Fluorescent tag Sporothrix brasiliensis: a tool for host-pathogen interaction studies

UMinho | 2022 Universidade do Minho Escola de Medicina Jorge Carlos Dias de Sousa Filho Fluorescent tag Sporothrix brasiliensis : a tool for host-pathogen interaction studies Fluorescent tag Sporothrix brasiliensis : a tool for host-pathogen interaction studies Jorge Carlos Dias de Sousa Filho outubro de 2022 Julho de 2021 Universidade do Minho Escola de Medicina Sónia Beatriz Alves Gomes Title Dissertação de Mestrado Mestrado em Ciências da Saúde Trabalho efetuado sob a orientação da Doutora Susana Cristina Roque Oliveira e da Doutora Palmira da Conceição de Araújo Barreira da Silva outubro de 2022 Universidade do Minho Escola de Medicina Jorge Carlos Dias de Sousa Filho Fluorescent tag Sporothrix brasiliensis : a tool for host-pathogen interaction studies Dissertação de Mestrado Mestrado em Ciências da Saúde Trabalho efetuado sob a orientação de Doutor Fernando José Santos Rodrigues e de Doutor Ricardo Jorge Leal Silvestre ii 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/ iii Agradecimentos Aos meus orientadores, Doutor Fernando Rodrigues e Doutor Ricardo Silvestre, pela orientação e pela oportunidade de poder aprender convosco e pelos inúmeros ensinamentos que muito contribuíram para o meu crescimento profissional e pessoal. À doutora Cristina Cunha, pela orientação, ajuda disponibilizada e pelos inúmeros ensinamentos que muito contribuíram para o meu crescimento profissional e pessoal. Ao Relber Gonçalves e Ana Frias, grandes amigos e brilhantes pesquisadores que doaram seu tempo, suor e sangue para a realização de diversos experimentos realizados na construção dessa tese. Sem vocês dois, certamente a conclusão dessa difícil etapa seria impossível. A Daniela Antunes, Rita Gomes e demais pesquisadores que me disponibilizaram imenso apoio científico e ajuda pessoal. Esta jornada foi seguramente mais fácil graças a vocês. Ao Ehsan, Sara, Sônia, Stephanie, Consuello, Ian, Cláudio entre diversos outros pesquisadores com quem desenvolvi uma amizade no laboratório, ajudando a tornar todo o processo menos difícil. Agradeço ao ICVS – Instituto de Ciências da Vida e Saúde pela oportunidade e experiência. E também a Fundação para a Ciência e a Tecnologia pelo apoio financeiro dado a esse estudo - FCT2022.03348.PTDC - Sporob_SiA: Elucidation of the role of sialidase 1 in the severity of feline sporotrichosis caused by Sporothrix brasiliensis. E por último, mas não menos importante, à minha família. Aos meus pais, tia e meu namorado, pessoas indispensáveis ao longo da minha trajetória. Tudo o que alcancei se deve a vocês. Serei eternamente grato por todas as oportunidades que me concederam, todo o afeto e por todos os esforços e sacrifícios que fizeram por mim ao longo do caminho. iv 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. v Fluorescent tag Sporothrix brasiliensis : a tool for host-pathogen interaction studies Abstract Sporotrichosis is considered an emerging health problem, being the world's most prevalent subcutaneous mycosis, occurring on mammal hosts, which facilitates zoonotic transmission . Agrobacterium tumefaciens -mediated transformation (ATMT) is a broadly technique used for both plants and fungus transformation. Here, we report the establishing of an ATMT system for S. brasiliensis , considering different strains of A. tumefaciens and S. brasiliensis and several conditions of co-cultivation. Our results point to 72h of co-cultivation at 26°C, the AGL-1 bacterial strain, and the 2:1 ratio (bacteria:fungi) as ideal conditions for a high number of transformants. In these conditions, we obtained 3179 ± 1171 mutants/co-cultivation. PCR analysis showed that all Hygromycin B resistant clones tested harboured a copy of the HPH gene. FACS analysis showed high mitotic stability of the GFP gene in pGAPDH::GFP mutants. The S. brasiliensis pGAPDH::GFP and pGAPDH::H2A::GFP strains were respectively used to evaluate the phagocytosis index and fungicidal activity,. pGAPDH::GFP cytoplasm expression of GFP allowed a strong fluorescence visualization inside monocytes/macrophages in both microscopic and FACS analysis. The phagocytosis index was 64.25 ± 9.96% at the MOI of one peripheral blood mononuclear cell (PBMC) to five pGAPDH::GFP yeast cells, with two hours of infection. The pGAPDH::H2A::GFP S. brasiliensis strain failed to provide a correlation between loss of fungal viability or fungal death and loss of GFP fluorescence in all fungicidal experiments performed. Furthermore, its GFP fluorescence was not visible upon monocytes/macrophages engulfment in both microscopic and FACS analysis. Our results showed, firstly, an efficient genetic toolbox to create large-scale transformant libraries for S. brasiliensis, and secondly, the possibility to use the pGAPDH::GFP strain in phagocytosis assays without fluorophore stain prior to infection. Together, these results can help provide new insights to better understand the host-pathogen interactions and virulence mechanism for the Sporothrix spp.. Keywords: ATMT, fluorescent tag strains, sporotrichosis, Sporothrix brasiliensis . vi Marcadores fluorescentes em Sporothrix brasiliensis : uma ferramenta para estudos de interação entre hospedeiro e patógeno Resumo A esporotricose é um problema de saúde emergente e a micose subcutânea mais prevalente no mundo. Esta doença ocorre em hospedeiros mamíferos, o que facilita a sua transmissão zoonótica. A transformação mediada por Agrobacterium tumefaciens (ATMT) é uma técnica amplamente utilizada para a transformação de plantas e fungos. No presente trabalho, estabelecemos um sistema de ATMT para S. brasiliensis , considerando diferentes estirpes de A. tumefaciens e S. brasiliensis, bem como várias condições de co-cultivo. Os nossos resultados apontam para 72h de co-cultivo a 26°C, a estirpe bacteriana AGL-1 e o rácio de 2:1 (bactérias:fungos) como condições ideais para a obtenção de um elevado número de transformantes. Nestas condições, obtivemos 3179 ± 1171 transformantes/cocultivo. A análise de RTPCR demostrou a presença de uma cópia do gene HPH . A análise de FACS mostrou alta estabilidade mitótica do gene GFP dos isolados da estirpe pGAPDH::GFP. Os mutantes pGAPDH::GFP e pGAPDH::H2A::GFP de S. brasiliensis foram respetivamente usados para calcular o índice de fagocitose e a atividade fungicida. A expressão citoplasmática de GFP da estirpe de pGAPDH::GFP permitiu uma forte visualização de fluorescência dentro dos monócitos/macrófagos em análises de microscópica e FACS. Relativamente aos mutantes pGAPDH::H2A::GFP S. brasiliensis não foi possível obter uma correlação entre a perda de viabilidade fúngica ou morte fúngica e a perda de fluorescência GFP em todos as experiências realizadas no que diz respeito à avaliação da capacidade fungicida dos monócitos/macrófagos. Para além disso, não foi possível detetar a sua fluorescência GFP após ter sido fagocitado pelos monócitos/macrófagos em análises de microscópica e FACS. Em primeiro lugar, este trabalho permitiu a criação de uma ferramenta genética eficiente para construir bibliotecas de transformantes em larga escala para S. brasiliensis . Em segundo lugar, os nossos resultados demostraram que o mutante pGAPDH::GFP pode ser usado em ensaios de fagocitose sem a necessidade de coloração com fluoróforo antes da infeção. Em soma, os nossos resultados podem ajudar a entender melhor as interações hospedeiro-patógeno e o mecanismo de virulência da Sporothrix spp.. Palavras-chaves: ATMT, esporotricose, marcadores fluorescentes, Sporothrix brasiliensis. vii Table of Contents Licença concedida aos utilizadores deste trabalho................................................................ ii Agradecimentos .............................................................................................................................. iii Abstract ............................................................................................................................................ v Resumo ............................................................................................................................................ vi List of abbreviations ....................................................................................................................... x List of figures ................................................................................................................................. xiii List of tables................................................................................................................................... xv CHAPTER 1 Introduction .............................................................................................................. 1 1.1. Sporothrix-schenckii complex and sporotrichosis ...................................................... 2 1.2. Sporotrichosis epidemiological aspects ...................................................................... 5 1.3. Sporotrichosis treatment and control strategies ....................................................... 9 1.4. Components and virulence factors ............................................................................. 10 1.5. Immune response in Sporothrix infection ................................................................. 11 1.6. Agrobacterium tumefaciens-mediated transformation (ATMT) .............................. 13 CHAPTER 2 Aims and Outline ..................................................................................................... 15 CHAPTER 3 Optimization of Agrobacterium tumefaciens -mediated transformation on Sporothrix brasiliensis ................................................................................................................ 18 Abstract ..................................................................................................................................... 19 3.1. Introduction ................................................................................................................... 19 3.2. Materials and Methods ................................................................................................ 20 3.2.1. Microorganisms and culture conditions ...................................................................... 20 3.2.2. Inoculum count optimization for future experiments in vitro ........................................ 22 3.2.3. Sporothrix brasiliensis sensitivity analysis to Hygromycin B and Chlorimuron ethyl ...... 22 3.2.4. Agrobacterium tumefaciens -mediated transformation protocol of Sporothrix brasiliensis……. ........................................................................................................................ 23 xiv Figure 20 Schematic of S. brasiliensis pGAPDH::GFP::H2A loss of GFP fluorescence upon cell death………………………………………………………………………………………… 43 Figure 21 Schematic of the gating strategy used to analyse phagocytosis in FACS …… 46 Figure 22 Analysis of the correlation between loss of GFP fluorescence and cell death by Macrophage-fungicidal assay ……………………………………………………. 48 Figure 23 Analysis of the correlation between loss of GFP fluorescence and cell death by Heat-fungicidal assay ………………………………………………………………… 49 Figure 24 Stimulation of PBMCs with S. brasiliensis wild-type and pGAPDH::GFP cells results in high production of cytokines……………………………………………………… 50 Figure 25 Phagocytosis assay analysis using S. brasiliensis pGAPDH::GFP strain to infect PBMCs and hMDMs…………………………………………………………………….…. 51 xv List of tables Table 1 S. brasiliensis strains used in this work……………………………………………………….. 20 Table 2 Primers list from copy number analysis………………………………………………………. 26 Table 3 Table explaining the details from both S. brasiliensis GFP mutants used in this thesis…………………………………………………………………………………………………… 40 1 CHAPTER 1 Introduction 2 Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has become a public health concern that will likely worsen in the future (1). It is an acute or chronic granulomatous mycosis with wide geographic distribution, whose incidence and etiological agent vary according to geographic region, mostly based on case report observations (2). The etiologic agents of this disease are fungi often present in the pathogenic clade of the Sporothrix genus, but some cases can be caused by the environmental clade, associated with the sapronoses form of sporotrichosis (1,3). Among the Sporothrix genus, S. brasiliensis is reported to be the most virulent, exhibiting the worst clinical manifestations (4,5). Although sporotrichosis etiological agents have a susceptible response profile to antifungal drugs, several reports of drug resistance have already been recently reported (6,7). Nowadays Rio de Janeiro state in Brazil is the greatest feline zoonotic transmission epicentre, with more than 4,000 human and 4,000 feline diagnosed cases (8). Similar epidemics are also occurring in São Paulo and Rio Grande do Sul states, with a high prevalence of S. brasiliensis infections (5). Cats play an important role in the S. brasiliensis infection, due to their susceptibility to sporotrichosis and highly transmissibility to both felines and humans (9,10). Despite the increasing incidence of sporotrichosis, it is unclear which virulence traits are involved in the establishment, development, and severity of this disease. 1.1. Sporothrix-schenckii complex and sporotrichosis Sporotrichosis was firstly reported in 1898 by the medical student Benjamin Schenck (11), and nowadays is the world's most prevalent and dispersed subcutaneous mycosis (12), being able to affect both immunodeficient (13) and immunocompetent hosts (14). Although inhalation of conidia can be a source of infection, humans are usually infected when the fungi reach subcutaneous tissue through a skin trauma, either by contaminated plant material or through the bites or scratches of cats with sporotrichosis (6). It is a subacute or chronic infection, which promotes a skin lesion at the inoculation site, resulting in an ulcerated nodule. Through the lymphatic system, the infection eventually spreads to other host organs, causing a more severe and disseminated version of the disease (15). The sporotrichosis etiological agents belong to the Sporothrix schenckii complex (12), characterized by several ascomycetous thermally dimorphic fungi from the genus Sporothrix inside the Ophiostomatales order (16). As illustrated in Figure 1, they grow as cigar-shaped yeast-like cells at 37◦C or while infecting mammals and grow as sessile dematiaceous conidia along with hyaline sympodial conidia in its filamentous form at 25◦C or in the environment in substrates like living and decaying vegetation, animal excreta, and soil (2,5). Even though organisms that are part of the Ophiostomatales 3 order are normally associated with a plant or insect lifestyle, the Sporothrix genus exhibits pathogenicity to mammals. The mechanisms responsible for this are still poorly described (5). These fungi are abundant in soil, wood, and moss, reaching mammals through skin lesion inoculation, a situation common to gardeners and farmers (12,17). After traumatic inoculation, the fungi transition to the parasitic yeast form. How efficient this morphological transition occurs and consequently the fungi pathogenicity varies accordingly to the Sporothrix species (5,17,18). (A) (B) Figure 1. Thermal dimorphism morphology schematization and microscopic demonstration of Sporothrix brasiliensis. (A) Saprophytic morphology schematization and microscopic image. The scale bar equals 10μm. During the saprophytic (25-30°C) phase of Sporothrix , it develops septate hyaline hyphae with erect conidiophores. Two types of conidia are produced: the primary or sympodial conidia, with a hyaline thin wall, or the secondary or sessile conidia, with a thick wall of dark brown colour. (B) Parasitic morphology schematization and microscopic image. The scale bar equals 10μm. During the pathogenic/parasitic phase (35–37°C), morphology is characterized by oval or spherical yeasts (2–6µm), sometimes elongated in a cigar shape. Image adapted from Rodrigues et al., 2020. 4 Although sporotrichosis had been for an entire century exclusively attributed to the single species, Sporothrix schenckii , the development of advanced phylogenetic molecular techniques over the last decade allowed to address the role of other species such as S. brasiliensis , S. globosa , and S. luriei , S. mexicana , S. pallida and S. schenckii stricto sensu . This revolution highlighted significant differences in morphological, physiological, genetic, epidemiological, virulence traits and antifungal susceptibility among pathogenic Sporothrix spp (4,7,18–23). It elucidated that few of these Sporothrix species are successful mammal pathogens and therefore clinically relevant, such as S. schenckii, S. brasiliensis, S. globosa , and S. luriei (6,17,24). Since a species complex is defined as a monophyletic clade of species with equivalent clinical relevance, the S. schenckii complex term has been classified as no longer appropriate. There is currently a suggestion to adopt the terms ‘‘clinical clade’’ or ‘‘pathogenic clade’’ to refer to S. brasiliensis , S. schenckii , S. globosa and S. luriei , species often isolated from human and animal cases, while the remaining Sporothrix species would be nested in an ‘‘environmental clade,’’ due to their often association with the sapronoses form of sporotrichosis (1,3). Sporothrix spp. is strongly present in the genus Ophiostoma. Despite the members from this order are primarily associated with Protea or plant pathogens, Sporothrix spp. have never been observed as plant pathogens (25). They grow abundantly on dead wood, being omnipresent in the environment in dead wood, mosses, cornstalks, soil, and hay, and require a temperature of 22◦C–27◦C, 90% humidity, soil rich in cellulose and with a pH between 3.5 and 9.4 (25,26). Sporotrichosis is widely prevalent in warm-blooded animals and several mammals are prone to infection, such as humans, dogs, cats, rats, mice, chimpanzees, parrots, fishes, and dolphins (25,27). This large spectrum of hosts facilitates animal horizontal and zoonotic transmission of sporotrichosis (27). The route of infection varies with Sporothrix species. As illustrated in Figure 2, the species within Sporothrix environmental clade are generally associated with an environmental route of transmission, while the pathogenic clade , especially the S. brasiliensis species, is normally transmitted between infected animals where dissemination to humans can happen (1,5). The human disease has a broad range of clinical manifestations and can be classified into fixed cutaneous, lymphocutaneous, disseminated cutaneous, and extracutaneous sporotrichosis (28). Cats are relevant zoonotic transmitters of sporotrichosis in Brazil and usually may present three clinical forms upon infection: cutaneous, lymphocutaneous, and disseminated (9). The cutaneous form is the most common, and ulcers are the main clinical signs observed (8). However, respiratory, ophthalmic, osteoarticular and central nervous system sporotrichosis infection and hypersensitivity reactions have 5 also been reported (12,27). Although in humans host immunodeficiency is often associated with the worst clinical presentations (12), such correlation has not been proven in felines yet (29,30). Figure 2. Transmission routes in human and animal sporotrichosis. The purple route (animal horizontal transmission) illustrates S. brasiliensis association with large epizooties during animal horizontal transmission. Although less frequent, the same route applies to S. schenckii . The zoonoses route (red) represents sporotrichosis which can be transmitted to humans via animal deep scratching and biting, especially from infected cats. Lastly, the green route (sapronoses) is associated with S. schenckii and S. globosa infections. The probability of involvement (high, medium, or low) is proportional to the size of the species’ circumference in each transmission route. Image from Rodrigues et al., 2016. 1.2. Sporotrichosis epidemiological aspects Although human sporotrichosis is generally responsive to standardized antifungal strategies, this disease is now considered an emerging health problem (8). Since sporotrichosis is not a reportable 6 disease, its exact prevalence is unknown (25), whereas the incidence is mostly based on case report documentation (2). As illustrated in Figure 3 and 4, this mycosis has wide geographic distribution and the etiological agent and disease burden vary from country to country and geographic region (2). It is estimated that in most regions of the world, more than 80% of cases are caused by a predominant species (1). In Asia, S. globosa counts for 99,3% of sporotrichosis cases and is a common subcutaneous infection in Japan, China, India, and Malaysia (1). In Australia and South Africa, it is estimated that S. schenckii has a predominance of 94%, while in south-eastern South America, S. brasiliensis is the etiological agent in 88% of cases. The case reports from sporotrichosis in humans in Africa still refer to sapronotic transmission, with some sporadic cases of infection in animals, despite the HIV/AIDS epidemic (1,31). Lastly, in western South America, Central and North America S. schenckii is the most predominant (89%), while S. luriei and S. mexicana are restricted to Africa and Mexico, respectively. (10,12,25,28). The disease is less prevalent in Europe, except for the unique outbreak that occurred in France, at the beginning of the nineteenth century (10,32). Figure 3. Recent global distribution of the species from the pathogenic ( S. brasiliensis , S. schenckii and S. globosa ) and the environmental clade of the Sporothrix species. S schenckii has almost worldwide distribution, while S. brasiliensis is restricted to the South and Southeast of Brazil. S. globosa is found less frequently in the Americas and Europe but is an emerging species in Asia. Sporotrichosis caused by the environmental clade is less frequent and spread worldwide. Image adapted from Rodrigues et al., 2020. 7 Figure 4. Distribution and relative burden of sporotrichosis in the world. Image from Chakrabarti et al., 2015. Sporotrichosis is currently a hyperendemic disease in regions of countries like China, Mexico, Brazil, Peru, Venezuela, Colombia, and South Africa (1,10,12,27). For decades until the mid-1990s, feline sporotrichosis in Brazil appeared only as sporadic, self-limiting clusters. However, over the last decades in Brazil, the sporotrichosis epidemiological scenario changed drastically and became the most endemic country in the world (1,10). Clinical sporotrichosis in mammals results from two major infection routes: animal transmission and plant origin. Historically, sporotrichosis was a rare sapronoses (plant origin), caused by S. schenckii that present the classic and sporadic route of transmission by traumatic inoculation of the etiologic agent while handling organic matter, similar to the rest of the world. It was considered an occupational disease, occurring when workers are exposed to a high incidence of injuries with contaminated material (33). Nowadays, feline sporotrichosis caused by S. brasiliensis has led to a large epidemic with zoonotic transmission in Brazil, becoming the main etiological agent of feline and human sporotrichosis in the region (1,5,25,34). Cats are very susceptible to sporotrichosis, and through deep scratching and biting, the disease is highly transmissible to both felines and humans, as an epizooty (cat-cat) and zoonosis (cat-human), respectively. Feline sporotrichosis challenging treatment, combined with the population's poor socioeconomic background and scarce access to human and veterinary health services are important factors that can explain the cause of this ongoing epidemic in Brazil (20,24,25). Currently, cases of human sporotrichosis occur in 25 of the 26 Brazilian states. Zoonotic 8 sporotrichosis cases have been expanding rapidly toward the northeast regions of Brazil in recent years, which is directly linked to the feline sporotrichosis epizootic, as shown in Figure 5. This public health concern will likely worsen in the future since S. brasiliensis infection is projected to expand its biogeographic domains and host range and become more virulent (1). Rio de Janeiro state is the greatest feline zoonotic transmission epicentre, with more than 4,000 human and 4,000 feline cases diagnosed at Fundação Oswaldo Cruz between 1998 and 2012 (8). Similar epidemics are occurring in São Paulo and Rio Grande do Sul states, also with a high prevalence of S. brasiliensis infections (5). Urban areas with high feline population densities seem to be important drivers of epizootics S. brasiliensis transmission, and the epidemiological profile group usually is people with poor socioeconomic conditions with direct and frequent contact with these animals, namely children, the elderly, and women. However, outside urban areas, the classical sapronoses transmission type prevails (5,35). Figure 5. Geographic distribution of human and feline sporotrichosis in recent years in Brazil. (A) Cases of human sporotrichosis have been reported in 25 of 26 Brazilian states, with significant differences in their frequency. (B) The South and Southeast states of Brazil show the largest epizootic feline sporotrichosis in the world. Currently, the zoonotic sporotrichosis driven by S. brasiliensis is expanding rapidly in Northeast Brazil. Image from Rodrigues et al ., 2020. 15 CHAPTER 2 Aims and Outline 16 The mechanism involved in Sporothrix brasiliensis increased virulence and aggressiveness are yet to be clarified. Targeted fungal transformation is an important tool which permits the identification of molecular mechanisms and cellular processes involved directly in the virulence of the target microorganism, and thus, can clarify the role of gene functions in vitro and in vivo . The first step to achieve this goal is the development of an easy, quick, and reproducible methodology for transformation of S. brasiliensis. Secondly, the design and creation of strains with fluorescent proteins is an important technology which could facilitate the execution of in vitro and in vivo infection assays with S. brasiliensis . Therefore, our main objectives were to: 1. Establish a protocol and optimizations for an efficient Agrobacterium tumefaciens - mediated transformation (ATMT) of S. brasiliensis . 2. Design, create and categorize S. brasiliensis strains with fluorescent proteins. 3. Perform human monocyte-derived macrophages (MDMs) infections with fluorescent strains S. brasiliensis and evaluate its applicability. The work presented in this thesis aims to contribute for the development of molecular tools to unravel the virulence mechanisms of S. brasiliensis , as well as to create fluorescent strains and evaluate their applicability in in vitro models of infection. Chapter 1 presents an introduction to the state of art of sporotrichosis, its etiologic agents, the immune response to Sporothrix spp. and the ATMT technique. Firstly, a brief description of the disease is presented, its clinical forms, epidemiology, diagnosis, and treatment. Secondly, the virulence factors and the immune response to Sporothrix spp. are also described. Lastly, the state of art of the ATMT technique is introduced. Chapter 2 describes the aims of this dissertation and its outline. Chapter 3 focus on the establishment of an ATMT protocol to produce S. brasiliensis transformants. The effects of several parameters were evaluated on the transformation efficiency, such as, time, ratio bacteria:fungi, sterile membranes and strains used in the co-cultivation. To summarize, from the analysis of the tested conditions, we concluded that 26ºC for 72h, using a 2:1 ratio (bacteria:fungi), using the A. tumefaciens AGL-1 and S. brasiliensis ATCC 4823 are the best conditions for ATMT of S. brasiliensis . Overall, we were able to develop a protocol for transformation of S. brasiliensis using ATMT with high transformation rates and mitotic stability. In Chapter 4, S. brasiliensis pGAPDH::GFP and pGAPDH::H2A::GFP strains with fluorescent proteins in the cytoplasm and the nucleus, respectively, were used assay to assess several antifungal mechanisms. The pGAPDH::GFP strains still had a detectable amount of GFP fluorescence upon 17 phagocytosis and thus, can be used without fluorophore stain prior to infection. On the other hand, the loss of GFP fluorescence by the pGAPDH::H2A::GFP strain inside the macrophages preclude there use in downstream assays since we were unable to correlate the loss of GFP fluorescence of the pGAPDH::H2A::GFP strain with the loss of viability and/or death after monocytes/macrophages engulfment in both microscopic and FACS analysis. Chapter 5 presents a broad discussion and future perspectives regarding this thesis. 18 CHAPTER 3 Optimization of Agrobacterium tumefaciens -mediated transformation on Sporothrix brasiliensis 19 Abstract Sporothrix brasiliensis is the most virulent etiological agent that causes sporotrichosis, the most prevalent mycosis worldwide and a hyperendemic in Brazil. The virulence factors which result in the S. brasiliensis worst phenotype, when compared to the rest of the Sporothrix pathogenic clade, remain poorly understood. The development of genetic engineering techniques for S. brasiliensis transformation is urgently needed to better understand the association of specific genes and functions, unravelling this microorganism pathophysiology. In this work, we optimized the protocol for its genetic manipulation using the Agrobacterium tumefaciens -mediated transformation (ATMT) system. Several conditions already preestablished in other fungi were tested its effect on the ATMT efficiency. Our results point to 72h of cocultivation, the AGL-1 strain and the 2:1 ratio (bacteria:fungi) as ideal conditions for a high number of transformants. In these conditions, we obtained 3179 ± 1171 transformants/co-cultivation. No statistical differences were found between the Hybond™-C e Hybond™-N+ sterile membranes used. Additionally, the ATCC 4823 and 4858 S. brasiliensis strains produce more transformants. Only the co-cultivation temperature of 26°C was used. The ATMT methodology used is an efficient tool for mutagenesis in S. brasiliensis . 3.1. Introduction Sporotrichosis is the world's most prevalent and distributed subcutaneous mycosis. This disease etiological agents belong to the Sporothrix pathogenic clade (1,3), characterized by several thermodimorphic fungi from the genus Sporothrix growing as yeast in infects mammals (16). Among all species pathogenic to mammals, the most virulent and with the most impactful clinical manifestations is S. brasiliensis (18,48–51). However, there is yet a limited genetic manipulation tool repertoire for the Sporothrix genus (110), and despite the role of dimorphism, thermo-tolerance, and melanin, Sporothrix virulence factors are still not clearly understood (54). Agrobacterium tumefaciens -mediated transformation (ATMT) offers an efficient tool for random insertional mutagenesis. This bacterium can transform intact cells, such as conidia, mycelium and yeasts while still producing many mutants due to its non-sequence-specific manner of T-DNA integration within the host genome (98). Although ATMT has already been standardized for S. schenckii (98–100) , the information about the use of this technique in S. brasiliensis is scarce. ATMT technique has proven to be a useful genetic tool to unveil several pathogen pathophysiology mechanisms (50,99), thus an attempt to standardize this technique on Sporothrix brasiliensis is justifiable. 20 Here, we report the establishment of an ATMT system of S. brasiliensis , with an analysis into the important factors affecting the transformation efficiency. We report an efficient, simple, and reproducible transformation method which enabled us to obtain many T-DNA insertional mutants within a two-week experimental period. 3.2. Materials and Methods 3.2.1. Microorganisms and culture conditions The S. brasiliensis ATCC MYA-4823, ATCC MYA-4824, and HUPE 114158 were used for fungal transformation. Their origin is better described in Table 1. The ATCC MYA-4823 strain was cultured at 37°C for 72h in yeast extract peptone dextrose (YPD) medium (2% glucose, 2% peptone, 1% yeast extract; for solid medium 1.5% agar was added; pH = 7.8), while both the ATCC MYA-4824 and HUPE 114158 strains were cultured in Brain Heart Infusion Broth (BHI) medium (3.7% Biolife™ BHI; for solid medium 1.5% agar was added; pH = 7.8). All cultures were maintained at 4°C for short-term storage and were routinely sub-cultured every 4–6 weeks. Yeasts were obtained through sterile gaze filtration. Table 1. S. brasiliensis strains used in this work. Isolate Another name Isolation Source Citation Virulence Origin ATCC MYA-4823 5110 Brazil Feline skin lesion (48,54,109,111) High (48) Bought at ATCC© ATCC MYA-4824 IPEC 17943 Brazil Feline skin lesion (48,112) Low (48) Kindly donated by Lopes-Bezerra, L. M. HUPE 114158 Ss58 Brazil Human cutaneous with facial destruction (48,109) High (48) Kindly donated by Lopes-Bezerra, L. M. The A. tumefaciens strains AGL-1 (113,114), EHA105 (114), and LBA1100 (103,109), harboring the commercially available pUR5750 plasmid (Figure 6A) (96), were used to perform ATMT and evaluate its efficiency, due to this plasmid hygromycin B phosphotransferase (HPH) gene. All A. tumefaciens cells were cultured at 26°C in solid LC Broth medium supplemented with the antibiotics rifampicin (20μg/ml) and kanamycin (100μg/ml) and maintained at 4°C for short-term storage. The A. tumefaciens strains used were kanamycin-resistant and harboured two distinct plasmids, as schematized in Figure 6B. The first plasmid was the Ti plasmid, which carries the spectinomycin resistance gene along with virulence promoting genes. The phenolic compound acetosyringone (AS) induces the expression of the Vir domain inside the Ti plasmid, which leads to the generation, transference, and random integration of the T-DNA, present in the constructed plasmid, into the recipient cell’s genome (88,92,115). The binary vector used 21 was the plasmid pUR5750 (Figure 6B). It had the gene of resistance to kanamycin in A. tumefaciens and Escherichia coli , and the T-DNA constructed to insert a specific gene inside the host genome, in this case, the HPH gene. (C) (D) Figure 6. Schematic representation of the system used for A. tumefaciens-mediated transformation. (A) The A. tumefaciens carried two distinct plasmids and chromosomal DNA. Image adapted from Michielse et al., 2008. (B) The binary vector used was the plasmid pUR5750. (C) Schematic representation of the pPZP201BK::SUR::gpdA::Kat::TrpC plasmid. Image from Sbaraini et al., 2021. (D) Schematic representation of the pGAPDH::GFP plasmid. The second plasmid used, pPZP201BK::SUR::gpdA::Kat::TrpC (Figure 6C), was designed by Sbaraini (116) to produce transformants that express and accumulate the far-red fluorescent protein TURBOFP635/Katushka in the cytoplasm. This plasmid uses the selection marker Magnaporthe grisea acetolactate synthase enconding gene (SUR gene), which confers resistance to Chlorimuron ethyl (116,117). Using two selection markers allow the production of mutants with two fluorescent proteins upon consecutive ATMTs. The last plasmid used in the ATMT was the pPST608::pGAPDH::GFP (Figure 6D), to produce strains that express and accumulate the GFP in the cytoplasm, for the mitotic stability analysis. 22 3.2.2. Inoculum count optimization for future experiments in vitro Several protocols performed in this thesis required a quick and accurate way to obtain S. brasiliensis inoculums for experiments in vitro . To optimize the inoculum concentration, exponential growth cells were acquired through culture at 37°C for 24h in liquid YPD (pH 7.8) medium and sterile gaze filtration. Samples had their optic density (OD660nm) adjusted to several values ranging from 0.1 up to 1 and the number of cells was counted by both the Neubauer chamber counting technique and by counting the number of Colony Forming Unit (CFU) after 7 days of culture at 37°C in a YPD (pH 7.8) solid plates. Linear regression was performed to obtain the linear correlations between the concentration of exponential growth S. brasiliensis ATCC 4823 yeast cells, the Neubauer chamber count and the optic density (OD660nm). 3.2.3. Sporothrix brasiliensis sensitivity analysis to Hygromycin B and Chlorimuron ethyl Transformation systems can take advantage of the resistance markers, such as the HPH and SUR resistance genes (117,118), which confer resistance to Hygromycin B (HygB) and Chlorimuron ethyl (CE), respectively. To create mutants with these resistance markers, the minimum inhibitory concentrations of these antibiotics for S. brasiliensis ATCC 4823 were first evaluated. To analyze cell growth in selective YPD (pH 7.8) solid and liquid medium (100), exponential growth S. brasiliensis ATCC 4823 wild-type cells were acquired through culture at 37°C for 24h in liquid non-selective YPD (pH 7.8) medium and sterile gaze filtration. To analyze the resistance to Hygromycin B and Chlorimuron ethyl, cell growth was evaluated respectively in both liquid and solid medium of the selective YPD (pH 7.8) and Yeast Nitrogen Base (YNB) liquid medium (0.16% BD Difco™ YNB, 2% glucose, 0.5% ammonium sulfate, 25 µg/mL of leucine, 25 µg/mL of histidine, 25 µg/mL of methionine and 25 µg/mL of uracil) (100,116,117). Exponential growth S. brasiliensis ATCC 4823 wildtype cells were acquired through culture at 37°C for 24h in liquid nonselective YPD (pH 7.8) medium and sterile gaze filtration. Cell density was adjusted to an OD660nm of 0.1 by the Genesys™ 20 Spectrophotometer (Sigma-Aldrich, model Z376035) in both mediums. Fungal growth was evaluated by measuring absorbance at 660nm after incubation at 37°C for 24 hours with reciprocal shaking at 200rpm in several antibiotic concentrations. To define the minimum inhibitory concentration of HygB (Formedium™, Hunstanton, England) and CE (Santa Cruz Biotechnology, Dallas, USA) in S. brasiliensis in solid YPD and YNB mediums, cells were diluted to three concentrations: 108, 107, and 106. Those concentrations were then inoculated into 23 mini plates containing solid medium YPD (pH 7.8), with different concentrations (µg/mL) of each antibiotic. HygB plates had the concentrations of no antifungal present, 25, 50, 75, 100,150, 200, and 250µg/mL. The CE plates had concentrations of no antifungal present, 5, 10, 15, 20, 25, and 30µg/mL. For each mini plate, 5µL of each dilution was inoculated: 108 on the left border, 107 on the middle, and 106 on the right border. All the mini plates were put at 37°C for seven days, and a visual (qualitative) comparison was made at the end. 3.2.4. Agrobacterium tumefaciens -mediated transformation protocol of Sporothrix brasiliensis The transformation procedure was based on previously described protocols (92,98,99,109), with some modifications, and schematized in Figures 7 and 8. Figure 7. Schematic of S. brasiliensis ATMT technique. 24 Each A. tumefaciens strain cells were cultured at 28°C while shaking (200 rpm) in 10 mL LC broth supplemented with antibiotics for 6-10h. Subsequently, the culture was centrifuged at 4000 rpm for 6 min and the pelleted cells were resuspended with Induction Medium (IM) (92) with 200μM of acetosyringone (AS), to an optic density (OD660nm) of 0.3. The culture grew overnight at 26°C with shaking to an OD660nm of 0.6–0.8, to acquire virulence. In parallel, exponential growth fungal cells were cultured at 37°C for 24h while shaking (200 rpm) in 50mL YPD (pH 7.8), or BHI in the case of the ATCC 4858 strain, and yeast cells were obtained through sterile gaze filtration. Each A. tumefaciens strain was mixed with S. brasiliensis cells, at two different ratios. Inside the 1:1 ratio, 5 x 107 cells of each species were used, while the 2:1 ratio had double the amount of A. tumefaciens cells (1 x 108). Those mixtures were centrifuge and had their supernatant removed to an approximate final volume of 100μL, and were inoculated on a sterile membrane, onto solid IM plates. Four co-cultivation periods in the dark (24h, 48h, 72h, and 96h) and two different sterile membranes were compared (Hybond™-C and Hybond™-N+, 0.45μm pore, GE Healthcare Limited, Amersham, UK). Before incubation, the plates were air-dried in a safety cabinet with the lights off for approximately 30 minutes (103). Next, after each period of co-cultivation, the membranes were transferred to falcon tubes and the cells dislodged into a 5mL YPD medium (pH 7.8) containing cefotaxime (200μg/mL, Formedium™, Hunstanton, England), for growth inhibition of A. tumefaciens . Lastly, after 6h of cultivation at 37ºC, while shaking at 200 rpm, 1mL were centrifuged, 800μL of supernatant was removed and the final volume of 200μL was inoculated on a selective YPD solid medium (hygromycin B, 150 μg/mL, Formedium™, Hunstanton, England). Since the ATCC 4824 and 4858 strains did not grow properly in YPD, their cells had to be instead respectively inoculated in the selective BHI medium (with hygromycin B, 150 μg/mL, Formedium™, Hunstanton, England). Only to produce transformants with the plasmid pPZP201BK::SUR::gpdA::Kat::TrpC, the final volume of 200μL was inoculated on a selective Yeast Nitrogen Base (YNB) solid medium (0.16% BD Difco™ YNB, 2% glucose, 0.5% ammonium sulphate, 25 µg/mL of leucine, 25 µg/mL of histidine, 25 µg/mL of methionine and 25 µg/mL of uracil), with Chlorimuron ethyl (30μg/mL, Santa Cruz Biotechnology ™, Dallas, USA) instead of hygromycin. Fungal cells without the transforming agent A. tumefaciens were inoculated into selective and non-selective YPD mediums, as a positive and negative control, respectively. Lastly, after cultivation for a week at 37ºC, the number of transformants was counted, scaled to the total amount of selective medium used (5mL), and compared between each condition. To compare different conditions for the ATMT protocol, only the S. brasiliensis ATCC 4823 was used. 31 between the usage of Hybond™-N+ or Hybond™-C membrane for co-cultivation ( p =0,1824, Figure 13). The co-cultivation temperatures of 26°C showed more transformants ( p <0.0001) and increased biofilm production than 28°C (Figure 14). Hybond™-C Hybond™-N+ 0 500 1000 1500 2000 2500 3000 3500 4000 4500 Number of transformants ns Figure 13. Effects of co-cultivation with different sterile membranes in S. brasiliensis ATMT efficiency. Effect on the efficiency of S. brasiliensis ATMT between the use of Hybond™-C or Hybond™- N+ in co-cultivation. Bars depict the mean ± SEM number of transformants per condition (n≥5). No statistical difference was observed between the membranes by the student’s t-test (p=0,1496). (A) 26C 28C 0 50 100 150 1000 1500 2000 2500 3000 3500 Number of transformants ✱✱✱✱ (B) 26°C 28°C Figure 14. Effects of co-cultivation temperature in S. brasiliensis ATMT efficiency. (A) Effect of co-cultivation temperature in the ATMT efficiency. Bars depict the mean ± SEM number of transformants per condition (n≥10). Statistically significant data was determined by student’s t-test ( p <0,0001) (B) Filter pieces transferred to recover medium with different co-cultivation temperatures show different biofilm production. 32 Lastly, the efficiency of three different S. brasiliensis strains was compared (Figure 15). The S. brasiliensis strains ATCC 4823 and ATCC 4858 were able to grow rapidly both in solid and liquid mediums. However, the ATCC 4824 strain had a low growth, which difficulted the ATMT protocol, possibly impacting its efficiency. While the ATCC 4824 strain showed fewer transformants, both ATCC 4823 and ATCC4858 strains showed the same transformation efficiency ( p =0,6672, n≥6). All strains had different biofilm production after 72h of incubation at 26°C. (A) HUPE 114158 ATCC MYA-4823 ATCC MYA-4824 0 25 50 75 100 1000 2000 3000 4000 Number of transformants ns ✱✱✱✱ ✱✱✱ (B) ATCC 4823 ATCC 4824 ATCC 4858 Figure 15. ATMT efficiency using different S. brasiliensis strains. (A) The effect of the strain S. brasiliensis on transformation efficiency. Bars depict the mean number of transformants ± SEM per S. brasiliensis strain (n ≥ 6). Statistically significant data was determined by One-Way ANOVA by Tukey's multiple comparisons tests (*p<0.05). (B) Filter pieces were transferred to recover medium with different S. brasiliensis strains, showing different biofilm production. 3.3.4. Characterization of isolated transformants To investigate and confirm the integration of a single copy of the T-DNA into the S. brasiliensis genome after the ATMT, 5 transformants were randomly selected for DNA Extraction and subsequent qPCR analysis. DNA extraction samples ranged from 109 to 220,1ng/µL and were not contaminated. The copy numbers revealed for each of the genes closely correlated to the known copies in the genome sequence, thus confirming the validity of the described qPCR technique (Figure 16). Furthermore, transformants had a single copy of the HPH gene, thus confirming the success of the ATMT technique. 33 Sb_1 Sb_2 Sb_3 Sb_4 Sb_5 0.0 0.5 1.0 1.5 2.0 HPH copy number HPH / Actin- HPH / GAPDH Figure 16. HPH gene screening of S. brasiliensis transformants via HPH copy number analysis. T-DNA copy number of S. brasiliensis isolates, based on glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as a single-copy reference gene and hygromycin phosphotransferase (HPH) as a proxy for T-DNA insertion event Isolates resistant to Chlorimuron ethyl after transformation were characterized by microscopy by the presence of the far-red Kat-fluorescence protein, TURBOFP635/Katushka. The wild-type strain, ATCC MYA-4823, showed low levels of autofluorescence, while the transformed strain expressed a sharp and bright fluorescent signal (Figure 17). Bright Field GFP Katushka Merge (A) (B) Figure 17. Fluorescence microscopic analysis of randomly-selected transformant and wildtype S. brasiliensis cells. The scale bar equals 10μm for all images. (A) Yeast cells, subsequently transformed with the plasmids pPST608::pGAPDH::H2A::GFP and pPZP201BK::SUR::gpdA::Kat::TrpC, with high levels of GFP fluorescence in the nucleus and TURBOFP635/Katushka fluorescence in the cytoplasm. (B) Hyphae and conidia transformants cells, subsequently transformed with the plasmids pPST608::pGAPDH::H2A::GFP and pPZP201BK::SUR::gpdA::Kat::TrpC, presented as, with high levels of GFP fluorescence in the nucleus and TURBOFP635/Katushka fluorescence in the cytoplasm. 34 3.3.5. Mitotic stability characterization Transformants must be mitotically stable for an effective mutagenesis system. All five transformants presented high mitotic stability in all five generations. In five restreaks, after 12h of incubation the log (OD660nm) went, on average, from 0.1 to 0.362 ± 0.032 and the mean percentage of transformant cells that expressed GFP across all generations was 99,38% ± 0.45% (Figure 18). Overall, these results suggest that 26ºC for 72h of co-cultivation, using a 2:1 (bacteria:fungi) ratio is sufficient to promote the insertion of the T-DNA into the genome of S. brasiliensis cells yeast cells resulting in mitotically stable transformants. Figure 18: Mitotic stability measurement through GFP gene stability analysis. (A) Five randomly-selected transformant colonies were restreak five times in a non-selective medium. The data represent the means of log (OD660nm) ± SEM after five restreaks of 12h of incubation. µ and td stand for the specific growth rate of the microorganism and time required for cell duplication, respectively. (B) Percentage of transformant cells that expressed GFP across all five generations, each one with a twelvehour incubation period in a non-selective medium. Bars depict the mean of MFI ± SEM percentage of negative cells per restreak (n = 5). (C) Dot plot of side scatter (SSC-A LOG) versus green fluorescence 35 intensity (FITC-A), showing the higher MFI of GFP positive cells ( S. brasiliensis, transformed with pUR5750) when compared with wild-type cells. (D) Representative ( S. brasiliensis, transformed with pUR5750) dot plot of side scatter (SSC-A LOG) versus green fluorescence intensity (FITC-A) with a selective gate defined around the cellular subpopulation which is GFP positive. 3.4. Discussion and conclusion Sporotrichosis is nowadays the most prevalent and distributed subcutaneous mycosis worldwide. The most virulent etiological agent of this disease is S. brasiliensis , but the virulence mechanism behind it is still unclear (50). There is yet a limited genetic manipulation tool repertoire for the Sporothrix genus (110), hence the development of a system for its genetic manipulation was the main goal of this work. Agrobacterium tumefaciens -mediated transformation (ATMT) offers an efficient tool for random insertional mutagenesis and had already been performed in several different fungal species, including Sporothrix schenckii (89,92,98,103,116). Although ATMT has already been standardized for S. schenckii (98–100), besides Ferreiras’ work (109), the information about the use of this technique in S. brasiliensis is scarce (109). Therefore, we decided to develop and establish this methodology in S. brasiliensis . Several co-cultivation conditions already pre-established in other fungi were tested to evaluate their effect on S. brasiliensis ATMT efficiency, such as the effect of time, temperature, bacteria:fungi ratio, strains used, and type of sterile membrane used. All those variables can be of extreme importance for the transformation efficiency because they change the co-cultivation environment in which the transference of DNA from bacteria to fungal cells occurs (115). Concerning feasibility, some co-cultivation conditions had to be based on published studies. The bacteria:fungi mixture placed in membranes was air dried for 30 min in the flow chamber before incubation according to previous work (103). Moreover, it has been reported that T-DNA can be inserted not only into protoplasts but also into intact cells by ATMT (89), including S. schenckii cells (98–100), which avoided the laborious protoplasts preparation process. Additionally, yeast cells were used in the co-cultivation instead of conidia, since yeasts has being reported as easily transformed using the ATMT technique (115). The chromosomal background of the A. tumefaciens strain also plays an important role in the ATMT system efficiency. In addition to the function of the vir region, the recognition and binding of A. tumefaciens to the host surface also depends on the genes encoded in the bacterial genome (121). In addition to the A. tumefaciens strain, the ideal bacteria:fungi ratio depends on the transformation system and fungi species since the addition of both bacterial or fungal cells can decrease or enhance the ATMT 36 efficiency (115). Several experiments have proven those claims empirically, even with S. schenckii ATMT (92,98,116). The three A. tumefaciens strains used, LBA1100, EHA105 and AGL-1, harboring the plasmid pUR5750, showed different efficiencies during the ATMT procedure. However, there were no significant efficiency differences between strains in the 1:1 ratio of co-cultivation, only when the 2:1 ratio was applied. In this case, co-cultivation with AGL-1 strain showed more transformants (p<0.0001). This result is similar to Zhang’s work (98), indicating that the strain of A. tumefaciens used affects the efficiency of S. brasiliensis transformation, where AGL-1 is the more efficient one. Although EHA105 often showed more transformants than LBA1100 at the 2:1 ratio, it was not statistically significant. The co-cultivation time is a critical step in the transformation procedure, which varies according to Agrobacterium strains of and host species (122,123). Fewer transformants were observed with a 24h co-cultivation time ( p <0.0104), as reported before (98,116,120). The incubation periods of 48h and 72h showed more transformants. In the ATMT technique with thermos-dimorphic fungi, the cells can be mixed for co-cultivation above different membrane materials, such as cellophane and Hybond™-N+ (102,104,105,116,120). S. schenckii has already been transformed using both cellophane and Hybond™-N+ membranes (98,99), while the only transformation protocol for S. brasiliensis until this present work used the Hybond™-N+ membrane (109). No statistical differences were observed between the sterile membranes Hybond™-N+ and Hybond™-C. The temperature is often described to affect ATMT efficiency, and optimal temperatures frequently range between 20ºC and 28ºC (103). ATMT protocols using S. schenckii were reported using the co-cultivation temperature from 25°C to 28°C (98–100) and in Ferreira’s work (109), between 25ºC and 27ºC, the last one produced more S. brasiliensis transformants. The chosen temperatures to be tested were 26°C and 28°C, where the colder one produced more transformants and biofilm. Lastly, all S. brasiliensis strains available in the laboratory were used to compare ATMT efficiency. The S. brasiliensis strain ATCC MYA-4824 did not grow properly in YPD, BHI, or BD Sabouraud Glucose, which difficulted the implementation of the ATMT protocol, possibly impacting its efficiency. While the ATCC MYA-4824 strain showed few transformants, ATCC MYA-4823 and HUPE 114158 strains showed, on average, thousands of transformants, with no statistical difference between them. Our results point to 72h of co-cultivation at 26°C, the AGL-1 strain and the 2:1 ratio (bacteria:fungi) as ideal conditions for a high number of transformants. In these conditions, we obtained 3179 ± 1171 transformants/co-cultivation. Our PCR analysis showed that all HygBR clones harboured the HPH gene. 37 Transformants mitotic stability is usually measured by analysing the stability of resistance gene marker used to transform the fungi. Traditionally, randomly-selected transformants are successively cultured on plates without the selective marker for three to five generations, to then be plated again with selective medium. Mitotic stability is quantified by the percentage of transformants that survive and grow in this selective medium by maintaining the resistance gene inside its genome (99,116,117,124). Our approach relied in the same principle, where mitotic stability was measured by the persistence of the GFP gene inside the genome of the pGAPDH::GFP transformants. All the selected transformants were mitotically stable. In conclusion, the present work contributes to the implementation of an efficient ATMT protocol for the generation of a S. brasiliensis mutant library, a valuable asset to uncover the association between gene functions and virulence traits of this emerging and highly virulent pathogen. 38 CHAPTER 4 Macrophage infection and fungicidal assays with Sporothrix brasiliensis fluorescent strains 39 Abstract Inside the Sporothrix pathogenic clade, S. brasiliensis is the most virulent species. Since the discovery of autofluorescence proteins, strains expressing GFP have become a powerful tool, which can help better understand the pathophysiology of several pathogens, including S. brasiliensis . Strains expressing GFP in the nucleus (H2A::GFP) and the cytoplasm (pGAPDH::GFP) created in our laboratory were used to assess their applicability as tools for the evaluation of several in vitro assay antifungal mechanisms of immune cells. No statistical difference in cytokine production was observed between stimulation with wildtype or pGAPDH::GFP strain. The pGAPDH::GFP strain was used to infect both PBMCs and hMDMs for 2 hours, at the MOI of 1:5. In both cases, we were able to visualize a strong GFP fluorescence inside the monocytes/macrophages, in both microscopic and FACS analysis. These results demonstrate the applicability of the pGAPDH::GFP strain in infection experiments. On the other hand, the results obtained with pGAPDH::H2A::GFP S. brasiliensis strain were not promising. In all the fungicidal experiments performed, this strain failed to provide a binary correlation between the loss of nucleus GFP fluorescence and loss of fungal viability or fungal death. Further experiments are required and may provide a correlation between the percentage of mean GFP intensity lost and cell viability markers. Additionally, upon monocytes/macrophages' engulfment, the GFP fluorescence from this strain became undetectable by FACS and microscopy. 4.1. Introduction Sporotrichosis has become a public health concern as is the most prevalent subcutaneous mycosis worldwide (1). Within the Sporothrix pathogenic clade, the most virulent species with the most impactful clinical manifestations is S. brasiliensis (1,18,48–51,125). The limited repertoire of genetic manipulation tools for the Sporothrix genus limits the research in S. brasiliensis pathophysiology (54,110). Autofluorescent proteins, such as the green fluorescent protein (GFP), are convenient tools that assist experimentation and research with intact living cells and organisms in fields ranging from cell biology to biomedicine (126,127). Their use has already been reported on a broad spectrum of cells, such as protozoa, fungi, plants, animals, and viruses (128–132). Even though several methods to detect apoptosis are currently available, many are laborious process that often require additional use of dyes, specific substrates, enzymes, or antibodies. The use of GFP strain cells has already been used to detect the induction of apoptosis, necrosis and cytotoxicity, which can help avoid other work-intensive and resource-intensive screening procedures (133,134). 40 The exact role of immune cells in Sporothrix spp. infection is not yet fully elucidated, but macrophages are probably the most important immune cells for containing and terminating sporotrichosis (68,69) since their phagocytic activity plays a crucial role in surveillance and clearance of fungal pathogens (70,71). GFP-expressing strains have also been used to study the interaction of macrophages with bacteria and fungi (135–138). Although S. schenckii strains expressing GFP have already been produced (99), currently there is no report of S. brasiliensis strains expressing GFP, and their interaction with macrophages. 4.2. Materials and Methods 4.2.1. Ethics statement. The functional experiments involving cells isolated from the peripheral blood of healthy volunteers at Hospital of Braga, Portugal, were approved by the Ethics Subcommittee for Life and Health Sciences (SECVS) of the University of Minho, Portugal (no. 014/015). Experiments were conducted according to the principles expressed in the Declaration of Helsinki, and participants provided written informed consent. 4.2.2. Microorganism and cell culture conditions S. brasiliensis strains expressing GFP in the cytoplasm (pGAPDH::GFP, Figure 19A) and the nucleus (pGAPDH::H2A::GFP, Figure 19B) were designed and produced in the lab by collaborators (Table 3). To that end, plasmid DNA extraction, recombinant DNA manipulation and E. coli and A. tumefaciens transformation procedures were performed as reported elsewhere (139,140). S. brasiliensis strains expressing GFP were produced through the ATMT protocol established in the previous chapter. All S. brasiliensis strains used in this chapter were cultured at 37°C in a selective YPD (pH = 7.8) medium with HygB (150 µg/mL), thus ensuring the presence of the GFP gene. All cultures were maintained at 4°C for short-term storage and were routinely sub-cultured every 4–6 weeks. Exponential growth yeast cells were obtained through sterile gaze filtration after a 24h incubation at 37°C in a selective YPD medium (pH = 7.8). Table 3. Table explaining the details from both S. brasiliensis GFP strains used in this thesis. Mutant name Fluorescence Promotor Region of expression Origin pGAPDH::GFP GFP pGAPDH Nucleus Figure 19a pGAPDP::H2A::GFP GFP pGAPDH Cytoplasm Figure 19b 47 To perform the microscopic phagocytosis analysis, plates had their supernatant removed, were washed with PBS and each well had the number of internalized fungi inside macrophages counted using the Olympus Widefield Upright Microscope BX61, by either bright-field (upper panel) or fluorescent microscopy (lower panels). All images were captured using 395nm/509nm for excitation and emission, respectively, and exposition time to the laser beam was automatically set. Images were treated in the ImageJ© v1.8 software (https://imagej.nih.gov/ij/). The percentage of phagocytosis was measured based on the GFP fluorescence of the pGAPDH::GFP strain and according to this subsequent formula: 𝐏𝐡𝐚𝐠𝐨𝐜𝐲𝐭𝐨𝐬𝐢𝐬 %=(number of Monocytes/macrophages with internalized yeast number of Monocytes/Macrophages in the same field )X 100% An additional step was performed in the infection protocol of macrophages. To measure the number of adhered yeast to the macrophage cell membrane, each well was stained with calcofluor (CFW, 0.1mg/mL, Sigma-Aldrich, 10 minutes at RT) before the infection period. Lastly, the percentage of adhered yeast cells was compared to the total amount of macrophages, by FACS and microscopy. 4.2.6. Statistical analysis Data from the evaluation of phagocytosis indexes and fungicidal activities were obtained on a BD FACS LSRII instrument (B.D. Biosciences) and an Olympus Widefield Upright Microscope BX61. Data were processed using FlowJo© v.10 software (Tree Star Inc., https://www.flowjo.com/) and ImageJ© v.1.8 software (https://imagej.nih.gov/ij/). Data are reported as the mean ± standard error of the mean (SEM) of at least two independent assays with two or three replicates. The statistical analysis was performed using GraphPad Prism Software version 8.0 (GraphPad Software Inc, USA, https://www.graphpad.com/scientific-software/prism/). The normality assumptions were assessed in all cases using the Shapiro-Wilk test. Comparing phagocytosis indexes and the percentage of dead cells upon fungicidal experiments were made by either One-Way ANOVA or Two-Way ANOVA. Statistically significant values are indicated as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001. 4.3. Results 4.3.1. Analysis of the correlation between loss of GFP fluorescence and cell death Microscopic analysis showed that, upon phagocytosis, the pGAPDH::H2A::GFP fluorescence intensity was not strong enough to be distinguish from the macrophages autofluorescence (data not shown). Additionally, after 1 hour of phagocytosis and 2 hours of fungicidal activity, three cell death 48 parameters were compared (Figure 22). Even though 73.24 ± 7.27% of the cells still had their GFP fluorescence, only 45.11 ±12.38% of them were alive (PI-negative). Additionally, from an inoculum containing 4,625 x 104 yeast cells, only 23.96 ± 2.154% of cells appeared as CFU in the YPD plates after the fungicidal procedure, despite 73.24± 7.27% of them still had a GFP-fluorescence above the wildtype autofluorescence threshold. These results demonstrated that the loss of cell viability or cell death does not correlate with the loss of GFP fluorescence while using the pGAPDH::H2A::GFP S. brasiliensis strain. Further fungicidal assays were performed to evaluate this hypothesis. CFU FITC+ PI0 20 40 60 80 100 Cell Survival (%) ✱✱✱ ✱✱✱✱ ✱✱✱✱ Figure 22: Analysis of the correlation between loss of GFP fluorescence and cell death by Macrophagefungicidal assay. The PI-negative bar depicts the percentage of cell survival ± SEM (n=7). The FITC positive bar shows the percentage of cells ± SEM which express GFP fluorescence (n=7). The CFU bar represents the percentage of CFU ± SEM present in the plated YPD medium (n=7). Statistically significant data was determined by One-Way ANOVA by Tukey's multiple comparisons test. As shown in Figure 23, in fungicidal assays with ultraviolet radiation and incubation with high temperatures, the promotion of cell death once again did not correlate with the loss of GFP fluorescence by pGAPDH::H2A::GFP S. brasiliensis strain. When those cells were submitted to the incubation temperatures of 50 and 65°C and ultraviolet radiation, although no CFU was present in the YPD plates, meaning cells were no longer viable, at least 95% of those cells still had the GFP fluorescence beyond the wild-type autofluorescence threshold. Even after incubation of 30 minutes with the higher temperatures 49 of 75 and 85°C, at least 11 and 7% of cells respectively still emitted a GFP fluorescence intensity above the wild-type autofluorescence threshold while no CFU was present in the YPD plates. 0 20 40 60 80 100 Number of cells (%) 0 10 20 30 120 180 Time (min) 50C 65C 75C 85C 0 20 40 60 80 100 Number of cells (%) (A) 0 20 40 60 80 100 Number of cells (%) 0 60 120 180 240 Ultraviolet Time (min) 0 20 40 60 80 100 Number of cells (%) (B) Figure 23: Analysis of the correlation between loss of GFP fluorescence and cell death by Heat-fungicidal assay. The pGAPDH::H2A::GFP S. brasiliensis strain was used in all assays. (A) Analysis of the correlation between loss of GFP fluorescence and cell death by several heat fungicidal assays. Bars depict the percentage of yeast cells present in the plated YPD medium before (white bar, at the left y axis) and after (black bars, at the right y axis) 30 and 180 minutes of incubation (n=4). Dots represent the percentage of cells that expressed GFP fluorescence throughout the incubation periods, for each incubation temperature (n=4). (B) Analysis of the correlation between loss of GFP fluorescence and cell 50 death by Ultraviolet-killing. Bars depict the percentage of cells present in the plated YPD medium before (white) and after (black) the U.V. radiation (n≥4). Green dots represent the percentage of cells that expressed GFP fluorescence throughout the same period (n≥4). 4.3.2. Cytokines measurement The production of pro and anti-inflammatory cytokines in human PBMCs were evaluated after 24h of exposure to yeasts of wild-type and pGAPDH::GFP S. brasiliensis cells. As demonstrated in Figure 24A, B and C, the levels of TNF, IL-6 and IL-10 was higher in stimulated wells (p≤0.05), regardless of the fungal strains used, wildtype or pGAPDH::GFP. No statistical difference in cytokine production was observed between stimulation with wildtype or pGAPDH::GFP yeast cells in those three cytokines. No stimuli Wildtype pGAPDH::GFP 0 100 200 3000 6000 9000 12000 TNF- pg/mL ✱✱✱✱ ✱✱✱✱ No stimuli Wildtype pGAPDH::GFP 0 100 200 2000 4000 6000 8000 10000 12000 IL6 pg/mL ✱ ✱ No stimuli Wildtype pGAPDH::GFP 0 5 10 15 20 25 30 35 IL-10 pg/mL ✱✱ ✱ (A) (B) (C) Figure 24. Stimulation of PBMCs with S. brasiliensis wild-type and pGAPDH::GFP cells results in high production of cytokines. Bars depict the concentration of TNF-α (A), IL-6 (B) and IL-10 (C) after 20h of stimulation with wild-type (black) and pGAPDH::GFP (green) cells. The data were expressed as mean ± SEM. Statistically significant data was determined by One-Way ANOVA by Tukey's multiple comparisons test (n≥3). 4.3.3. Phagocytosis analysis using the pGAPDH::GFP strain The GFP fluorescence of the pGAPDH::GFP strain was used as parameter to measure the percentage of phagocytosis in the infection assays with PBMCs and hMDMs. Infection occurred for two hours in a MOI of 1:5. Analysing by FACS, the percentage of phagocytosis with CD14+ PBMCs was 56.18 ± 1.78%, and 61.48 ± 4.32% with hMDM. Analysing by microscopy, the percentage of phagocytosis of PBMCs (CD14+) and hMDM were, respectively, 54.83 ± 2.20 and 64.25 ± 4.07. No statistical difference was observed between the percentage of phagocytosis (n≥6, Two-Way ANOVA by Tukey's multiple 51 comparisons test), as depicted in Figure 25A. Furthermore, pGAPDH::GFP S. brasiliensis cells exhibited a strong GFP autofluorescence upon phagocytes engulfment (Figure 25B, C). Only in the hMDM infection protocol, calcofluor-white was used post-infection to stain the fungal cell adhered to the macrophages’ cell membrane. In that case, the percentage yeast cells adhered was 9.34 ± 2.42% and 6.39 ± 1.90%, according to the FACS and microscopic analysis, respectively (Figure 25D). Figure 25C illustrates the blue layer (CFW staining) overlaying with the FITC layer, revealing the nonphagocytosed but adhered yeast cells. (A) Microscopy FACS Microscopy FACS 0 20 40 60 80 100 Phagocytosis (%) PBMCs (CD14+) hMDMs ns (D) 0 5 10 15 20 30 40 50 60 70 80 90 100 Phagocytosis (%) Phagocyte d Adhered FACS Microscopy ns ns (B) (C) Figure 25. Phagocytosis assay analysis using S. brasiliensis pGAPDH::GFP strain to infect PBMCs and hMDMs. (A) Comparison of phagocytosis between human monocytes and macrophages of S. brasiliensis pGAPDH::GFP yeast cells at 52 the MOI of 1:5. Both FACS and microscopic techniques to count phagocytosis were compared. Bars depict the percentage of phagocytosis ± SEM (n≥6). No statistical difference was observed. (B) The green layer and bright fields were merged to visualize GFP fluorescence of the pGAPDH::GFP strain inside cd14+ PBMCs. (C) Blue layer (CFW staining) overlaid with the FITC layer revealing the difference between phagocytosed and non-phagocytosed but adhered yeats. (D) Percentage of phagocytosis obtained by infecting hMDM at an MOI of 1:5 for 2 hours. The green bars depict the mean percentage ± SEM of internalized/adhered GFP-expressing yeast cells, while the blue bars show the mean percentage ± SEM of adhered cells within the GFP-positive macrophages, relative to the total macrophage population. Internalization and adherence percentages are compared between from FACS and microscopy analyses. Scale bars equal 50 μm for all images. 4.4. Discussion and conclusion Several fungicidal assays evaluated the loss of cell viability or cell death with the loss of GFP fluorescence of the pGAPDH::H2A::GFP S. brasiliensis cells (nucleus GFP expression). Firstly, the fungicidal assay performed with hMDMs failed make this correlation (Figure 22), thus requiring further inquiry. With that in mind, fungicidal experiments by incubation high temperatures were realized and failed to demonstrate a correlation between the loss of GFP fluorescence and cell viability (Figure 23). Despite all of them having no cell viability after the incubation period at 50 and 65°C, the GFP fluorescence was still present. Only in the higher temperatures of 75 and 85°C, which are known to cause GFP denaturation (141), the GFP fluorescence decayed. Since the DNA is main molecule damaged by ultraviolet radiation (142,143), pGAPDH::H2A::GFP cells were submitted to this radiation in a fungicidal assay, to confirm if the MFI decayed due to the protein denaturation instead of the loss of cell viability. The results were like the fungicidal assays by temperatures of 50 and 65°C, meaning cell viability was completely impaired while GFP still emitted fluorescence. These results corroborate with authors who claim the GFP may not show a significant difference between live and apoptotic populations, thus requiring a multiparametric assay for a more precise interpretation of cell viability (144). Perhaps, a thorough analysis could correlate the percentage of mean GFP intensity lost with cell viability markers (133,134), instead of the binary approach used in the gating strategy of the FACS in this thesis. Nevertheless, the GFP expression of pGAPDH::H2A::GFP strain was not intense enough to be distinguished from the macrophages/monocytes autofluorescence, which makes both the accounting of the phagocytosis index and the binary analysis of cell death not viable for this strain. S. brasiliensis pGAPDH::GFP yeasts (cytosolic GFP expression), were used to analyse phagocytosis. However, there is a small change the random insertion site of the Ti DNA inside the genome can alter the microorganism virulence and immunological interaction with the host (145). To evaluate that, the production of cytokines in human PBMCs were compared after 24h of exposure to yeasts of 53 wild-type and pGAPDH::GFP S. brasiliensis cells. As demonstrated in Figure 23, no statistical difference in cytokine production was observed between stimulation with wildtype or pGAPDH::GFP strain, supporting the conclusion that this GFP tag strain is not immunologically inert and therefore, can be used in immunological assays. Monocytes can differentiate into either macrophages or dendritic cells, whose three main functions are phagocytosis, antigen presentation and cytokine production. (146). These cell types, together with neutrophils and mast cells are recognized to function as professional phagocytes (147). S. brasiliensis pGAPDH::GFP yeasts (cytosolic GFP expression), were used to analyse phagocytosis and, like other studies, the GFP fluorescence was intense enough to be visible after phagocytosis both in the microscopic and FACS analysis (138,148–150). Few articles have reported the phagocytosis index while using S. brasiliensis . Ferreira's work (109) obtained percentages of phagocytosis ranging from 70 to 90% while using the MOI (hMDM:fungi) of 1:3 and an incubation period of 2 hours. Our experiments used the MOI of 1:5 and found a lower phagocytosis percentage of 64.25 ± 9.96%. In Rossato’s experiments (77,78), when using BMDMs at 3h of incubation and an MOI of 1:5, the phagocytosis index was on average 70%, but the method used to count the inoculum was not clarified. These differences in the phagocytosis index might be caused by divergent Neubauer chamber counting methodologies. In the Neubauer chamber, the strategy for our work was to count each cell individually, despite any cell cluster. Since another possible methodology is to consider cell clusters as single counts, the resulting divergent inoculum concentration can difficult comparison between phagocytosis indexes. Concluding, from the two strains tested, only the pGAPDH::GFP S. brasiliensis strain proved to be useful in infection assays of monocytes/macrophages. Its cytoplasm GFP expression allowed a strong fluorescence visualization inside monocytes/macrophages, in both microscopic and FACS analysis. The pGAPDH::H2A::GFP S. brasiliensis strain, however, failed to provide a correlation between loss of cell viability or cell death and loss of GFP fluorescence, and its GFP fluorescence was not visible upon monocytes/macrophages engulfment. 54 CHAPTER 5 Discussion and Future prospects 55 Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has become a public health concern that will likely worsen in the future (Rodrigues et al., 2020). The etiologic agents of this disease are fungi often present in the pathogenic clade of the Sporothrix genus, inside the Ophiostomatales order, but some cases can be caused by the environmental clade, associated with the sapronoses form of sporotrichosis (1,3,16). Among the Sporothrix genus, S. brasiliensis is reported the most virulent, exhibiting the worst clinical manifestations (4,5). Infection mechanisms and virulence factors of Sporothrix organisms are not completely understood, while antifungal resistance reports and zoonoses cases are rising (7,151). For decades until the mid-1990s, feline sporotrichosis in Brazil appeared only as sporadic, self-limiting clusters. However, over the last decades in Brazil, the sporotrichosis epidemiological scenario changed drastically and became the most endemic country in the world (Rodrigues et al., 2020; Y Zhang et al., 2015). Rio de Janeiro state in is the greatest feline zoonotic transmission epicentre, with more than 4,000 human and 4,000 feline diagnosed cases (Isabella D F Gremião et al., 2015). Similar epidemics also are occurring in São Paulo and Rio Grande do Sul states, with a high prevalence of S. brasiliensis infections (Rodrigues et al., 2016). Cats play an important role in the S. brasiliensis infection, due to their susceptibility to sporotrichosis and highly transmissibility to both felines and humans (Lloret et al., 2013; Y Zhang et al., 2015). Despite the increasing incidence of sporotrichosis, it is unclear which virulence traits are involved in the establishment, development, and severity of this disease. Therefore, in the long term we aim to contribute for the better understanding of this mycosis and S. brasiliensis virulence mechanisms. A widely used methodology to unravel virulence mechanisms for organism is the generation of mutant libraries that allow posterior studies of genes function (152). The genetic manipulation of thermodimorphic fungi is challenging due to the occurrence of non-legitimate recombination. However, genetic manipulation of thermodimorphic fungi have already been stablished through successful ATMT protocols, including Sporothrix spp. (98,103,109,153). Part of our work is an extension of Ferreira’s thesis (109), further optimizing the ATMT protocol for the S. brasiliensis species (Chapter 3), which can provide a mutant library to help uncover associations between gene functions and virulence traits of this emerging and highly virulent pathogen. We managed to produce mutants using three different S. brasiliensis strains. The ideal co-cultivation conditions for maximum transformation rates were 72h of cocultivation at 26°C while using the AGL-1 strain at ratio of 2 bacteria to 1 yeast cell. In these conditions, we obtained 3179 ± 1171 transformants/co-cultivation. The exact role of immune cells in Sporothrix infection is not yet fully elucidated. Still, macrophages are probably the most important immune cells for containing and terminating sporotrichosis (68,69) since 56 phagocytic activity plays a crucial role in the surveillance and clearance of fungal pathogens (70,71). With optimized ATMT protocol for S. brasiliensis , our group in the laboratory managed to produce strains that express GFP in the nucleus (pGAPDH::H2A::GFP) and the cytoplasm (GAPDH::GFP), to perform several fungicidal and phagocytosis experiments, respectively (Chapter 4). Phagocytosis protocols were inspired by Ferreira’s in vitro models (109). Our results using the pGAPDH::H2A::GFP S. brasiliensis strain in cell death assays, such as ultraviolet radiation, incubation with high temperatures, and macrophage-fungicidal, showed no correlation between the loss of cell viability and the loss of GFP fluorescence, as reported before (144). However, some authors managed to correlate the percentage mean GFP intensity lost with cell viability markers instead of the binary approach used in the gating strategy of the FACS in this thesis (133,134). Perhaps, future experiments further correlating pGAPDH::H2A::GFP MFI-loss with cell viability markers could also show the same result. Furthermore, upon phagocyte’s engulfment, the GFP intensity of the pGAPDH::H2A::GFP cells became indistinguishable from macrophages/monocytes autofluorescence. These results make both phagocytosis and fungal death count not viable for this strain without the use of external stains. The GAPDH::GFP mutant proved to be a helpful tool for analysing phagocytosis in vitro . Like other studies with several microorganisms, the GFP expression from this mutant was intense enough to be visible after phagocytosis, both during the microscopic and FACS analysis (138,148–150). The phagocytosis index was, on average, 59.13 ± 7.01, a bit lower than Ferreira’s work (109), which divergent Neubauer chamber counting methodologies might cause. In conclusion, the ATMT protocol for S. brasiliensis was further optimized, allowing the creation of several mutants expressing fluorescent proteins. From these, two mutants were tested. The GAPDH::GFP S. brasiliensis mutant proved to be useful in infection assays of monocytes/macrophages. Its cytoplasm GFP expression allowed a strong fluorescence visualization inside monocytes/macrophages, visible in microscopic and FACS analyses. The pGAPDH::H2A::GFP S. brasiliensis mutant failed to provide a correlation between loss of cell viability or cell death and loss of GFP fluorescence, and its GFP fluorescence was not visible upon monocytes/macrophages engulfment. In the future, we intend to further transform fluorescent tag strains with mutations in their genome that could influence S. brasiliensis virulence. Our group has recently performed a dual RNA-sequencing on macrophages infected with S. brasiliensis and S. schenckii aiming to identify potential virulence factors that provide a competitive advantage to S. brasiliensis . This approach has revealed a specific transcriptome signature of S. brasiliensis absent in S. schenckii , where sialidase-1 was identified on the 63 https://doi.org/10.1146/annurev.immunol.17.1.593 71. Lee WL, Harrison RE, Grinstein S. Phagocytosis by neutrophils. Microbes Infect. 2003 Nov;5(14):1299–306. 72. Jain N, Moeller J, Vogel V. Mechanobiology of Macrophages: How Physical Factors Coregulate Macrophage Plasticity and Phagocytosis. Annu Rev Biomed Eng. 2019 Jun;21:267–97. 73. Meier A, Kirschning CJ, Nikolaus T, Wagner H, Heesemann J, Ebel F. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell Microbiol. 2003 Aug;5(8):561–70. 74. Netea MG, Sutmuller R, Hermann C, Van der Graaf CAA, Van der Meer JWM, van Krieken JH, et al. Toll-like receptor 2 suppresses immunity against Candida albicans through induction of IL-10 and regulatory T cells. J Immunol. 2004 Mar;172(6):3712–8. 75. Negrini T de C, Ferreira LS, Alegranci P, Arthur RA, Sundfeld PP, Maia DCG, et al. Role of TLR-2 and fungal surface antigens on innate immune response against Sporothrix schenckii. Immunol Invest. 2013;42(1):36–48. 76. Hoving JC. Pneumocystis and interactions with host immune receptors. PLoS Pathog. 2018 Feb;14(2):e1006807. 77. Rossato L, Santos SS Dos, Ferreira LG, de Almeida SR. The importance of Toll-like receptor 4 during experimental Sporothrix brasiliensis infection. Med Mycol. 2019 Jun;57(4):489–95. 78. Rossato L, Silvana Dos Santos S, Ferreira LG, Rogério de Almeida S. The impact of the absence of Toll-like receptor-2 during Sporothrix brasiliensis infection. J Med Microbiol. 2019 Jan;68(1):87–94. 79. Bessa J, Bachmann MF. T Cell-dependent and -Independent IgA Responses: Role of TLR Signalling. Immunol Invest [Internet]. 2010 Jan 1;39(4–5):407–28. Available from: https://doi.org/10.3109/08820131003663357 80. Romani L. Immunity to fungal infections. Nat Rev Immunol. 2011 Apr;11(4):275–88. 81. Nascimento RC, Almeida SR. Humoral immune response against soluble and fractionate antigens in experimental sporotrichosis. FEMS Immunol Med Microbiol. 2005;43(2):241–7. 82. Carlos IZ, Sgarbi DBG, Santos GC, Placeres MCP. Sporothrix schenckii lipid inhibits macrophage phagocytosis: involvement of nitric oxide and tumour necrosis factor-alpha. Scand J Immunol. 2003 Mar;57(3):214–20. 83. de Almeida JRF, Kaihami GH, Jannuzzi GP, de Almeida SR. Therapeutic vaccine using a monoclonal antibody against a 70-kDa glycoprotein in mice infected with highly virulent Sporothrix schenckii and Sporothrix brasiliensis. Med Mycol. 2015 Jan;53(1):42–50. 84. Maia DCG, Gonçalves AC, Ferreira LS, Manente FA, Portuondo DL, Vellosa JCR, et al. Response of Cytokines and Hydrogen Peroxide to Sporothrix schenckii Exoantigen in Systemic Experimental Infection. Mycopathologia. 2016 Apr;181(3–4):207–15. 85. Villalobos-Duno HL, Barreto LA, Alvarez-Aular Á, Mora-Montes HM, Lozoya-Pérez NE, Franco B, et al. Comparison of Cell Wall Polysaccharide Composition and Structure Between Strains of Sporothrix schenckii and Sporothrix brasiliensis. Front Microbiol. 2021;12:726958. 86. Guzman-Beltran S, Perez-Torres A, Coronel-Cruz C, Torres-Guerrero H. Phagocytic receptors on macrophages distinguish between different Sporothrix schenckii morphotypes. Microbes Infect. 2012 Oct;14(12):1093–101. 87. Dale J (Jeremy W., Schantz M von., Plant N. From genes to genomes : concepts and applications of DNA technology [Internet]. 3rd Edition. Wiley-Blackwell; 2012 [cited 2022 Aug 19]. 386 p. Available from: https://www.wiley.com/enus/From+Genes+to+Genomes%3A+Concepts+and+Applications+of+DNA+Technology%2C+3rd+ Edition-p-9780470683866 88. Li D, Tang Y, Lin J, Cai W. Methods for genetic transformation of filamentous fungi. Microb Cell 64 Fact [Internet]. 2017;16(1):168. Available from: https://doi.org/10.1186/s12934-017-0785-7 89. dos Reis MC, Pelegrinelli Fungaro MH, Delgado Duarte RT, Furlaneto L, Furlaneto MC. Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana. J Microbiol Methods. 2004 Aug;58(2):197–202. 90. Smith EF, Townsend CO. A Plant-Tumor of Bacterial Origin. Science (80- ) [Internet]. 1907 Apr 26;25(643):671–3. Available from: https://doi.org/10.1126/science.25.643.671 91. De Cleene M, De Ley J. The host range of crown gall. Vol. 42, The Botanical Review. 1976. p. 389–466. 92. Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ. Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc. 2008;3(10):1671–8. 93. Newell CA. Plant transformation technology. Developments and applications. Mol Biotechnol. 2000 Sep;16(1):53–65. 94. Lacroix B, Tzfira T, Vainstein A, Citovsky V. A case of promiscuity: Agrobacterium’s endless hunt for new partners. Trends Genet. 2006 Jan;22(1):29–37. 95. Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJ. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 1995 Jul;14(13):3206–14. 96. de Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM. Agrobacterium tumefaciensmediated transformation of filamentous fungi. Nat Biotechnol [Internet]. 1998;16(9):839–42. Available from: https://doi.org/10.1038/nbt0998-839 97. Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V. Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci U S A. 2001 Feb;98(4):1871–6. 98. Zhang Y, Li G, He D, Yu B, Yokoyama K, Wang L. Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens. J Microbiol Methods [Internet]. 2011;84(3):418–22. Available from: http://dx.doi.org/10.1016/j.mimet.2011.01.017 99. Lozoya-Pérez NE, Casas-Flores S, Martínez-Álvarez JA, López-Ramírez LA, Lopes-Bezerra LM, Franco B, et al. Generation of Sporothrix schenckii mutants expressing the green fluorescent protein suitable for the study of host-fungus interactions. Fungal Biol [Internet]. 2018;122(10):1023–30. Available from: https://doi.org/10.1016/j.funbio.2018.07.004 100. Tamez-Castrellón AK, Romo-Lucio R, Martínez-Duncker I, Mora-Montes HM. Generation of a synthetic binary plasmid that confers resistance to nourseothricin for genetic engineering of Sporothrix schenckii. Plasmid [Internet]. 2018;100:1–5. Available from: https://doi.org/10.1016/j.plasmid.2018.09.006 101. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 2003 Aug;301(5633):653–7. 102. Sugui JA, Chang YC, Kwon-Chung KJ. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol. 2005 Apr;71(4):1798–802. 103. Almeida AJ, Carmona JA, Cunha C, Carvalho A, Rappleye CA, Goldman WE, et al. Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet Biol. 2007 Dec;44(12):1387–98. 104. Fernandes FF, Oliveira AF, Landgraf TN, Cunha C, Carvalho A, Vendruscolo PE, et al. Impact of Paracoccin Gene Silencing on Paracoccidioides brasiliensis Virulence. MBio. 2017 Jul;8(4). 105. Menino JF, Almeida AJ, Rodrigues F. Gene Knockdown in Paracoccidioides brasiliensis Using Antisense RNA BT - Host-Fungus Interactions: Methods and Protocols. In: Brand AC, MacCallum DM, editors. Totowa, NJ: Humana Press; 2012. p. 187–98. Available from: https://doi.org/10.1007/978-1-61779-539-8_12 106. Wang J-Y, Li H-Y. Agrobacterium tumefaciens-mediated genetic transformation of the 65 phytopathogenic fungus Penicillium digitatum. J Zhejiang Univ Sci B. 2008 Oct;9(10):823–8. 107. Zemska O, Rappleye CA. Agrobacterium-mediated insertional mutagenesis in Histoplasma capsulatum. Methods Mol Biol. 2012;845:51–66. 108. Gauthier G, Sullivan T, Gallardo S, Brandhorst T, Wymelenberg A, Cuomo C, et al. SREB, a GATA Transcription Factor That Directs Disparate Fates in Blastomyces dermatitidis Including Morphogenesis and Siderophore Biosynthesis. PLoS Pathog. 2010 Apr 1;6:e1000846. 109. Ferreira BH. Sporothrix brasiliensis – From the genome to the phenotype. University of Minho; 2018. 110. Mora-Montes HM, da Silva Dantas A, Trujillo-Esquivel E, de Souza Baptista AR, Lopes-Bezerra LM. Current progress in the biology of members of the Sporothrix schenckii complex following the genomic era. FEMS Yeast Res. 2015;15(6):1–10. 111. Ferreira BH, Ramírez-Prado JH, Neves GWP, Torrado E, Sampaio P, Felipe MSS, et al. Ploidy determination in the pathogenic fungus Sporothrix spp. Front Microbiol. 2019;10(FEB). 112. Martínez-Álvarez JA, García-Carnero LC, Kubitschek-Barreira PH, Lozoya-Pérez NE, BelmonteVázquez JL, de Almeida JRF, et al. Analysis of some immunogenic properties of the recombinant Sporothrix schenckii Gp70 expressed in Escherichia coli. Future Microbiol [Internet]. 2019 Mar 1;14(5):397–410. Available from: https://doi.org/10.2217/fmb-2018-0295 113. Jones HD, Doherty A, Wu H. Review of methodologies and a protocol for the Agrobacteriummediated transformation of wheat. Plant Methods. 2005 Sep;1(1):5. 114. Wang S, Chen H, Wang Y, Pan C, Tang X, Zhang H, et al. Effects of Agrobacterium tumefaciens strain types on the Agrobacterium-mediated transformation efficiency of filamentous fungus Mortierella alpina. Lett Appl Microbiol [Internet]. 2020 May 1;70(5):388–93. Available from: https://doi.org/10.1111/lam.13286 115. Michielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet. 2005;48(1):1–17. 116. Sbaraini N, Tomazett MV, Penteriche AB, Gonçales RA, Camargo M da S, Bailão AM, et al. An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae). Genet Mol Biol. 2021;44(3):e20210073. 117. Lin L, Wang F, Wei D. Chlorimuron ethyl as a new selectable marker for disrupting genes in the insect-pathogenic fungus Metarhizium robertsii. J Microbiol Methods. 2011 Nov;87(2):241–3. 118. Blochlinger K, Diggelmann H. Hygromycin B phosphotransferase as a selectable marker for DNA transfer experiments with higher eucaryotic cells. Mol Cell Biol. 1984 Dec;4(12):2929–31. 119. Larionov A, Krause A, Miller W. A standard curve based method for relative real time PCR data processing. BMC Bioinformatics [Internet]. 2005;6(1):62. Available from: https://doi.org/10.1186/1471-2105-6-62 120. Du Z, Zong Q, Gao H, Guo Q, Liu T, Chen W, et al. Development of an Agrobacterium tumefaciensmediated transformation system for Tilletia controversa Kühn. J Microbiol Methods [Internet]. 2021;189(March):106313. Available from: https://doi.org/10.1016/j.mimet.2021.106313 121. Koukolíková-Nicola Z, Hohn B. How does the T-DNA of Agrobacterium tumefaciens find its way into the plant cell nucleus? Biochimie. 1993;75(8):635–8. 122. Talhinhas P, Muthumeenakshi S, Martins J, Oliveira H, Sreenivasaprasad S (Prasad). Agrobacterium-Mediated Transformation and Insertional Mutagenesis in Colletotrichum acutatum for Investigating Varied Pathogenicity Lifestyles. Mol Biotechnol. 2008 May 1;39:57–67. 123. Kummasook A, Cooper C, Vanittanakom N. An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei. Med Mycol. 2010 May 1;48:1066–74. 124. Figueiredo JG, Goulin EH, Tanaka F, Stringari D, Kava-Cordeiro V, Galli-Terasawa L V, et al. Agrobacterium tumefaciens-mediated transformation of Guignardia citricarpa. J Microbiol Methods 66 [Internet]. 2010;80(2):143–7. Available from: https://www.sciencedirect.com/science/article/pii/S0167701209003807 125. Orofino-Costa R, de Macedo PM, Bernardes-Engemann AR. Hyperendemia of Sporotrichosis in the Brazilian Southeast: Learning From Clinics and Therapeutics. Curr Fungal Infect Rep [Internet]. 2015;9(4):220–8. Available from: https://www.embase.com/search/results?subaction=viewrecord&id=L605999676&from=expor t 126. Misteli T, Spector DL. Applications of the green fluorescent protein in cell biology and biotechnology. Nat Biotechnol [Internet]. 1997;15(10):961–4. Available from: https://doi.org/10.1038/nbt1097-961 127. Matz M V, Lukyanov KA, Lukyanov SA. Family of the green fluorescent protein: journey to the end of the rainbow. Bioessays. 2002 Oct;24(10):953–9. 128. Wu L, Yuan Y-W, Jiang X-G, Fu X-L, Lu Z-X, Tu G-H, et al. [Stable green fluorescent protein expression in Toxoplasma gondii mutant]. Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese J Parasitol Parasit Dis. 2011 Dec;29(6):439–42. 129. McDevitt JJ, Lees PSJ, Merz WG, Schwab KJ. Use of green fluorescent protein-expressing Aspergillus fumigatus conidia to validate quantitative PCR analysis of air samples collected on filters. J Occup Environ Hyg. 2005 Dec;2(12):633–40. 130. Perez JT, García-Sastre A, Manicassamy B. Insertion of a GFP reporter gene in influenza virus. Curr Protoc Microbiol. 2013;Chapter 15:Unit 15G.4. 131. Sheen J, Hwang S, Niwa Y, Kobayashi H, Galbraith DW. Green-fluorescent protein as a new vital marker in plant cells. Plant J. 1995 Nov;8(5):777–84. 132. Lipták N, Bősze Z, Hiripi L. GFP transgenic animals in biomedical research: a review of potential disadvantages. Physiol Res. 2019 Aug;68(4):525–30. 133. Strebel A, Harr T, Bachmann F, Wernli M, Erb P. Green fluorescent protein as a novel tool to measure apoptosis and necrosis. Cytometry [Internet]. 2001 Feb 1;43(2):126–33. Available from: https://doi.org/10.1002/1097-0320(20010201)43:2%3C126::AID-CYTO1027%3E3.0.CO 134. Steff A-M, Fortin M, Arguin C, Hugo P. Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: An assay amenable to high-throughput screening technologies. Cytometry [Internet]. 2001 Dec 1;45(4):237–43. Available from: https://doi.org/10.1002/1097-0320(20011201)45:4%3C237::AID-CYTO10024%3E3.0.CO 135. Gille C, Spring B, Tewes L, Poets CF, Orlikowsky T. A new method to quantify phagocytosis and intracellular degradation using green fluorescent protein-labeled Escherichia coli: comparison of cord blood macrophages and peripheral blood macrophages of healthy adults. Cytometry A. 2006 Mar;69(3):152–4. 136. Li W, Houston KD, Houston JP. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry. Sci Rep. 2017 Jan;7:40341. 137. Weingart C, Broitman-Maduro G, Dean G, Newman S, Peppler M, Weiss A. Fluorescent Labels Influence Phagocytosis ofBordetella pertussis by Human Neutrophils. Infect Immun [Internet]. 1999 Aug 1;67(8):4264–7. Available from: https://doi.org/10.1128/IAI.67.8.4264-4267.1999 138. Wasylnka JA, Moore MM. Uptake of Aspergillus fumigatus Conidia by phagocytic and nonphagocytic cells in vitro: quantitation using strains expressing green fluorescent protein. Infect Immun. 2002 Jun;70(6):3156–63. 139. den Dulk-Ras A, Hooykaas PJ. Electroporation of Agrobacterium tumefaciens. Methods Mol Biol. 1995;55:63–72. 140. Green Sambrook, Joseph., Sambrook, Joseph., MR. Molecular cloning : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press; 2012. 141. Kaur H, Nguyen K, Kumar P. Pressure and temperature dependence of fluorescence anisotropy 67 of green fluorescent protein. RSC Adv. 2022;12(14):8647–55. 142. Mullenders LHF. Solar UV damage to cellular DNA: from mechanisms to biological effects. Photochem Photobiol Sci Off J Eur Photochem Assoc Eur Soc Photobiol. 2018 Dec;17(12):1842–52. 143. Griffiths HR, Mistry P, Herbert KE, Lunec J. Molecular and cellular effects of ultraviolet lightinduced genotoxicity. Crit Rev Clin Lab Sci. 1998 Jun;35(3):189–237. 144. Csepregi R, Temesfői V, Poór M, Faust Z, Kőszegi T. Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration. Vol. 23, Molecules. 2018. 145. de Vallée A, Bally P, Bruel C, Chandat L, Choquer M, Dieryckx C, et al. A Similar Secretome Disturbance as a Hallmark of Non-pathogenic Botrytis cinerea ATMT-Mutants? Front Microbiol. 2019;10:2829. 146. Germic N, Frangez Z, Yousefi S, Simon H-U. Regulation of the innate immune system by autophagy: monocytes, macrophages, dendritic cells and antigen presentation. Cell Death Differ. 2019 Mar;26(4):715–27. 147. Hünniger K, Kurzai O. Phagocytes as central players in the defence against invasive fungal infection. Semin Cell Dev Biol [Internet]. 2019;89:3–15. Available from: https://www.sciencedirect.com/science/article/pii/S1084952117305402 148. Miura T, Moriya H, Iwai S. Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells. FEMS Microbiol Lett. 2017 Jul;364(12). 149. Deeney AS, Maglennon GA, Chapat L, Crussard S, Jolivet E, Rycroft AN. Mycoplasma hyopneumoniae evades phagocytic uptake by porcine alveolar macrophages in vitro. Vet Res. 2019 Jun;50(1):51. 150. Vance J, Santos A, Sadofsky L, Morice A, Cervantes J. Effect of High Glucose on Human Alveolar Macrophage Phenotype and Phagocytosis of Mycobacteria. Lung. 2019 Feb;197(1):89–94. 151. Borba-Santos LP, Rodrigues AM, Gagini TB, Fernandes GF, Castro R, de Camargo ZP, et al. Susceptibility of Sporothrix brasiliensis isolates to amphotericin B, azoles, and terbinafine. Med Mycol [Internet]. 2015 Feb 1;53(2):178–88. Available from: https://doi.org/10.1093/mmy/myu056 152. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. Studying gene expression and function. In: Molecular Biology of the Cell. 4th Editio. New York: Garland Science; 2002. 153. Lozoya-Pérez NE, Casas-Flores S, Martínez-Álvarez JA, López-Ramírez LA, Lopes-Bezerra LM, Franco B, et al. Generation of Sporothrix schenckii mutants expressing the green fluorescent protein suitable for the study of host-fungus interactions. Fungal Biol [Internet]. 2018;122(10):1023–30. Available from: https://www.sciencedirect.com/science/article/pii/S1878614618301739