embryo is usually at rest, the early larva gradually begins to swim about actively and starts moving its jaws, pectoral fins, and eyes, which produce prompt escape responses and indicate respiration, the seeking of prey and feeding [122]. The growth rate of zebrafish is rapid during the first 3 months of age, followed by a decrease over time until it ceases when reaching around 18 months of age. Zebrafish typically reach sexual maturity around 3 months of age, though zebrafish maturation is more associated with the size than with the age, being that when the specimen reaches around 23 mm it can already demonstrate some sexual traits or even be fully sexually mature, which allows for their use in breeding experiments [119], [122]. To date and to the best of our knowledge, there have been no reported studies detailing the lifespan of zebrafish in the wild. In a laboratory environment, zebrafish can live up to 5 years although after 2 years of age it is common for specimens to start evidencing muscular disorders and spinal curvatures [123]. 22 1.7.1.2 Reproduction In nature, zebrafish exhibit an annual reproductive behavior that commences with the spawning season just before the onset of the monsoon. This phenomenon allows for the main period of specimens rapid growth to take place during the monsoon months (June - September), a period of elevated temperatures and food availability [114]. Zebrafish are asynchronous, batch spawners that breed in small groups, with females scattering clutches of eggs over the substratum for them to be fertilized by the male once they are laid [119]. Egg spawning is dependent on the presence of a male and their odors considering that females kept in isolation for extended periods of time or older females often develop a plug, consisting of necrotic clumped eggs, which clogs the oviduct and prevents any further successful spawning. Moreover, it has been revealed that females exposed to male pheromones for several days prior to spawning produce more eggs, and a lower proportion of non-viable embryos than females isolated for several days [124]. The spawning in domesticated zebrafish at a laboratory atmosphere is interconnected with photoperiod in order to allow reproduction throughout the year [114]. Their spawning activity usually commences within the first minute of exposure to light following darkness, continuing for about an hour. Courtship behavior in the male is triggered by female pheromones and consists of a male chasing the female rapidly, often nudging her flanks with his snout aiming to lead her to a spawning site, and once over a spawning site, the sexual mates swim side by side triggering oviposition in the female and sperm release in the male, simultaneously. This sequence of behaviors is repeated throughout the spawning period but it is the most active within the first 30 min [125]. 1.7.1.3 Embryonic development The embryonic development of zebrafish has been fully described since 1995 [122]. Seven broad periods of embryogenesis were defined to highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization: the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods (Figure 6). The developmental process starts with the newly fertilized egg in the zygote period for about 40 min until the first cleavage occurs and the first cell is formed. This is followed by the cleavage period which comprises six cleavages where cell divisions occur synchronously every 15 min until the initial single cell is divided in 64 cells, around 2.25 hpf. During the blastula period, the embryo enters midblastula transition at the 512-cell stage, the yolk syncytial layer forms, and epiboly begins and reaches 30%. In the gastrula period, the primary germ layers and the embryonic axis are produced as well as the 23 primary structures for the head and the tail, and the epibolic process is completed. Reaching 10 hpf, the embryo enters in the segmentation period, where a variety of morphogenetic movements now occur, the somites develop, the rudiments of the primary organs become visible, the tail bud becomes more prominent, and the embryo elongates. Moreover, the first cells differentiate morphologically, and the first body movements appear. Throughout the pharyngula period, which begins at 24 hpf, the embryo head straightens out, the nervous system is being developed, and the circulatory system forms with well-delineated chambers and the heart beginning to beat just at the onset of the period. At 48 hpf, the embryo enters in the hatching period where morphogenesis of the primary organs systems is completed, the cartilage in the head and pectoral fin develops, and the embryo begins to hatch from their chorions. By day 3, the hatched larva has completed most of its morphogenesis and continues to grow rapidly. Prominent changes during the next day include the inflation of the swim bladder and the continued anterior-dorsal protrusion of the mouth. Whereas during the hatching period the embryo is usually at rest, the early larva gradually begins to exhibit free swimming, and demonstrating food-seeking and active avoidance behaviors. Figure 6. Stages of zebrafish embryonic development. Adapted from [122]. 1-cell Zygote Period 8-cell Cleavage Period 512-cell Blastula Period 30%-epiboly Blastula Period 75%-epiboly Gastrula Period 6-somite Segmentation Period 21-somite Segmentation Period High pec Pharyngula Period Pec fin Hatching Period Protruding mouth Hatching Period 24 1.7.2 Zebrafish as a research model In the recent years, zebrafish has been gaining acceptance as a vertebrate model host for the understanding of host-pathogen interactions, creating a bridge between simple assays based on cell culture and biological validation in traditional mammalian models such as rodents (Figure 7) [65]. The zebrafish has more similarities to mammals than the invertebrate models used in research in terms of anatomical structures, physiology, genetics and an immune system that comprises both innate and adaptive immune functions with a high degree of conservation with mammals, including humans [126], [127], [128]. Their embryonic development and organs functionality are similar to those of humans, and their genome, fully sequenced since 2011, reveals a high similarity with human genetics by having 70% similarity to human genes and 82% human disease-causing proteins, which is a considerable advantage in modelling human disease. Other advantages attributed to this model are the small size, ease of care and manipulation, low maintenance cost, high reproductive rate, rapid development and maturation, comprehensive molecular tools, and the fact that it allows for a more ethical in vivo analysis [97]. In addition to adult fish, zebrafish embryos also offer unique advantages as a host for the study of infections, such as a rapid and external development [129] and its optical transparency, which enables real-time visualization to track the progress of the pathogens and the responsive immune cells simultaneously [111]. Importantly, the use of zebrafish in research goes accordingly with the 3Rs of animal research which stand for Replace, Reduce, and Refine whose main goal is to alter traditional animal testing practices so that animal experimentation is reduced to a minimum whenever possible, attempting as well to lower animal distress and suffering during research to a minimum [130]. Studies have confirmed that fish have a less developed nervous system comparing to other mammalian models and that, although they are neurologically equipped with nociceptors (sensory receptors that send signals that cause pain perception) and unconscious emotional responses, they do not suffer pain and awareness feelings [131]. Moreover, in line with the European Commission Directive of 2010, (Directive 2010/63/EU), experiments with the earliest stages of zebrafish, up to 5 dpf, are not regulated as animal studies since these have not yet achieved free living. Therefore, although there are many promising advances in alternative research models, zebrafish offer a unique opportunity to make use of a living organism but still follow the principles of the 3Rs, reducing the number of animals needed to perform testing as well as the time and resources compared to other animal models. 31 o Amicon® Stirred Cell - a pressure-based sample concentration device with 1,000 Dalton molecular weight cutoff filters o Amicon® Ultra 15 mL centrifugal device with Ultracel® 3,000 Dalton molecular weight cutoff filters Concentrated samples were further subjected to a filtration through pore size of 0.2 µm sterile nitrocellulose membrane filters to ensure sterility and absence of other cellular elements but exosomes in the samples. The media used to grow the yeast cultures were subjected to the same procedure that C. albicans -free supernatants were as an experimental control. 2.3 Exosomes analysis Samples size and polydispersity were determined by Dynamic Light Scattering (DLS) and particles surface charge measured by Electrophoretic Light Scattering (ELS). Exosomes production and purification was assessed by western blot protocol, and proteins quantified by Pierce™ BCA Protein Assay Kit. The morphology of the exosomes was assessed by Scanning Transmission Electron Microscopy (STEM). 2.3.1 Sample size and polydispersity determination by dynamic light scattering The size and polydispersity of samples were measured using a Zetasizer Nano ZS particle analyzer and analyzed with the Zetasizer Software (Malvern Instruments) according to the manufacturer’s protocol. This technique involves the measurement of the scattered light from small particles, measuring their diffusivity when undergoing Brownian motion, which can provide information about the particle’s size and their state of motion. The Brownian motion of the particles causes the scattering light to fluctuate randomly. Small particles move faster than large ones which makes this random fluctuation of the scattering light also faster [151], [152]. The measured particles will have a certain level of heterogeneity. To determine the uniformity degree of the samples in terms of the particle’s size, the polydispersity index (PDI) is used. Samples are considered monodisperse if the PDI is below 0.1 and polydisperse for higher values, however, many factors may affect this parameter such as the particles’ heterogeneity, aggregation or contamination. High polydispersity values can lead to unreliable results, far from the real size of the particles [151]. In summary, the analysis of the size and PDI was carried out by placing 1000 µl of the solutions in a polystyrene cuvette, setting an attenuator between 6 and 9, and a count rate always superior to 150 counts. 32 2.3.2 Surface charge measurement by electrophoretic light scattering The ELS method is based on electrophoretic principles: when a solution is inserted into a cell with two electrodes and an electrical field is applied into those, the existing particles that have a net charge will migrate towards the oppositely charged electrode. This migration has a specific velocity, which is known as the mobility of the particles, which is related to their zeta potential. With this technique, it is possible to determine the particle charge in the solution. This approach measures both the outer layer and the solvation layer of the particle and the charge between these two layers is called zeta potential (Figure 8). Such characteristic leads to a wide range of implications, with the most significant one being the possibility of the real charge of particles being mitigated by the solvent. For this measurement, an aliquot of 800 µl of the solutions prepared for the DLS analysis was placed in the zeta cuvette. 2.3.3 Scanning transmission electron microscopy analysis The morphology of the exosomes was assessed by STEM analysis, a hybrid electron microscopy technique used for imaging and morphological characterization with atomic-scale resolution. In summary, after the concentration of the C. albicans SC5314 -free supernatant, C. albicans 124A -free supernatant and the YEPD medium using the Amicon® Ultra-15 Centrifugal Filter Unit and subsquent filtration, a 200-mesh copper grid coated with carbon was immersed in each sample. After 1 min of immersion, the samples were left to dry, and were posteriorly analyzed in an ultra-high-resolution scanning electron microscope (NanoSEM FEI Nova 200 (FEG/SEM)). The microscope was operated at 15 kV. Figure 8. Schematic representation of a dip cell. Retrieved from [163]. 33 2.3.4 Protein concentration determination Protein was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) according to the manufacturer’s instructions. 2.3.5 Protein gel electrophoresis and Western blot techniques Firstly, samples were subjected to 12% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE), where a broad range SDS-PAGE molecular weight marker (MWM) (11-245 kDa) was used (NZYTech). In order to optimize the experimental conditions, two different protein concentrations were tested: o Gel loaded with 8 µg of protein directly from samples o Gel loaded with 20 µg of protein, whose samples were submitted to trichloroacetic acid (TCA) protein precipitation (PanReac AppliChem, ITW Reagents) Control gels were stained with Coomassie Brilliant Blue G250 and Silver Nitrate and remaining gels were transferred to nitrocellulose blotting membranes (GE Healthcare Whatman™ Protran™). The membranes were blocked with 5% non-fat dry milk (w/v) in Tris-saline buffer containing 0.05% Tween (v/v) for 2 h at room temperature. Afterwards, membranes were incubated overnight at 4 °C with a pool of antisera (1:1000) from immunized rabbits and bound antibodies were detected with Goat anti-Rabbit IgG HRP conjugate (1:20000). In this work, four different exosome antibodies were used (EXOAB products): anti-CD9, anti-CD63, anti-CD81 and anti-Hsp70. Detection was fulfilled by the enhanced chemiluminescent method using Immobilon® ECL Ultra Western HRP Substrate and a ChemiDoc™ MP imaging system. 2.4 Macrophage studies The in vitro studies were performed using the murine macrophage cell line J774A.1 that was cultured and maintained in complete medium, Dulbecco’s Modified Eagle Medium (DMEM) composed by high glucose, Lglutamine, and 25 mM HEPES supplemented with 10% FBS and 1 mM sodium pyruvate (ThermoFisher Scientific) at 37 °C in a 5% CO2 atmosphere. After confluent growth, macrophage cells were recovered, washed, and resuspended in complete medium to the desired final cell concentration. 34 2.4.1 Cytotoxicity analysis Macrophages were plated onto 96-well tissue culture plates (Orange Scientific) at 1x105 cells/well and incubated to adhere overnight at 37 °C in a humidified atmosphere of 5% CO2. Different stimuli were added to the cells: the concentrated samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit concentration system, specifically C. albicans SC5314 -free supernatant and C. albicans 124A -free supernatant whose growth was performed in minimal medium, alongside with minimal medium used as an experimental control, and lipopolysaccharide (LPS) used as a positive control since it induces host inflammatory responses and cell injury. Cell viability was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) assay after 24 h of incubation, according to the manufacturer’s instructions. The MTT assay is a colorimetric assay to assess cell metabolic activity, where MTT is a water-soluble tetrazolium salt that can be modified into an insoluble formazan that has a purple color, by cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. Enzymatic activity was quantified using a solution of DMSO:ethanol (1:1) to solubilize MTT formazan and measuring absorbance at 570 nm. As a control of viability (100%) untreated cells were used while for control of cytotoxicity (100%) cells treated with Tris base were utilized. 2.4.2 Phagocytosis analysis 2.4.2.1 Preparation of yeast cells In this protocol, wild type strain of Candida albicans SC5314 was used. Firstly, a pre-inoculum was prepared and incubated at 26 °C in YEPD medium with the agitation of 160 rpm for 24 h. The following day, cell culture was prepared by diluting the pre-culture to an OD640 of approximately 0.1 and grown until OD640 reached approximately 0.5, reaching the exponential phase. Once the cell culture reached the desired OD, cells were collected by centrifugation at 5000 rpm for 3 min at 4 °C and washed in sterile 1x phosphate buffered saline (PBS) buffer. To fix cell wall PAMPs, yeast cells were incubated for 15 min in formol/ethanol (1:9), washed two times in sterile 1x PBS buffer for removal of formol/ethanol solution and resuspended in sterile 1x PBS buffer. To proceed with the assay, yeast cells were incubated for 10 min with 1 µM SYTOX Green at room temperature (RT) in the dark. After incubation, cells were collected by centrifugation at 5000 rpm for 3 min at 4 °C and resuspended in complete DMEM. Serial dilutions were performed to count the 35 cells on a hemocytometer and a final working solution was achieved by diluting the solution with complete DMEM to a final concentration of 5x106 cells/mL. 2.4.2.2 Preparation of J774A.1 macrophage cell line Macrophages were cultured in complete DMEM at 37 °C in a 5% CO2 atmosphere. After confluent growth was achieved, macrophage cells were recovered, washed, and resuspended in complete DMEM to a final concentration of 1x106 cells/mL. For fluorescence microscopy assays, 500 µl of the resulting cell suspension was transferred to 24-well tissue culture plates containing clean sterile glass coverslips (Ø13 mm). Afterwards, different stimuli were added to the cells: the concentrated samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit concentration system, specifically C. albicans SC5314 -free supernatant and C. albicans 124A -free supernatant whose growth was performed in minimal medium, alongside with minimal medium used as an experimental control, and LPS used as a positive control. Finally, cells were incubated for 24 h at 37 °C in a 5% CO2 atmosphere, to allow macrophage adherence and incubation with the stimuli. In the following day, phagocytic cells were promptly used for phagocytic assays. Duplicates were done for each plate. 2.4.2.3 Phagocytosis of Candida cells Macrophages were incubated with the labeled yeast suspension at a multiplicity of infection (MOI) of 1M:5Y for 45 min, at 37 °C and 5% CO2. After incubation, samples were kept on ice to stop phagocytosis and incubation with propidium iodide (PI) at a final concentration of 0.001 µg/mL for 5 min at RT was conducted. Cells were analyzed by fluorescence microscopy (Leica DM5000B), and subsequential microscopy images were analyzed using ImageJ cell counter software. 2.4.3 Cytokine production Macrophages were plated onto 96-well tissue culture plates (Orange Scientific) at 1x105 cells/well and incubated with different stimuli for 24 h: the concentrated samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit concentration system, specifically C. albicans SC5314 -free supernatant and C. albicans 36 Figure 9. Freshwater aquaria system layout. 124A -free supernatant whose growth was performed in minimal medium, alongside with minimal medium used as an experimental control, and LPS used as a positive control. The following day, macrophages were incubated with labeled yeast suspension at a MOI of 1M:5Y for 45 min. The release of pro-inflammatory cytokines TNF-α and IL-10 was quantified after the 24 h of incubation with the stimuli and after the 45 min incubation with the yeast suspension. The Mouse TNF-α Elisa Kit (Sensitivity: 8 pg/mL) and the Mouse IL-10 Elisa Kit (Sensitivity: 32 pg/mL) (Thermo Fisher Scientific) were used according to the manufacturer’s instructions. Macrophages alone were used as a negative control. 2.5 Zebrafish studies 2.5.1 Zebrafish maintenance and husbandry For the animal well-being, various maintenance activities were executed daily such as feeding, monitoring of freshwater physicochemical properties (pH, chlorine, nitrate and nitrite) and aquaria sanitation. The aquaria set, illustrated in Figure 9, was supplied by a UV lamp sterilized filtering and an activated carbon filter. 37 Figure 10. Stock aquarium. 2.5.2 Parental zebrafish feeding To achieve reproductive fitness with a high rate of viable egg spawning, the zebrafish breeding stock requires a good nutrition. Every day, animals were fed three times with two different types of food: solid food as commercial Nutrafin Max flakes, or live food as nauplii of brine shrimp Artemia sp. cultured in laboratory. Live food is of particular importance due to the inherent higher nutritional value and to the fact that it acts as a stimulus factor for fish to “hunt” it, which incentive their predatory instinct. For live food preparation, Artemia was incubated in artificial salt water at 35 ppm, 28.0 ± 1.0 ºC in a tapered cylindrical container at the bottom, where dry cysts were left to hatch for 24 to 48 h. When hatched, 24 h nauplii were used to feed developing larvae and 48 h nauplii were used to feed zebrafish adults, which usually corresponded to the first meal of the day or to the last on pre-spawning days. At the weekend, parental fish were just fed with a solid flakes by an automatic feeder, twice a day. 2.5.3 Stock aquarium maintenance The stock aquarium is the aquarium used to house the zebrafish adults exploited as breeding specimens (F0, parental), which had a total capacity of 100 L (Figure 10). To maintain the optimal freshwater conditions for zebrafish, several parameters needed to be daily verified and adjusted if necessary: temperature around 25.0 ± 2.0 °C; pH of 7.40 ± 0.5; chlorine at zero level, the nitrates and nitrites at values close to zero. Moreover, the tank was renewed, and filtered by activated carbon filtering, 50% daily automatically by peristaltic pumps, under an open circulation system. Optimal environment conditions were also ensured by water aeration through air stones placed at the aquarium corners and by removal of fecal waste twice a day manually, using hand nets. 38 A B C Figure 11. Breeding and Hatching aquaria. (A) Breeding system assembled; (B) Hatching aquarium; (C) Aquarium for microplates incubation. 2.5.4 Breeding & Hatching aquaria The breeding system is composed of an aquarium with capacity of about 30 L, that it is equipped with a spawning cage to proceed with the breeding protocol (Figure 11A). This cage features a net at the bottom which allows for the eggs to deposit on the base of the aquarium, where the breeding fish have no direct contact, and as a result cannibalism behavior by the parental animals is avoided. To mimic the zebrafish natural habitat and induce spawning, marbles were added to the bottom of the cage [153]. Zebrafish females tend to rub their genital papilla on the marbles, which serves as substrate supporting the releasing of oocytes to the freshwater, a behavior that males capture as a signal to release their sperm, and consequently external fertilization occurs [114]. In order to secure a stable environment for reproduction, the breeding system was always assembled in the mornings, and used in late afternoons to allow time for the water to reach the optimal conditions. When successful reproduction occurs, with about 1000 embryos spawned, the remainder embryos are incubated in a 5 L aquarium, under the same physical-chemical conditions of the stock aquarium. This small tank is placed inside a 30 L aquarium that serves as a water bath for fish development, a complex that is designated as hatching aquarium (Figure 11B). Throughout the embryos development, dead embryos were verified and removed daily to prevent microbiological cross-contamination, and the freshwater was renewed daily to ensure optimal oxygenation. For the FET protocol, a 30 L aquarium was adapted in order to allow incubation of microplates containing the developing embryos exposed during the assay with optimal conditions. This system, designated as “microplates aquarium” (Figure 11C), consists of a foam support platform that permits the microplates to be partially immersed in acclimated freshwater at 28 °C, which is an essential requirement of this protocol that was ensured from the beginning (2 hpf) to the end (80 hpf) of the exposure. 39 2.5.5 Photoperiod Zebrafish is a photoperiodic animal, so the periods of light and dark should be regular and constant without interference or disturbance, especially on pre-breeding days [114]. When fish are isolated for the breeding protocol, the dark period serves to accommodate the animals in the new environment which is at the breeding aquarium, and when the lights turn on, the zebrafish begins its mating behavior that ends with a spawning event. At the NANODANIO laboratory, a timer was set to enable 12 h of light and 12 h of darkness. 2.5.6 Infection of zebrafish embryos with Candida cells 2.5.6.1 Preparation of yeast cells For this protocol, wild type strain of C. albicans 124A was used. Firstly, a pre-inoculum was prepared and incubated at 30 °C in YEPD medium with the agitation of 200 rpm overnight. The following day, cell culture was prepared by diluting the pre-culture to an OD640 of approximately 0.1 and grown until OD640 reached approximately 0.5, reaching the exponential phase. Once the cell culture reached the desired OD, cells were collected by centrifugation at 5000 rpm for 3 min at 20 °C, washed two times in sterile deionized 𝐻2𝑂, and resuspended in 5 mL of sterile deionized water. Subsequently, cells were counted on a hemocytometer to allow the making of an initial working solution with 1x108 cells/mL, by diluting the cell solution with sterile deionized water. Using this initial solution, serial dilutions were performed to obtain the remainder of the working solutions with different Candida cells concentrations: from 1x107 cells/mL to 1x104 cells/mL. 2.5.6.2 FET protocol The FET protocol begins with the parental fish isolation at the breeding cage at the end of the day, using a sex ratio of 3 females and 5 males, and can be done once or twice during the week. The next day, 1 h after the onset of the light period, zebrafish embryos were collected and washed, and viable embryos separated from the dead with the naked eye, as viable embryos are transparent, while unviable eggs are opaque and white. For this assay, the FET protocol was adjusted (Figure 12). At 2 hpf, the embryos were transferred to a 24-well plate with a final volume of 2 mL per well, with HEPES E3 buffer as the chosen medium since it was previously demonstrated that it induces enhanced zebrafish embryos survival and, as a saline solution, it offers the best compromise between ensuring the optimum pH for zebrafish embryos, i.e. pH 7.4, and avoiding sublethal effects throughout development. Embryos co-incubation with Candida cells was performed 40 Figure 12. Scheme of FET to evaluate the effect of zebrafish embryos co-incubation with Candida cells on their survival rate. Adapted from [164]. at 6 hpf, by removing the original medium and adding 1900 uL of HEPES E3 buffer along with 100 uL of the specific Candida working solution to the wells, which results in incubations with 1x107 to 1x103 Candida cells. As experimental control, zebrafish embryos were incubated with 1900 uL of HEPES E3 buffer along with 100 uL of deionized water, since it is the solvent used in the yeast cells solutions. The test conditions and the defined control had 2 replicates, each well representing one replicate with 10 embryos, and survival analysis in each condition was performed at 8, 32, 56 and 80 hpf. For FET to be reliable, the mortality rate from the defined control must be below 10%. 47 • Mean size (nm): • PDI: • Populations (nm): 1.681 ± 0.409 representing 21.1% ± 5.1 of total population 440.0 ± 76.87 representing 74.6% ± 2.7 of total population • Mean size (nm): 105.2 ± 4.244 • PDI: 0.603 ± 0.039 • Populations (nm): 17.01 ± 5.740 representing 8.3% ± 2.3 of total population 225.1 ± 19.02 representing 88.6% ± 3.7 of total population 3699 ± 2070 representing 2.3% ± 1.3 of total population • Mean size (nm): 98.99 ± 49.33 • PDI: 0.488 ± 0.238 • Populations (nm): 1.352 ± 0.021 representing 45.8% ± 0.7 of total population 333.0 ± 73.39 representing 51.9% ± 3.0 of total population 2913 ± 2655 representing 2.4% ± 2.5 of total population • Mean size (nm): 53.98 ± 63.09 • PDI: 0.592 ± 0.282 • Populations (nm): 2.693 ± 0.95 representing 15.6% ± 3.8 of total population 48.72 ± 73.45 representing 33.5% ± 5.6 of total population 159.9 ± 99.32 representing 46.6% ± 2.8 of total population C. albicans SC5314 -free supernatant concentrated C. albicans SC5314 -free supernatant residues C. albicans 124A -free supernatant concentrated C. albicans 124A -free supernatant residues • Mean size (nm): 176.0 ± 15.96 • PDI: 0.708 ± 0.164 • Populations (nm): 10.99 ± 6.3 representing 4.7% ± 2.8 of total population 40.07 ± 26.7 representing 8.6% ± 4.8 of total population 443.5 ± 26.06 representing 83.4% ± 6.4 of total population • Mean size (nm): 630.3 ± 117.7 • PDI: 0.623 ± 0.142 • Populations (nm): 1.461 ± 0.034 representing 32.0% ± 2.4 of total population 509.8 ± 71.28 representing 65.9% ± 3.5 of total population 3325 ± 3035 representing 2.0% ± 2.1 of total population YEPD medium concentrated YEPD medium residues Figure 16. DLS size distributions of samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit, where C. albicans SC5314 and C. albicans 124A -free supernatant whose growth was performed in YEPD medium were used. YEPD medium was used as control. The mean size, PDI and populations size are indicated. 48 When comparing the DLS analysis of samples whose growth was conducted in YEPD medium and samples whose growth was conducted in minimal medium, differences can be observed. The DLS analysis of the C.albicans -free supernatants whose growth was supported by minimal medium displayed a mean particle size of approximately 100 nm for both C. albicans strains and lower values for the polydispersity parameter (Figure 17). This indicates that distribution of the particles is less broad than it was when in YEPD medium, nonetheless, as previously noted, a distribution analysis should be more accurate than the mean size values since it is possible to analyze the different populations present in the samples and more accurately characterize the exosomes populations. In Figure 18, different size populations are clearly detected, being that the one with the highest concentration of particles in both C. albicans -free supernatants concentrated should illustrate the exosomes present in the samples in similarity with the DLS analysis in Figure 16. Nonetheless, the other populations in these samples have a much lower presence and, therefore, a reduced interference with the exosomes population and the overall analysis. Accordingly, this can also be perceived through the DLS analysis of both media, in which YEPD medium has more different and dispersed particles populations when comparing to the minimal medium. Overall, the results indicate that growth performed in minimal medium induces an optimized analysis due to less particles populations inherited from the medium, therefore concentrated samples whose growth was carried out in minimal medium were the ones chosen to perform all of the following assays. The ζ-potential measurement of these samples, represented in Figure 17, indicates negatively charged exosomes for both of C. albicans strains in agreement with the previous ELS analysis. Particularly, the overall analysis indicates negatively charged (-3.5 ± 0.87 mV) exosomes with a mean size of 157.1 ± 7.786 for C. albicans SC5314 and negatively charged (-5.3 ± 0.78) exosomes with a mean size of 147.6 ± 9.915 for C. albicans 124A . C. albicans SC5314 -free supernatant C. albicans 124A -free supernatant Minimal medium C. albicans SC5314 -free supernatant C. albicans 124A -free supernatant Minimal medium Concentrated samples Residues Figure 17. Representative mean size, PDI and ζ-potential of samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit, where C. albicans SC5314 and C. albicans 124A -free supernatant whose growth was performed in minimal medium were used. Minimal medium was used as control. 49 Minimal medium residues • Mean size (nm): 98.16 ± 1.279 • PDI: 0.389 ± 0.045 • Populations (nm): 6.838 ± 9.457 representing 1.8% ± 2.5 of total population 157.1 ± 7.786 representing 97.4% ± 3.5 of total population 1598 ± 2214 representing 0.76% ± 1.2 of total population • Mean size (nm): 799.1 ± 111.9 • PDI: 0.363 ± 0.165 • Populations (nm): 0.554 ± 0.311 representing 4.4% ± 2.5 of total population 595.9 ± 133.0 representing 95.6% ± 2.5 of total population • Mean size (nm): 296.5 ± 306.1 • PDI: 0.487 ± 0.246 • Populations (nm): 0.904 ± 0.07 representing 60.7% ± 5.0 of total population 249.8 ± 61.49 representing 34.8% ± 5.1 of total population 4079 ± 2315 representing 4.5% ± 3.3 of total population C. albicans SC5314 -free supernatant concentrated C. albicans SC5314 -free supernatant residues • Mean size (nm): 425.1 ± 39.82 • PDI: 0.274 ± 0.084 • Populations (nm): 303.9 ± 17.54 representing 100% ± 0.0 of total population C. albicans 124A -free supernatant concentrated C. albicans 124A -free supernatant residues • Mean size (nm): 102.8 ± 4.721 • PDI: 0.289 ± 0.008 • Populations (nm): 11.68 ± 10.83 representing 2.7% ± 2.6 of total population 147.6 ± 9.915 representing 97.3% ± 2.6 of total population • Mean size (nm): 263.6 ± 49.57 • PDI: 0.516 ± 0.070 • Populations (nm): 61.65 ± 30.14 representing 24.3% ± 13.5 of total population 378.0 ± 158.0 representing 74.1% ± 14.9 of total population 3275 ± 2990 representing 1.6% ± 1.7 of total population Minimal medium concentrated Figure 18. DLS size distributions of samples resulting from the Amicon® Ultra-15 Centrifugal Filter Unit, where C. albicans SC5314 and C. albicans 124A -free supernatant whose growth was performed in minimal medium were used. Minimal medium was used as control. The mean size, PDI and populations size are indicated. 50 These results are in agreement with what was previously reported for C. albicans SC5314 exosomes in [82], where most vesicles had sizes between 50 and 100 nm, and a smaller percentage had larger sizes reaching more than 200 nm. Slight differences in the distribution of EVs particle size between different strains have been reported, as in [83], but for these specific C. albicans strains no significant differences were noted. 3.3 Analysis of exosomes isolation by Western blotting In order to further validate exosomes isolation, immunoblotting assays were performed using antibodies recognizing four different molecular markers of exosomes: anti-CD9, anti-CD63, anti-CD81 and anti-Hsp70. For this analysis, C. albicans SC5314 -free supernatant and C. albicans 124A -free supernatant concentrated samples whose growth was performed in minimal medium were used to evaluate the exosomes isolation, alongside with minimal medium used as control and the residues of both supernatants samples to verify if particles loss occurred during the process. As mentioned before, two different protein concentrations were tested for the purpose of optimizing the protocol: for each antibody one gel was loaded with 8 µg of protein directly from the samples and other gel was loaded with 20 µg of protein, whose samples were submitted to TCA protein precipitation. In Figure 19 is represented the western blot analysis using the anti-Hsp70 exosome antibody for both protein concentrations tested. B A Figure 19. Western blot analysis of C. albicans -free supernatants concentrated samples alongside with their residues using anti-Hsp70 exosome antibody. Minimal medium (MM) was used as control. (A) gel loaded with 8 µg of protein directly from the samples; (B) gel loaded with 20 µg of protein, whose samples were submitted to TCA protein precipitation. 51 The results show the presence of the vesicle marker Hsp70 antigen epitope in both of C. albicans -free supernatants concentrated samples, while reaction is negative in the control, minimal medium, and a minimal presence of this vesicle was detected in the residues samples which implicates a minimal loss during the experimental process. The positive result in the concentrated samples indicates the presence of C. albicans exosomes, which goes in agreement with previous reports that also detected this protein in C. albicans vesicles, and further validates the isolation protocol [76]. The protein band for the heat shock protein Hsp70 is reported to be located at a molecular weight of circa 55 kDa – 70 kDa, however our results demonstrate detection starting at a molecular weight above 245 kDa until circa 70 kDa which can possibly be explained by protein glycosylation that causes detections in higher molecular weight than expected. In terms of protein quantities, no significant differences were noted in this assay. The western blot assays using the anti-CD9, anti-CD81, and anti-CD63 antibodies revealed the absence of these proteins on the exosomes. These tetraspanins are considered to be biomarkers of extracellular vesicles by several authors, but unfortunately, proteomic analyses of fungal EVs have revealed that many of the markers that have been so useful for the isolation and study of mammalian EVs are not present in fungal EVs [154], [155], [156]. In C. albicans research, two promising putative markers have been suggested for C. albicans EVs, the plasma membrane proteins Sur7 and Evp1, which are indicated to have potential of being fungal equivalents of the tetraspanins markers due to their topological similarity to CD9, CD81 and CD63, but antibody-based validation is required and commercial antibodies for these are not yet available [83]. Our results are in accordance with this hypothesis, nonetheless there are reports revealing the detection of these specific tetraspanins in the fractions of Candida exosomes demonstrating inconsistency among the literature, which can be due to different experimental conditions, species and strains specificity used that could lead to different EVs composition and characterization [157]. 3.4 The effect of exosomes in cytotoxicity, uptake and cytokine production by macrophages One of the main concerns before studying the interaction of these EVs with any cell line is the evaluation of its cytotoxicity. In this sense, the effect of the C. albicans SC5314 and C. albicans 124A exosomes on the metabolic activity of J774A.1 cells was evaluated using the MTT assay after 24 h of co-incubation, alongside with minimal medium used as an experimental control, and LPS used as a positive control since it is a highly conserved cell wall component of gram-negative bacteria known to activate immune cells [158]. As represented in Figure 20, a slight decrease in cell metabolic activity is observed with the C. albicans -free 52 supernatants, particularly for the C. albicans SC5314 strain. Nonetheless, both exosomes samples were well tolerated by the macrophage cells, showing viability values above 80% and 90% for C. albicans SC5314 -free supernatant and C. albicans 124A -free supernatant , respectively, which indicates that both of C. albicans exosomes are not cytotoxic to these cells. In order to elucidate the effect of exosomes in the cellular uptake of C. albicans , J774A.1 cells were firstly incubated with both of the exosomes samples for 24 h at 37 °C in a 5% CO2 atmosphere. On the following day, the activated phagocytic cells were incubated with formol-killed C. albicans cells previously stained with Sytox Green for 45 min and then contra-stained with PI, according to our previous developed phagocytosis assay [159]. This specific protocol takes advantage of PI quenching effect of the non-internalized yeast cells, since PI is not permeant to live cells, and of the integrity of the Sytox Green fluorescence of C. albicans cells inside live macrophages, since Sytox Green is a high affinity nuclear stain that penetrates cells and intercalates with nucleic acids. Therefore, this protocol enabled the differentiation of the non-labeled phagocytes, representing phagocytes with no interaction with yeast cells, the Sytox Green labeled phagocytes, representing phagocytes that have internalized yeast, and the dead phagocytes that become stained with PI . For this phagocytosis analysis, fluorescence microscopy images were taken, and subsequently analyzed using ImageJ cell counter software to assess the percentage of macrophages with and without internalized yeast cells, and dead macrophages (Figure 21). Results revealed lower percentages of macrophages with Control LPS MM C. albicans SC5314 -free supernatant C. albicans 124A -free supernatant Figure 20. Viability of J774A.1 cells after 24 h incubation with C. albicans SC5314 and C. albicans 124A - free supernatants. Minimal medium (MM) was used as experimental control and LPS as positive control. Viability was assessed using the MTT assay after 24 h incubation. 53 Figure 21. Quantification of macrophage phagocytosis of yeast cells evaluated by fluorescence microscopy, where macrophages from test conditions were previously incubated with C. albicans SC5314 and C. albicans 124A -free supernatants for 24 h. ( ) Macrophages without internalized yeast cells; ( ) Macrophages with internalized yeast cells; ( .) Dead macrophages. internalized yeast cells in both test conditions where macrophages were previously incubated with C. albicans SC5314 and C. albicans 124A exosomes when compared with the control condition, which suggests that the presence of C. albicans exosomes affects the phagocytic capacity of macrophages to some extent. The percentage of macrophage phagocytosis was lower for the macrophages previously incubated with C. albicans SC5314 exosomes than with C. albicans 124A exosomes, nonetheless without a considerable difference. This difference in macrophage phagocytosis between the control and the test conditions is illustrated in Figure 22, which consists of fluorescence microscopy images taken of the control (Figure 22A) and of the test condition where macrophages were previously incubated with C. albicans SC5314 exosomes (Figure 22B). . A B Figure 22. Illustration of macrophage phagocytosis of yeast cells by fluorescence microscopy in the control (A) and the test condition where macrophages were previously incubated with C. albicans SC5314 -free supernatant for 24 h (B). ( ) Internalized yeast cells; ( ) Non-internalized yeast cells. 54 After the phagocytosis analysis, it was important to evaluate the effects that the presence of C. albicans exosomes could have in the macrophages activation. Accordingly, secretion of pro-inflammatory cytokines, namely TNF-α and IL-10, by the macrophages was quantified by ELISA after 24 h co-incubation with both C. albicans exosomes samples and after 45 min incubation with yeast cells, which corresponds to before and after infection, respectively (Figure 23). The inflammatory cytokine TNF-α is associated with the control of candidiasis, whereas IL-10 is described to facilitate an immuno-suppressive response favoring C. albicans dissemination [160]. The results obtained from the samples before infection indicate that the presence of both of C. albicans exosomes samples induced the production of the pro-inflammatory cytokine TNF-α in J774A.1 cells with considerably higher levels than the control, suggesting effective activation of macrophages by these vesicles. After infection, a reduction in TNF-α production is observed in all samples, except for LPS as expected, since it is known to induce host inflammatory responses. Nonetheless, TNF-α secretion levels by the macrophages that were pre-incubated with C. albicans exosomes remained higher than in the control but correlated inversely with the percentage of phagocytosis. Regarding the production of the pro-inflammatory cytokine IL10, no significative levels were secreted by J774A.1 cells to allow detection. Altogether, this data suggests Control LPS MM C. albicans SC5314 -free supernatant C. albicans 124A -free supernatant 24 h incubation with stimulus 45 min incubation with yeast cells Figure 23. Production of TNF-α by J774A.1 cells after 24 h incubation with C. albicans SC5314 and C. albicans 124A -free supernatants, and after 45 min incubation with C. albicans SC5314 yeast cells. Minimal medium (MM) was used as experimental control and LPS as positive control. 55 that C. albicans exosomes stimulate the host immune system inducing TNF-α secretion before infection while simultaneously reducing the phagocytic capacity of the macrophages. The presence of C. albicans exosomes in an infection situation seems to result in a balance within the inflammatory response and the survival of the pathogen, which supports the commensalism interactions between the yeast and the host. These results are in line with a previous report that demonstrated that C. albicans EVs are immunologically active and can potentially interfere with the host response in the setting of invasive candidiasis, where higher levels of TNF-α secreted by macrophages were also detected. Contrarily to our results, this report also detected high levels of IL-10 [85]. Another report demonstrated that C. albicans EVs followed the pattern exhibited by most fungal EVs, as they induced killing and did not induce the production of TNF-α and IL10 pro-inflammatory cytokines, which is discordantly with what our results indicate [78]. Nonetheless, different macrophages cell lines and strains of C. albicans were used in these studies, reinforcing the possibility that the biological activities of EVs could be model and strain specific, which is a hypothesis that has been suggested in earlier studies [77], [78]. 3.5 The effect of zebrafish embryos co-incubation with Candida cells on their survival rate After the characterization of C. albicans exosomes and the study of their role in an infection environment using a cell culture model, it is important to evaluate their role in the context of a whole organism where the complex host-fungus interactions can be investigated, and the disease development can be monitored right from the moment that fungal cells are introduced into the host. In this context, Danio rerio early life was the selected model for this analysis given the high similarity to mammals, as genotypic and embryonic development, at these early stages, and all the advantages that have been attributed to their use in research. To proceed with the exosomes studies, first it was necessary to establish the zebrafish embryo as an effective and feasible model to study hostCandida interactions. Therefore, the survival rates of infected zebrafish embryos with different doses of C. albicans cells were determined to evaluate the feasibility of using zebrafish early stages as a model host for C. albicans infections. Embryos co-incubation with Candida cells was performed at 6 hpf with different doses that ranged from 1x107 to 1x103 Candida cells. All of the conditions had 2 replicates, each well representing one replicate with 10 embryos, and survival analysis in each condition was performed at 8, 32, 56 and 80 hpf. In Figure 24 is represented the survival rates of zebrafish embryos co-incubated with Candida cells in three independent experiments. 56 Figure 24. The survival rates of zebrafish embryo infected with different doses of C. albicans SC5314 along 80 hpf in three independent replicates (A, B and C). As shown in Figure 24, the survival rates with the different Candida cells doses significantly differ in between replicates which indicated a problem in reproducibility for this assay. This inconsistency suggests that the inoculation doses were not consistent between replicates, which can be explained by some difficulties in the execution of the serial dilutions, and that a control with colony forming units (CFUs) should be performed to confirm the number of viable cells present in the samples. 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