UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara BEATRIZ HELENA DIAS PANARIELLO INFLUÊNCIA DE ALTERAÇÕES GENÉTICAS, DO FLUCONAZOL E DE ENZIMAS HIDROLÍTICAS NA MATRIZ EXTRACELULAR DE BIOFILMES DE Candida SUSCEPTÍVEL E RESISTENTE A FLUCONAZOL Araraquara 2017 UNESP - Universidade Estadual Paulista “Júlio de Mesquita Filho” Faculdade de Odontologia de Araraquara BEATRIZ HELENA DIAS PANARIELLO INFLUÊNCIA DE ALTERAÇÕES GENÉTICAS, DO FLUCONAZOL E DE ENZIMAS HIDROLÍTICAS NA MATRIZ EXTRACELULAR DE BIOFILMES DE Candida SUSCEPTÍVEL E RESISTENTE A FLUCONAZOL Tese apresentada ao Curso de Pós-Graduação em Reabilitação Oral- Área de Prótese, da Faculdade de Odontologia de Araraquara, Universidade Estadual Paulista “Júlio de Mesquita Filho”, para obtenção do título de Doutora em Reabilitação Oral. Orientadora: Profª. Drª. Ana Cláudia Pavarina Co-orientadora: Profª. Drª. Marlise Inêz Klein Araraquara 2017 Panariello, Beatriz Helena Dias Influência de alterações genéticas, do fluconazol e de enzimas hidrolíticas na matriz extracelular de biofilmes de Candida susceptível e resistente a fluconazol / Beatriz Helena Dias Panariello.-- Araraquara: [s.n.], 2017 116 f. ; 30 cm. Tese (Doutorado em Prótese) – Universidade Estadual Paulista, Faculdade de Odontologia Orientadora: Profa. Dra. Ana Cláudia Pavarina Co-orientadora: Profa. Dra. Marlise Inêz Klein 1. Candida 2. Biofilmes 3. Matriz extracelular 4. Resistência a medicamentos 5. Fluconazol 6. Mutação I. Título BEATRIZ HELENA DIAS PANARIELLO INFLUÊNCIA DE ALTERAÇÕES GENÉTICAS, DO FLUCONAZOL E DE ENZIMAS HIDROLÍTICAS NA MATRIZ EXTRACELULAR DE BIOFILMES DE Candida SUSCEPTÍVEL E RESISTENTE A FLUCONAZOL Comissão julgadora Tese para obtenção do grau de Doutora Presidente e Orientadora: Profª. Drª. Ana Cláudia Pavarina 2º Examinador: Profª. Drª. Maria José Soares Mendes Giannini 3º Examinador: Profª. Drª. Renata de Oliveira Mattos Graner 4º Examinador: Profª. Drª. Lívia Nordi Dovigo 5º Examinador: Profª. Drª. Janaína Habib Jorge Araraquara, 4 de agosto de 2017. DADOS CURRICULARES BEATRIZ HELENA DIAS PANARIELLO NASCIMENTO 16/02/1987- São Paulo, São Paulo. FILIAÇÃO Cibele Mara Dias Panariello Fábio Leonardo Panariello. 2006 – 2010 Graduação pela Faculdade de Odontologia de Araraquara- UNESP. 2008 Iniciação científica na disciplina de Prótese Parcial Removível da Faculdade de Odontologia de Araraquara- UNESP. 2009 Iniciação científica na disciplina de Odontopediatria da Faculdade de Odontologia de Araraquara- UNESP. 2010 Bolsista do projeto de extensão denominado “Programa de manutenção da saúde bucal em pacientes de 3ª idade usuários de Prótese Parcial Removível”- Faculdade de Odontologia de Araraquara- UNESP. 2011 Pós-Graduação em Reabilitação Oral, nível Mestrado, pela Faculdade de Odontologia de Araraquara- UNESP. Estágio docência na disciplina de Prótese Parcial Removível I. 2012 Estágio docência na disciplina de Prótese Parcial Removível II. 2013 Obtenção do Título de Mestre em Reabilitação Oral pela Faculdade de Odontologia de Araraquara- UNESP. Pós-Graduação em Reabilitação Oral, nível Doutorado, pela Faculdade de Odontologia de Araraquara- UNESP. 2014 Estágio docência na disciplina de Prótese Parcial Removível II. 2015 Estágio docência Prótese Parcial Removível II Estágio docência Prótese Total II 2016 Doutorado com período sanduíche em New York University College of Dentistry (NYU), Nova York, Estados Unidos. Dedico este trabalho Aos meus amados pais, Fábio Leonardo Panariello e Cibele Mara Dias Panariello, Pelo amor e carinho com que criaram a mim e meus irmão, educando-nos para a vida e nos proporcionando condições de perseguirmos nossos sonhos. A minha jornada de estudos até aqui não teria sido possível sem o apoio incondicional, e, acima de tudo, sem o amor de vocês. Aos meus pais, minha eterna gratidão e admiração. Ao meu marido e amor da minha vida, Éder Augusto Mastropietro Cavichioli, Companheiro desde a graduação, grande incentivador durante meu mestrado e, principalmente, durante o doutorado. Por viver comigo os meus sonhos e apoiá-los incondicionalmente. Por entender as minhas ausências para desenvolver este trabalho e me ajudar com as tarefas diárias. Por me dar forças para continuar e nunca ter medido esforços para me fazer feliz. Sou infinitamente grata por cada momento que vivemos e viveremos juntos. Esta conquista é nossa! Eu te amo! Agradecimentos especiais Agradeço de forma especial... À minha orientadora, professora Dra. Ana Cláudia Pavarina, exemplo de competência em tudo que faz. Excelente professora e pesquisadora que participou ativamente da minha formação acadêmica na graduação e na pós-graduação, e com quem tive a honra de aprender e compartilhar conhecimentos durante estes 4 anos de doutorado. Agradeço pela confiança em mim e no meu trabalho, pelos incentivos e pela paciência ao prestar ensinamentos. Sinto-me honrada em tê-la como orientadora! À minha co-orientadora, professora Dra. Marlise Inêz Klein, exemplo de excelência científica, pela paciência em me ensinar novas metodologias, por confiar e acreditar na minha capacidade, pela disponibilidade e dedicação na elaboração deste trabalho e pelo carinho com o qual sempre me tratou. Seus conhecimentos inspiraram meu doutorado, fazendo com que eu me apaixonasse ainda mais pela pesquisa. Seus ensinamentos foram fundamentais para que esta tese fosse concluída. É uma honra tê-la como co-orientadora! À professora Dra. Simone Duarte, pesquisadora brilhante e pessoa admirável, por ter me dado a oportunidade de viver um sonho ao aceitar me orientar, e por ter confiado em mim e no meu trabalho. Agradeço pelos ensinamentos e ajudas que excederam as fronteiras do laboratório, e pelo carinho e atenção que foram fundamentais para que minha experiência em Nova York fosse tão enriquecedora. Sinto orgulho por ter sido sua orientada durante o Doutorado Sanduíche! "Se enxerguei mais longe, foi porque me apoiei nos ombros de gigantes." Isaac Newton Agradecimentos Sou grata... Ao meu amado irmão, Fabrízio Dias Panariello, o primeiro grande presente que meus pais me deram na vida e que, recentemente, junto com a querida Amanda Tascone, presenteou-me com a alegria de ser tia do Pietro Tascone Panariello. À Thaís Helena Dias Panariello, minha amada irmã caçula, pela amizade e companheirismo, por se orgulhar de mim e sempre me incentivar a buscar meu sonhos. À minha sogra, Magda Regina Mastropietro, por todo o carinho com que sempre me tratou, por apoiar minhas decisões, pelos conselhos e pela inestimável ajuda enquanto estive em Nova York relizando parte desta tese de doutorado. À Faculdade de Odontologia de Araraquara da Universidade Estadual Paulista “Júlio de Mesquita Filho”, na pessoa responsável por sua direção, a Profª. Drª. Elaine Maria Sgavioli Massucato e ao seu Programa de Pós-graduação em Reabilitação Oral, na pessoa responsável por sua coordenação, a Profª. Drª. Ana Cláudia Pavarina, pela oportunidade de realizar minha pós-graduação. À FAPESP- Fundação de Amparo à Pesquisa do Estado de São Paulo, pela concessão de bolsa de doutorado regular (Processo FAPESP nº 2014/18804-1) e bolsa de estágio no exterior BEPE (Processo FAPESP nº 2016/00256-3). Aos amigos da pós-graduação, em especial Fernanda Alves, Juliana Cabrini Carmello, Lívia Jacovassi Tavares, Kássia de Carvalho Dias, Gabriela Caroline Alonso, Jeffersson Trigo Gutierrez, Bruna Pimentel, Carmélia Lobo, Maria Isabel Amaya, Camila de Foggi, Jéssica Bernegossi, Lucas Portela e Elkin Florez, pela parceria no laboratório e fora dele. Por alegrarem meus dias difíceis e tornarem mais leve a minha caminhada rumo a este título. Às bolsista de Apoio Técnico, , Bruna Novelli, Geisiane Bueno, Luana Sales e Lígia Sabino pela atenção, amizade e pelas ajudas no laboratório sempre com boa vontade e prontidão. Aos amigos Natalia Bertolo Domingues e Aion Mangino Messias que, mesmo fisicamente distantes, estiveram presentes todos os dias enquanto estive em Nova York. Obrigada pelo apoio e pela força que me ajudaram a passar por essa fase com alegria e tranquilidade. Às amigas da New York University College of Dentistry, Cecília Atem Gonçalves de Araújo, Paula Ventura da Silveira, Aline Rogéria Freire de Castilho e Adriana da Fonte Porto Carreiro. A amizade de vocês fez toda a diferença para que eu conseguisse seguir em frente com a pesquisa sem me abalar com as saudades de casa. Obrigada pela ajuda e companhia nos experimentos, pelo apoio, pelos conselhos e, especialmente para a Paula, meus sinceros agradecimentos pelo abrigo nas últimas semanas do doutorado em Nova York. E a todos aqueles que, de alguma forma, contribuíram para execução deste trabalho, muito obrigada! Panariello BHD. Influência de alterações genéticas, do fluconazol e de enzimas hidrolíticas na matriz extracelular de biofilmes de Candida susceptível e resistente a fluconazol [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017. RESUMO Biofilmes formados por Candida estão relacionados a infecções bucais, como a candidíase. Embora a resistência do biofilme seja multifatorial, a proteção exercida por sua matriz extracelular (MEC) é importante para os altos níveis de resistência a drogas antifúngicas. O conhecimento dos princípios estruturais da MEC possibilita maior compreensão de como atuar para desorganizá-la e melhorar a difusão de agentes antifúngicos para que atinjam mais eficientemente o biofilme, além de, futuramente, possibilitar o desenvolvimento de terapias mais eficazes para o controle da formação de biofilmes. Sendo assim, os objetivos principais deste estudo foram: (1) verificar a influência da inativação de genes envolvidos na filamentação (EFG1 e TEC1) em características estruturais dos biofilmes e na produção de componentes da MEC; (2) verificar a influência do fluconazol (FLZ) na MEC de biofilmes de Candida albicans ATCC 90028 (susceptível a fluconazol- CaS), C. albicans ATCC 96901 (resistente a fluconazol- CaR), Candida glabrata ATCC 2001 (susceptível a fluconazol- CgS) e C. glabrata ATCC 200918 (resistente a fluconazol- CgR) e (3) estudar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a MEC de biofilmes de CaS e CaR. Biofilmes maduros (48 horas) foram analisados através de contagem de unidades formadoras de colônia (ufc/mL), peso seco total, peso seco insolúvel e proteínas insolúveis. Os componentes da MEC- polissacarídeos solúveis em álcali (ASPs), polissacarídeos solúveis em água (WSPs), DNA extracelular (eDNA) e proteínas solúveis- foram quantificados. No estudo 1, foi observado que o conteúdo de ASPs é significativamente maior em cepa parental de C. albicans em comparação com as cepas mutantes Δ/Δ efg1 e Δ/Δ tec1, o que indica que a produção de ASPs pode estar relacionada à morfologia celular filamentosa em C. albicans. No estudo 2, observou-se que as biomassas totais e WSPs foram significativamente reduzidos pelo FLZ na MEC de CaS, CaR, CgS e CgR, mas as quantidades de eDNA e proteínas não foram influenciadas pela presença de FLZ nem pelo tipo de cepa. O FLZ interferiu na morfologia celular e na estrutura dos biofilmes, reduzindo a formação de hifas nos biofilmes de CaS e CaR e diminuindo o número de células nos biofilmes de CgS e CgR. No estudo 3, observou-se que exposição de biofilmes maduros à DNase por 5 minutos reduziu o conteudo de eDNA, polissacarídeos e proteínas solúveis da MEC de CaS e CaR, sendo um promissor adjuvante para terapias antibiofilme. A redução de polissacarídeos extracelulares e do conteúdo de proteínas pela DNase indicam que esses componentes estão interligados ao eDNA na MEC de CaS e CaR. Portanto, células filamentosas têm tendência de produzirem mais exopolissacarídeos, e estes componentes estão interligados ao eDNA e a proteínas solúveis na MEC de biofilmes de C. albicans. Para reduzir componentes da matriz e desorganizar a estrutura formada por eDNA-exopolissacarídeos-proteínas, a aplicação de enzima DNase por 5 min em biofilmes maduros de C. albicans se mostrou eficaz. Palavras chave: Candida. Biofilmes. Matriz extracelular. Resistência a medicamentos. Fluconazol. Mutação. Panariello BHD. Influence of genetic alterations, fluconazole and hydrolytic enzymes on the extracellular matrix of fluconazole-susceptible and -resistant Candida biofilms [Tese de Doutorado]. Araraquara: Faculdade de Odontologia da UNESP; 2017. ABSTRACT Biofilms formed by Candida are related to bucal infections, such as candidiasis. Although the biofilm resistance is multifactorial, the protection exerted by its extracellular matrix (ECM) is essential for its high levels of resistance to antifungals. The knowledge of the structural principles of the ECM permits a better understanding of how to disorganize the ECM and improve the diffusion of antifungal drugs to reach the biofilm. Moreover, the study of the ECM may enable the development of more effective therapies to control biofilm formation. Thus, the main objectives of this study were: (1) to verify the influence of the inactivation of genes involved in filamentation and structural characteristics of the biofilms (EFG1 and TEC1) on the production of ECM components; (2) to verify the influence of fluconazole (FLZ) on the biofilms’ ECM of Candida albicans ATCC 90028 (fluconazole-susceptible: CaS), C. albicans ATCC 96901 (fluconazole-resistant: CaR), Candida glabrata ATCC 2001 (fluconazole- susceptible: CgS) and C. glabrata ATCC 200918 (fluconazole-resistant: CgR) and (3) to study the action of hydrolytic enzymes (DNase, Dextranase and β-glucanase individually or in different combinations) on the ECM of CaS and CaR biofilms. Mature biofilms (48 hours) were analyzed by colony counting forming units (cfu/mL), total dry weight, insoluble dry-weight and total proteins. ECM components- alkali-soluble polysaccharides (ASPs), water-soluble polysaccharides (WSPs), extracellular DNA (eDNA) and soluble proteins- were quantified. In study 1, it was observed that ASPs content is significantly higher in C. albicans parental strain compared to the mutant strains Δ/Δ efg1 and Δ/Δ tec1, indicating that the production of ASPs may be related to the filamentous cell morphology in C. albicans. In study 2, it was observed that the total biomasses and WSPs were significantly reduced by FLZ in the ECM of CaS, CaR, CgS and CgR, but the amounts of eDNA and proteins were not influenced by the presence of FLZ nor by type of strain. FLZ interfered on the cellular morphology and structure of biofilms, reducing hyphae formation in CaS and CaR biofilms and reducing the number of cells in CgS and CgR biofilms. The study 3 demonstrated that exposure of mature biofilms to DNase for 5 minutes reduced the eDNA, polysaccharides and soluble proteins of the ECM of CaS and CaR, being a promising adjuvant for antibiofilm therapies. The reduction of extracellular polysaccharides and soluble proteins by DNase indicates that these components are intertwined to eDNA in the ECM of CaS and CaR. Therefore, filamentous cells tend to produce more exopolysaccharides, and these components are intertwined to eDNA and soluble proteins in the ECM of C. albicans biofilms. To reduce matrix components and disrupt the structure formed by eDNA-exopolysaccharide-proteins, 5 min exposure of mature biofilms to DNase showed to be effective. Keywords: Candida. Biofilms. Extracellular matrix. Drug resistance. Fluconazole. Mutation. SUMÁRIO 1 INTRODUÇÃO .......................................................................................... ...14 2 PROPOSIÇÃO ...............................................................................................17 3 PUBLICAÇÕES ......................................................................................... ...18 3.1 Publicação 1 ............................................................................................. ...18 3.2 Publicação 2 ............................................................................................. ...47 3.3 Publicação 3 .............................................................................................. ...78 4 DISCUSSÃO ............................................................................................... .104 5 CONCLUSÃO ............................................................................................. .112 REFERÊNCIAS ............................................................................................ .113 15 1 INTRODUÇÃO Os microrganismos do gênero Candida são fungos comensais da cavidade oral de indivíduos saudáveis que podem se tornar agentes patogênicos oportunistas em algumas situações, por exemplo, quando há alterações no sistema imunológico, disfunção metabólica ou em população de idade avançada (Abaci et al.1, 2010; Dagistan et al.7, 2009; Li et al.13, 2007; Luo, Samaranayake15, 2002; Pfaller, Diekema31, 2007; Samaranayake, Samaranayake38, 2001). Além disso, o uso crescente de antibióticos de amplo espectro, quimioterapias citotóxicas e transplantes também intensificam o risco de infecções por esses fungos oportunistas (Pfaller, Diekema31, 2007). Essas infecções são conhecidas como candidíase. A Candida albicans é a espécie mais prevalente associada a candidíase, seguida pela Candida glabrata, que é a espécie não-albicans mais prevalente que tem sido associada ao desenvolvimento de infecções orais (Abaci et al.1, 2010; Dagistan et al.7, 2009; Li et al.13, 2007; Luo, Samaranayake15, 2002; Samaranayake, Samaranayake38, 2001). A crescente importância de C. glabrata como um oportunista em indivíduos imunocomprometidos tem sido relatada (Bennet et al.3, 2001; Pfaller, Diekema31, 2007), principalmente devido a sua habilidade inata para adquirir resistência antifúngica (Mann et al.17 2009; Tsai et al.44, 2010). Infecções causadas por Candida estão frequentemente associadas à formação de biofilmes (Nobile, Mitchell26, 2007). O crescimento de biofilme se inicia quando células planctônicas aderem a um determinado substrato. Em seguida, ocorre profileração de células de levedura na superfície do substrato e o início do desenvolvimento de hifas. O passo final para o desenvolvimento do biofilme é o estágio de maturação, no qual o crescimento em forma de levedura é reprimido, o crescimento de hifas se eleva e matriz extracelular (MEC) encobre o biofilme (Blankenship, Mitchell5, 2006). A via de desenvolvimento de hifas é crítica para que haja formação significativa de biomassa de biofilme (Ramage et al.35, 2002). Mutantes com defeitos no fator de transcrição EFG1 (enhanced filamentous growth), o principal ativador do desenvolvimento de hifas, não foram capazes de formar nem mesmo uma monocamada de células sobre superfícies de poliestireno (Ramage et al.35, 2002). Além de EFG1, o fator de transcrição TEC1 também é necessário para a formação de hifas (Schweizer et al.40, 2000). Foi observado que biofilmes produzidos por cepa mutante com ausência do fator de transcrição TEC1 (Δ/Δ tec1) eram rudimentares, possuindo menos de 20 μm de espessura (Ramage et al.35, 2002). Cepas mutantes com defeitos em genes de filamentação são menos virulentas do que suas cepas parentais e apresentam menores níveis de infectividade de células endoteliais e catéteres (Lewis et al.12, 2002; Lo et al.14, 1997). 14 http://informahealthcare.com/action/doSearch?action=runSearch&type=advanced&result=true&prevSearch=%2Bauthorsfield%3A%28%29 16 Algumas espécies de Candida possuem resistência intrínseca a drogas antifúngicas, especialmente ao fluconazol (Pfaller, Diekma31, 2007; Tsai et al.44, 2010). Em contraste, a resistência pode ser desenvolvida pelo microrganismo após longos períodos de exposição a antifúngicos (Shapiro et al.41, 2011). Dessa forma, uma grande preocupação com os biofilmes de Candida é que suas células podem ter uma susceptibilidade reduzida contra azóis e polienenos, devido ao desenvolvimento de resistência (Pfaller et al.30, 2002). A resistência dos biofilmes de Candida é multifatorial e está associada ao estado fisiológico das células, à ativação de bombas de efluxo de drogas e ao efeito protetor dos β-glucanos presentes na MEC de C. albicans, que se ligam ao fluconazol e à anfotericina B (Nett et al.24, 2007, Nett et al.25, 2010), dificultando a penetração desses fármacos nos biofilmes (Vediyappan et al.45, 2010). Foi demonstrado que a MEC de biofilme de C. albicans possui grandes quantidades de β-1,6 glucanos e α-mananas, que interagem para formar um complexo manano-glucano (MGCx) (Mitchell et al.20, 2015; Zarnowski et al47., 2014). Esta interação de exopolissacarídeos foi considerada crucial para a proteção do biofilme contra tratamento medicamentoso (Mitchell et al.21, 2016). Além disso, demonstrou-se que o DNA extracelular (eDNA) contribui para a integridade estrutural do biofilme de C. albicans (Martins et al.18, 2012; Rajendran et al.34, 2014). Esforços para hidrolisar polissacarídeos e ácidos nucleicos da MEC têm sido eficazes na sensibilização de biofilmes de Candida e Aspergillus (Martins et al.18, 2012; Mitchell et al.20, 2015; Nett et al.24,2007; Rajendran et al.33, 2013). O acúmulo de α-mananos foi bloqueado com α-manosidase, uma enzima que catalisa a hidrólise de resíduos terminais não redutores de α-D- manose em α-D-manosídeos, aumentando a atividade do fluconazol contra os biofilmes de C. albicans (Mitchell et al.20, 2015). Além disso, biofilmes de 24 horas desafiados com RPMI contendo diferentes concentrações de antifúngicos isolados ou em combinação com DNase mostraram que a adição de DNase aumentou a susceptibilidade das células de C. albicans à anfotericina B (Martins et al.18, 2012). Ademais, demonstrou-se que a combinação de biofilmes com DNase associada a anfotericina B e caspofungina melhorou significativamente a susceptibilidade antifúngica em biofilme de Aspergillus fumigatus (Rajendran et al.33, 2013). A matriz extracelular é considerada um dos maiores desafios no controle do biofilme oral (Panariello et al.29, 2017). Sendo assim, o conhecimento dos princípios estruturais da matriz extracelular possibilita maior compreensão de como atuar para desorganizá-la e melhorar a difusão de agentes antifúngicos através do biofilme, a fim de que atinjam mais eficientemente as células de Candida. Além disso, possibilita que, futuramente, sejam desenvolvidas terapias mais eficazes para o controle da formação e patogenicidade de biofilmes de Candida. Portanto, os objetivos principais deste estudo foram: (1) caracterizar a matriz extracelular de biofilmes 15 17 de cepas mutantes (Δ/Δ efg1 e Δ/Δ tec1) e cepa parental (wild-type-WT) de C. albicans para verificar a influência da inativação de genes envolvidos na filamentação e em características estruturais dos biofilmes na produção de componentes da MEC; (2) caracterizar a MEC de biofilmes de C. albicans e C. glabrata susceptíveis e resistentes ao fluconazol na presença e na ausência desta droga para verificar sua influência na MEC dos biofilmes destas cepas e (3) estudar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a MEC de biofilmes de C. albicans susceptível e resistente a fluconazol. 16 18 2 PROPOSIÇÃO 1. Verificar a influência da inativação de genes envolvidos na filamentação e em características estruturais dos biofilmes (TEC1 e EFG1) na produção de componentes da MEC em C. albicans. 2. Verificar a influência do fluconazol na MEC de biofilmes de cepas de C. albicans e C. glabrata susceptíveis e resistentes a fluconazol. 3. Analisar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a matriz extracelular de biofilmes de C. albicans susceptível e resistente ao fluconazol. 17 19 3 PUBLICAÇÕES 3.1 Publicação 1 Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology Beatriz Helena Dias Panariello1, Marlise I. Klein1, Ana Claudia Pavarina1, Simone Duarte2 1 Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil. 2 Department of Cariology, Operative Dentistry and Dental Public Health, Indiana University School of Dentistry. Postal address: 1121 W Michigan St, # DS406, Indianapolis, IN, USA, 46202; Phone +1 (317) 278-4906; e-mail: siduarte@iu.edu *Corresponding author The studies were performed in the Department of Basic Science & Craniofacial Biology at New York University College of Dentistry where the supervisor Dr. Simone Duarte was working as associate professor before moving to Indiana University School of Dentistry. Artigo publicado no periódico Journal of Oral Microbiology VOL. 9, 1385372, 2017. DOI: 10.1080/20002297.2017.1385372 © 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 18 http://dx.doi.org/10.1080/20002297.2017.1385372 20 ABSTRACT Background: Infections caused by Candida spp. have been associated with formation of a biofilm, i.e. a complex microstructure of cells adhering to a surface and embedded within an extracellular matrix (ECM). Methods: The ECMs of a wild-type (WT, SN425) and two Candida albicans mutant strains, Δ/Δ tec1 (CJN2330) and Δ/Δ efg1 (CJN2302), were evaluated. Colony-forming units (cfu), total biomass (mg), water-soluble polysaccharides (WSPs), alkali-soluble polysaccharides (ASPs), proteins (insoluble part of biofilms and matrix proteins), and extracellular DNA (eDNA) were quantified. Variable-pressure scanning electron microscopy and confocal scanning laser microscopy were performed. The biovolume (μm3/μm2) and maximum thickness (μm) of the biofilms were quantified using COMSTAT2. Results: ASP content was highest in WT (mean ± SD: 74.5 ± 22.0 µg), followed by Δ/Δ tec1 (44.0 ± 24.1 µg) and Δ/Δ efg1 (14.7 ± 5.0 µg). The protein correlated with ASPs (r = 0.666) and with matrix proteins (r = 0.670) in the WT strain. The population in Δ/Δ efg1 correlated with the protein (r = 0.734) and its biofilms exhibited the lowest biomass and biovolume, and maximum thickness. In Δ/Δ tec1, ASP correlated with eDNA (r = 0.678). Conclusion: ASP production may be linked to C. albicans cell filamentous morphology. KEYWORDS: Candida albicans, biofilm, EFG1, TEC1, extracellular matrix 19 21 Introduction The microorganisms of the genus Candida are opportunistic fungi that are present in the oral cavity. When there is an imbalance in the immune system of the host, the oral microbiota is altered, and these microorganisms may invade the oral tissues [1,2]. Clinical manifestations of infections caused by Candida spp. can be superficial, such as oropharyngeal candidiasis (OPC) and/or systemic (e.g. candidemia). The OPC is an indicator of the development of AIDS and depending on the stage of the infection by HIV, about 90% of the patients show OPC [3,4]. Candida albicans is the most prevalent specie related to this infection [5-8]. Infections caused by Candida spp. are often associated with biofilm formation. Biofilm is a complex microstructure of cells adhered to a surface and embedded within an extracellular matrix (ECM) made up of secreted microbial and host-derived substances (i.e. saliva components) and cells lysis [9]. The ECM contributes to the biofilm build up, preservation of the biofilms’ architecture and maintenance of stable interactions between cell-cell and cell- surface and the environment [10]. Among the substances found in the ECM are polysaccharides, proteins, and nucleic acids, all of which play a major role in biofilms [9]. Three classes of conventional antifungals are used for treating infections caused by Candida: azoles (e.g. fluconazole), polyenes (e.g. amphotericin B) and echinocandins (e.g. casponfungin) [11]. However, antifungal drug resistance can arise from all drug classes and includes acquired resistance in susceptible strains and selection of innately less susceptible species [12]. Antifungals resistance in Candida biofilms is multifactorial and is associated with the physiological state of the cells, the activation of drug efflux pumps and the protective effect of the ECM performed by β-glucans (an alkali-soluble exopolysaccharide or ASP), which bind to fluconazole [13] and amphotericin B, preventing the penetration of drugs into the biofilm [14]. In addition to the protective effects of ECM performed by β-glucan, it has been shown that the extracellular DNA (eDNA) is another key component, contributing to the structural integrity of 20 22 C. albicans biofilm [15]. The ECM of C. albicans strain K1 was tested using in vitro and animal models, and the ECM composition was 55% protein, 25% carbohydrate, 15% lipid, and 5% nucleic acid, while β-1,3-glucan comprised only a small portion of the total matrix [16]. The “hyphal development pathway is critical for formation of significant biofilm mass” [17]. Mutants defective in the enhanced filamentous growth transcriptional factor (EFG1), a major activator of hyphal development, presented impaired formation of monolayer of cells on polystyrene surfaces [17]. This defect in biofilm development may be because of altered surface-protein composition and adherence properties of the EFG1 null mutant (Δ/Δ efg1) [18]. In addition, the lack of functioning EFG1 in C. albicans strains yielded only pseudohyphae on solid media and without growth in liquid media [19]. Tec1p is a TEA/ATTS transcription factor and it is required for hyphal formation [20]. Biofilm produced by the tec1 null mutant (Δ/Δ tec1) strain was rudimentary with less than 20 µm deep and composed exclusively of yeast cells [17], while its parental strain formed a biofilm 250–450 µm deep that included many hyphal filaments [17]. Therefore, mutant strains defective in the filamentation genes EFG1 and TEC1 are considered less virulent than their wild-type counterparts, because they present decreased levels of infectivity of endothelial cells and plasma-coated catheters [21, 22]. The study of C. albicans mutants with defective capability of forming biofilms is needed to evaluate the differences in the ECM components and structure when there is deficient formation of biofilm, facilitating the understanding of which components are related to a regular biofilm formation. Knowing the assembly principles of the matrix helps on deciding in which component or components new therapies should focus at to design effective treatments to control fungal biofilm formation and pathogenesis. Therefore, the aim of this study was to characterize the ECM of wild-type and mutant (Δ/Δ efg1 and Δ/Δ tec1) C. albicans strains. Materials and methods Biofilm formation and processing 21 23 The microorganisms used for this experiment were C. albicans SN425 (wild-type strain- WT), C. albicans CJN2302 and C. albicans CJN2330, the last two ones are mutant strains with deficient biofilm formation ability, Δ/Δtec1 and Δ/Δefg1 [23]. TEC1 is primarily an activator of its biofilm-relevant direct target genes and EFG1 is both activator and repressor [23]. The microorganisms stored at -80ºC were seeded onto Petri dishes with SDA (Sabourand dextrose agar) culture medium supplemented with chloramphenicol and incubated at 37°C for 48 h. Next, starter cultures containing about 5 colonies were grown using YNB medium (Yeast Nitrogen Base- DIFCO, Detroit, Michigan, USA) supplemented with 100 mM of glucose, and incubated at 37°C. After 16 h of incubation, the starter cultures were diluted with fresh YNB medium supplemented with 100 mM glucose (1:20 dilution). These inoculum cultures were incubated at 37°C until the three strains reached the mid-log growth phase (8 hours, Fig. S1). Then, the OD540nm of the inoculums was adjusted to reach 107 cells mL-1. Next, 1 mL of the inoculum of each strain was added to the wells of a 24-well polystyrene plate (Techno Plastic Products- TPP, Trasadingen, Switzerland). The culture plate was incubated at 37°C for cell adhesion to the substrate. After 90 min, the wells were washed twice with sterile 0.89% NaCl solution to remove non-adhered cells. Next, 1 mL of RPMI 1640 buffered with morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich, St. Louis, Missouri, USA) at pH 7 was added to each well. After 24 hours of biofilm formation, the culture medium was removed by aspiration and fresh RPMI buffered with MOPS (1 mL, pH 7.0) was added to each well. After 48 hours of biofilm formation, the wells were washed twice with 0.89% sterile NaCl solution. Biofilms were processed following the flowchart in Fig. 1. Briefly, biofilms were removed by scraping each well with a pipette tip and 2 mL of sterile 0.89% NaCl. From the biofilm suspension, 0.1 mL was used for the 10-fold serial dilution and culture on SDA plates for recovery of colony forming units (CFU) and 0.1 mL was used for dry weight (biomass) determination [24]. The 22 24 remainder of the volume was vortexed vigorously at high speed for 1 min for all samples during processing for mechanical disruption of the ECM and centrifuged at 5500 xg for 10 min (4°C). The supernatant was stored in another tube and the precipitate with the cells and the insoluble components of the ECM was washed twice with sterile milli-Q water (5500xg /10 min/ 4°C). From the stored supernatant, it was separated 1 mL for the quantification of water-soluble polysaccharides (WSP) [25], 0.650 mL for eDNA analysis [26] and 0.150 mL for protein tests [27]. The precipitate was re-suspended in water then 0.05 mL was separated for the quantification of protein [27] and 0.95 mL was separated for the determination alkali-soluble polysaccharide ASP [25]. Protein quantification Proteins in the ECM (soluble or supernatant) and in the insoluble part of biofilms were quantified. The proteins from the insoluble part were extracted by boiling at 100ºC for 1 h at 1000 rpm. Bovine serum albumin solution (P5369, Sigma-Aldrich, St. Louis, MO, USA) was prepared in saline buffer and the following concentrations were used as standard curve: 0 mg/mL, 0.03125 mg/mL, 0.0625 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL e 1.4 mg/mL. In 96 well plates, 200 µL of Bradford Reagent (B6916, Sigma-Aldrich, St. Louis, MO, USA) was mixed with 5 µL of each curve point and biofilm samples. The reaction was carried out during 30 min, and the absorbance at 595 nm was determined in a spectrophotometer. Water-soluble polysaccharides (WSP) analysis An aliquot of 1 mL per sample of the homogenized supernatant was transferred to sterile centrifuge tubes to which 2.5 volumes of 95% ethanol were added. The WSP were precipitated for 18 h at -20 °C and centrifuged at 9500 xg for 20 min at 4°C. After centrifugation, the supernatants were discarded. The samples were washed three times with ice-cold 75% ethanol 23 25 and the pellets were air-dried. Each pellet was resuspended with 1 ml of water, and total carbohydrates were quantified using the phenol-sulfuric acid method [25]. Glucose was used for standard curve (0, 2.5, 5, 10, 15, 20 and 25 μL glucose per tube). The method consists of adding 200 μL of 5% phenol to a glass tube containing 200 μL of the sample or standard curve point (triplicate per sample). After carefully mixing, one mL of sulfuric acid was added to each tube under agitation. After 20 min of reaction, samples were measured using a spectrophotometer (490 nm). Alkali-soluble polysaccharides (ASP) analysis Aliquots of 0.95 mL of each biofilm suspension were centrifuged (13000 xg/10 min /4 °C). The supernatant of each tube was carefully removed and discarded. The pellets were then dried in a desiccator for one week. The pellets were weighed and 300 µL of 1N NaOH per 1 mg of the dry-weight were added. The pellets with 1N NaOH were incubated for 2 h at 37°C and then were centrifuged at 13000 xg for 10 min. The supernatants were cautiously collected with a pipette and transferred to new microcentrifuge tubes, preserving just the pellet. Once more, the same previous volume of 1N NaOH was added to the tubes containing the pellets, and the same steps as above were repeated for the extraction of ASP. After incubation, samples were centrifuged (13000 xg /10 min) and the supernatants were carefully collected and added to the previously collected supernatant. For the third extraction, the same steps above were repeated, but this time, the samples were not incubated for 2 h before centrifugation. After three extractions, three volumes of cold 95% ethanol were added to each sample. The samples were then stocked at -20 °C for 18 h for precipitation of ASP. After precipitation of ASP, the tubes were centrifuged (9500 xg/20 min /4 °C), and the supernatants were discarded. Each resulting pellet was washed three times with ice-cold 75% ethanol and air-dried, following the procedures performed for WSP samples. The pellets were resuspended in the same total volume of the 24 26 original extraction with 1N NaOH. Finally, the samples were ready for quantification of total carbohydrates using the phenol-sulfuric acid method as described for WSP analysis. eDNA analysis Aliquots of 0.65 mL of the supernatant of biofilms suspensions were mixed with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and once with chloroform: isoamyl alcohol (24:1) for eDNA extraction. The aqueous phase of each sample was mixed with 3 volumes of isopropanol and 1/10 volume of 3M sodium acetate (pH 5.2) and stored at - 20º C for 18 h. The eDNA precipitated with isopropanol was collected by centrifugation (13000 xg/20 min/4 °C) and washed 3 times with ice-cold 70% ethanol, air dried, and then dissolved in 10 μL of TE buffer (Tris HCl/1 mM EDTA, pH 8.0). The amount of eDNA was determined using a spectrophotometer with light length of 260 nm. Variable pressure scanning electron microscopy protocol (VPSEM) After 48 h of biofilm formation, the samples were transferred directly to the VPSEM [Zeiss EVO 50 (Carl Zeiss Microscopy, LLC, Thornwood, NY) chamber and imaged at 100 Pa. VPSEM images were captured at a working distance of 6.5 and 7.0 mm and field widths of 10 µm and 20 µm [28]. Confocal scanning laser microscopy (CSLM) The biofilm morphology was determined by CSLM. Leica TCS SP5 microscope (Leica Lasertechnik GmbH, Heidelberg, Germany) with a HCX APOL U-V-I 40X/0.8-numerical- aperture water immersion objective was used. The biofilms were stained with a live/dead viability kit (Molecular Probes. Invitrogen, Eugene, Oregon. USA). The stain was prepared by diluting 1.5 μL of SYTO 9 and 1.5 μL of propidium iodide in 1.0 mL of sterile 1% phosphate buffered solution (pH 7.4) [29]. The plates were incubated at room temperature in the dark for 15 min and examined under a CSLM. The biovolume (μm3/μm2) and maximum thickness (μ) 25 27 [30] of the biofilms were quantified using COMSTAT2- http://www.comstat.dk; and the images were rendered in the Amira software (Mercury Computer Systems Inc., Chelmsford, MA). Statistical analyses All the experiments were repeated on three separated occasions, with four replicates (n=12). Data was analyzed by one-way ANOVA with Tukey post-hoc test (α = 0.05). A Pearson’s correlation test (r) was applied to check correlations between the different ECM components. Correlation was considered significant at the 0.05 level (2-tailed). All the tests were performed using IBM SPSS Statistics 19. Results The quantitative data of population, dry weight, protein and extracellular matrix components are displayed in Table 1. The population data demonstrated significant differences between WT and Δ/Δ efg1 and Δ/Δ tec1 and Δ/Δ efg1 (p<0.001) while no statistical differences were observed between WT and Δ/Δ tec1 strains. The dry weight (total biomass) of the WT strain is higher than the mutant strains (p<0.005). The proteins are significantly higher (p<0.05) in the Δ/Δ efg1 mutant strain. The ASP data showed significant differences for all the strains (p=0.000), being the WT the strain that possess the higher amount of this ECM component (74.5 ± 22.0 µg), followed by Δ/Δ tec1 (44.0 ± 24.1 µg) and Δ/Δ efg1 (14.7 ± 5.0 µg). However, the other parameters, eDNA, WSP and matrix proteins, showed no significant differences among the strains (p>0.05; Table 1). Pearson’s Correlation was applied to compare the ECM components between each other. This analysis showed a significant correlation (p<0.05) between the ASP and protein content in the WT strain (r= 0.666) as well as for protein and matrix protein (r= 0,670) (Table 2). The population of Δ/Δ efg1 significantly correlated to the protein content of its ECM (r= 0.734) (Table 3). The Δ/Δ tec1 mutant strain showed a significant correlation between the ASP content and the eDNA in its ECM (r= 0.678) (Table 4). 26 http://www.comstat.dk/ 28 The WT strain present typical thick biofilm architecture in visual appearance (a) with the presence of abundant ECM (b and c), as observed by VPSEM. In contrast, Δ/Δ efg1 mutant shows sparse thin biofilm growth patterns (d) and morphologically distinct ECM (e, f) in comparison to the WT (b, c) and Δ/Δ tec1 strains (h, i). The Δ/Δ tec1 mutant present defects in visual appearance (g) compared to the WT strain (a), however, its ECM is morphologically similar to WT (h, i). CSLM representative images of the 48 h biofilms of C. albicans strains are shown in Fig. 3. Multidimensional imaging of live (green) cells can be observed at different depths of the biofilms. In addition, negligible amount of dead (red stained) cells were observed (not depicted in Fig. 3). The images show that the biofilm formed by the WT strain (Fig. 3a-1) has an elevated number of hyphae, contrary to the biofilm formed by Δ/Δ efg1 mutant strain, that did not exhibit hyphae (Fig. 3a-2). The biofilm formed by Δ/Δ tec1 mutant strain (Fig. 3a-3) exhibited hyphae, but in a smaller quantity than the WT strain (Fig. 3a-1). The orthogonal view of biofilms showed that the mutant strains biofilms of Δ/Δ efg1 (Fig. 3a-2) and Δ/Δ tec1 (Fig. 3a-3) are thinner than the WT (Fig. 3a-1), specially the Δ/Δ efg1, which possesses the thinner structure confirmed by the biovolume (Fig. 3b) and maximum thickness of biofilms (Fig. 3c). The profiles of the distribution of C. albicans WT and mutant strains in the 48 h-old biofilms are shown in Fig. 3d, where it can be observed that the biofilm profile of the WT and Δ/Δ tec1 strains are similar, while the Δ/Δ efg1 mutant showed a very low percentage of coverage. Discussion The biofilm architecture contributes to the maintenance of stable interactions between cell-cell, cell-surface, and the environment [31]. Furthermore, it protects against phagocytic cells and works as a scaffold for preserving biofilm integrity by limiting the diffusion of noxious substances into the biofilm [32]. Although biofilm resistance is multifactorial [33], the protection exerted by the ECM is a key supporter to the high levels of antifungal drug resistance 27 29 displayed by C. albicans biofilms [13, 14, 34]. It has been demonstrated that transcriptional regulatory genes in C. albicans, including TEC1 and EFG1, regulate biofilm formation [23, 35, 36]. Thus, understanding how these transcriptional regulatory genes influence the ECM composition is paramount to better prevent or impair biofilm formation. The present study demonstrated that ASP are major components of the ECM of the WT strain, and that these components are significantly reduced in the mutant strains with biofilm formation deficiency (Δ/Δ efg1 and Δ/Δ tec1), being Δ/Δ efg1 the strain that possess the smaller quantities of these components. A significant correlation (p<0.05) between the protein and the ASP content in the ECM of the WT strain (r=0.666) was present, so the production of ASP in this strain might be influenced by the proteins and vice-versa (Table 2). It appears that filamentous cells build-up ECM richer in ASP than non-filamentous cells. Early studies showed a correlation between ECM and levels of resistance against fluconazole and amphotericin B [37, 38]. An ECM component with a role in antifungal drug resistance is β-1,3-glucan (an ASP) which acts through a mechanism of drug sequestration. Delivery of β-1,3-glucan to the ECM is controlled by a glucan-modifying pathway composed of Bgl2p and Phr1p (glycosyltransferases) and Xog1p (glucanase) [39]. It has been shown that the deletion of any of the genes encoding these proteins resulted in at least 10-fold reduction in matrix β -1,3-glucan content and formed more vulnerable biofilms, that were easily disrupted [39]. Moreover, biofilms formed by these mutants showed less ability to sequester fluconazole and higher susceptibility to this drug [39]. In addition to drug sequestration, it has been demonstrated that the production of β-1,3-glucan by C. albicans biofilms hinders the production of reactive oxygen species by neutrophils and protects the cells in biofilm from neutrophil killing [40]. The β-1,3-glucan binds to fluconazole preventing this drug from reaching its cellular targets [13, 39, 41-42]. A similar effect was detected for amphotericin B [18] and for other classes of antifungal agents as well [41]. It has been demonstrated that EFG1 28 30 intermediates tolerance of C. albicans to azoles (i.e. fluconazole, ketoconazole, itraconazole) and polyenes, including amphotericin B [43]. Moreover, a mutant Δ/Δ efg1 C. albicans strain showed higher susceptibility to these drugs, including miconazole and caspofungin [43, 44]. Thus, the ASP of the ECM has a potential role in the tolerance to antifungals and biofilm structure, since it is much more accumulated in the ECM of the WT strain in contrast to the more susceptible mutant Δ/Δ efg1. Another ECM component with a role in antifungal drug resistance is eDNA. This component is a key matrix component of fungal and bacterial biofilms that enables adhesion to distinct surfaces and binds with other biopolymers, giving biofilms structural integrity and stability [34, 45, 46]. The addition of DNase increases the susceptibility of mature C. albicans biofilms against some antifungal agents [47]. The present study found a significant correlation between the eDNA and ASP content in the ECM of Δ/Δ tec1 mutant strain (r=0.678) (Table 4), indicating that the production of these components happens together on each other in this strain. Although the precise mechanism by which eDNA is released and contributes to drug resistance remains unclear [34], it has been suggested that eDNA may be released during hyphal growth [48]. However, the present study shows that independent of the presence of hyphae all the biofilms produced similar quantities of eDNA, contrary to ASP, where filamentous cells produces more ASP that non-filamentous cells. Therefore, the mutant strain Δ/Δ efg1 that does not form hyphae can produce eDNA and this indicates that the eDNA production may not be necessarily related to hyphal growth. The Δ/Δ efg1 population (Log10 CFU mL-1) is higher than WT and Δ/Δ tec1 cells. The smaller size and unicellularity can be considered reasons to explain differences in fungal population observed between mutant Δ/Δ efg1 and WT strains [49]. Moreover, cells lacking EFG1 genes showed increased colonization of the gastrointestinal tract of mice [50]. As the Δ/Δ efg1 mutant strain did not form hyphae, the smaller size of its cells might have influenced 29 31 in the higher number of viable cells for colony counting. In contrast, the strain Δ/Δ tec1 presented similar population to WT strain, which are statistically smaller than the Δ/Δ efg1 mutant strain. This might have happened probably because these strains present hyphae. However, the differences in population between all the strains is less than 1 log, meaning that this result may not be biologically significant. On the other hand, the biomass of the mutant strains (Δ/Δ efg1 and Δ/Δ tec1) is lower than the biomass of the WT strain, confirming that these mutants have defective capability of forming biofilm. In addition, the biovolume of the mutant strains is also lower than the WT one (Fig. 3). The quantitative analysis of proteins showed the mutant strain Δ/Δ efg1 possesses higher contents of proteins when compared to the WT and Δ/Δ tec1 mutant strain, which makes sense because Log10 (CFU mL-1) significantly influenced the protein in the biofilm of this strain (r=0.734) (Table 3). Pearson’s correlation test applied to the current data showed a significant (p<0.005) correlation between the protein and the matrix proteins (r= 0.670) in the WT strain (Table 2). Proteins encompass a large portion of the biomass in many microbial biofilms [31, 51, 52], having been shown to comprise more than half of the C. albicans ECM by weight [16]. A comparison of the matrix proteome of C. albicans and total matrix proteins identified several similarities between them, including a large amount of proteins involved in carbohydrate and amino acid metabolism [53, 54]. The known function of most proteins is related to metabolism [16], suggesting that the ECM might “function as an external digestive structure that disrupts extracellular biopolymers as an energy source” [16]. Thus, the higher amounts of protein related to the higher population values observed in the Δ/Δefg1 might represent an extra effort of these strain to obtain energy to arrange as a biofilm. Evaluation of intact biofilm architecture via VPSEM and CSLM provide valuable information of cells and ECM spatial organization. VPSEM preserves the ECM since it does not require sample dehydration process and high chamber vacuum [28]. The images show that 30 32 while WT strain present typical bulky biofilm architecture encased by extracellular matrix material (Fig. 2b and Fig. 2c), the Δ/Δ efg1 mutant strain is morphologically distinct from the WT (Fig. 2e and Fig. 2f). On the other hand, Δ/Δ tec1 mutant formed a biofilm with ECM comparable to the WT (Fig. 2h and Fig. 2i). Thus, the similarity between the ECM produced by the WT and Δ/Δ tec1 mutant strain can be related to the growth of pseudo hyphae and hyphae, which goes along with the production of ECM [55]. Moreover, the CLSM images corroborate the results obtained with VPSEM, showing that WT reference strain formed a thick and bulky biofilm on the surface of the polystyrene plate (Fig. 3a-1). However, the mutant strains tested showed defects in biofilm formation, especially Δ/Δ efg1, which is severely defective (Fig. 3a-2), while the Δ/Δ tec1 mutant had less pronounced defects (Fig. 3a-3). These results agree with a study that analyzed the same strains [23]. The measurements of the biovolume and maximum biofilm thickness obtained by COMSTAT2 confirms these results, showing that the Δ/Δ efg1 mutant strain has the lowest biovolume and maximum thickness (p>0.05) compared to the other studied strains. The biofilm profile of this strain is markedly different from the other studied strains, showing a small percentage of coverage area (Fig. 3d). In addition, previous data shows that Δ/Δ efg1 mutant strains have impaired hyphae growth under many conditions [19, 21]. Contrary to a previous report that found that biofilm produced by the tec1 null mutant (Δ/Δ tec1) strain was composed exclusively of yeast cells [17], in the present study it was observed that Δ/Δ tec1 is a defective mutant that exhibits hyphae; however, its biofilm displayed defective visual appearance and smaller quantity of hyphae when compared to the WT strain (Fig. 3a-3). Despite of the visual and microscopically differences between WT and Δ/Δ tec1 strains, they have a comparable biovolume and maximum biofilm thickness (p>0.05); in addition, the biofilm percentage coverage profile of these strains is also similar (Fig. 3d). 31 33 This study characterized the ECM of C. albicans WT and mutant strains derived from it. Despite that the Δ/Δ efg1 mutant strain present severe biofilm defects, its cells grow more in numbers than the WT and Δ/Δ tec1 mutant strains and it matches the higher quantity of proteins in biofilm. The amounts of eDNA, WSP and matrix soluble proteins are similar between the strains, but the eDNA correlates with the ASP content in the ECM of the Δ/Δ tec1 strain. On the other hand, the ASP content is significantly higher in the WT strain in comparison to the mutant strains, which indicates that ASP production may be linked to C. albicans cell filamentous morphology. Acknowledgements: We thank Dr. Alexander D. Johnson, Department of Microbiology and Immunology, UCSF, for his kind donation of the strains used in this study. Funding: This work was supported by the São Paulo Research Foundation (FAPESP, [Grant # 2014/18804-1 and 2016/00256-3]); and by the National Institute in Basic Optics and Applied to Life Sciences (FAPESP [Grant # 2014/50857-8] and National Counsel of Technological and Scientific Development – CNPq ([Grant # 465360/2014-9]). References 1. Eggimann P, Garbino J, Pittet D. Epidemiology of Candida species infections in critically ill non-immunosuppressed patients. Lancet Infect Dis. 2003; 3:685-702. 2. Sudbery P, Gow N, Berman J. 2004. The distinct morphogenic states of Candida albicans. Trends Microbiol. 2004; 12: 317-24. 3. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev. 2007; 20:133-63. 32 34 4. Maurya V, Srivastava A, Mishra J, Gaind R, Marak RS, Tripathi AK, Singh M, Venkatesh V. Oropharyngeal candidiasis and Candida colonization in HIV positive patients in northern India. J Infect Dev Ctries. 2013; 7: 608-13. 5. 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Proteomics. 2006; 6:5795–804. 54. Martínez-Gomariz M, Perumal P, Mekala S, Nombela C, Chaffin WL, Gil C. Proteomic analysis of cytoplasmic and surface proteins from yeast cells, hyphae, and biofilms of Candida albicans. Proteomics. 2009; 9: 2230–52. 55. Nobile CJ, Nett JE, Hernday AD, Homann OR, Deneault JS, Nantel A, Andes DR, Johnson AD, Mitchell AP. Biofilm matrix regulation by Candida albicans Zap1. PLoS Biol. 2009; 7: e1000133. 38 40 Table 1. Population and biochemical composition of C. albicans biofilms. Mean and standard deviations of Log10 (CFU mL-1), eDNA (µg), WSP (µg), ASP (µg), soluble (µg) and total protein (µg) for C. albicans SN 425 (wild-type), C. albicans CJN 2302 (Δ/Δ efg1) and C. albicans CJN 2330 (Δ/Δ tec1). Biofilm components ECM components Strains CFU mL-1 Dry weight (mg) Protein (μg) ASP (μg) WSP (μg) eDNA (μg) Matrix protein (μg) Log10 C. albicans SN 425 (WT) 6.89 A 24.5 A 47.8 A 74.5 A 47.7 A 20.1 A 13.8 A (0.07) (3.7) (1.5) (22.0) (13.4) (7.1) (9.8) C. albicans CJN 2302 (Δ/Δ efg1) 7.16 B 18.4 B 50.3 B 14.7 B 52.2 A 27.4 A 20.4 A (0.17) (2.8) (2.3) (5.0) (7.8) (7.6) (11.3) C. albicans CJN 2330 (Δ/Δ tec1) 6.83 A 18.5 B 46.5 A 44.0 C 46.1 A 34.2 A 17.4 A (0.18) (3.0) (1.4) (24.1) (11.3) (14.4) (4.3) Comparisons by one-way ANOVA and Tukey post-hoc test: means followed by the same letter in column are not significantly different from each other. 39 41 Table 2. Pearson correlation of Log10 (CFU mL-1) and ECM components for the WT strain (SN 425). WT strain (SN 425) Log10 ASP WSP Total Protein Matrix Protein eDNA Log10 Pearson Correlation 1 .372 .262 .173 .017 -.167 Sig. (2-tailed) .234 .411 .590 .957 .605 N 12 12 12 12 12 ASP Pearson Correlation 1 .184 .666* .361 .273 Sig. (2-tailed) .567 .018 .249 .391 N 12 12 12 12 WSP Pearson Correlation 1 .065 .017 -.326 Sig. (2-tailed) .841 .959 .301 N 12 12 12 Protein Pearson Correlation 1 .670* .376 Sig. (2-tailed) .017 .229 N 12 12 Matrix Protein Pearson Correlation 1 -.032 Sig. (2-tailed) .921 N 12 eDNA Pearson Correlation 1 Sig. (2-tailed) N * Correlation is significant at the 0.05 level (2-tailed) 40 42 Table 3. Pearson correlation of Log10 (CFU mL-1) and ECM components for the Δ/Δ efg1 mutant strain (CJN 2302) Δ/Δ efg1 Log10 ASP WSP Total Protein Matrix Protein eDNA Log10 Pearson Correlation 1 .246 .057 .734* -.286 -.315 Sig. (2-tailed) .442 .861 .010 .367 .318 N 12 12 12 12 12 ASP Pearson Correlation 1 .477 -.133 .252 .290 Sig. (2-tailed) .117 .696 .429 .361 N 12 12 12 12 WSP Pearson Correlation 1 .236 -.160 .080 Sig. (2-tailed) .485 .619 .805 N 12 12 12 Protein Pearson Correlation 1 -.205 .538 Sig. (2-tailed) .545 .071 N 12 12 Matrix Protein Pearson Correlation 1 -.247 Sig. (2-tailed) .463 N 12 eDNA Pearson Correlation 1 Sig. (2-tailed) N * Correlation is significant at the 0.05 level (2-tailed). 41 43 Table 4. Pearson correlation of Log10 (CFU mL-1) and ECM components for the Δ/Δ tec1 mutant strain (CJN 2330). Δ/Δ tec1 Log10 ASP WSP Total Protein Matrix Protein eDNA Log10 Pearson Correlation 1 -.080 .171 -.155 -.171 -.194 Sig. (2-tailed) .804 .596 .630 .595 .567 N 12 12 12 12 12 ASP Pearson Correlation 1 .285 .106 .190 .678* Sig. (2-tailed) .369 .743 .554 .022 N 12 12 12 12 WSP Pearson Correlation 1 -.002 -.535 .219 Sig. (2-tailed) .995 .073 .518 N 12 12 12 Protein Pearson Correlation 1 .000 .098 Sig. (2-tailed) 1.000 .775 N 12 12 Matrix Protein Pearson Correlation 1 .322 Sig. (2-tailed) .334 12 eDNA Pearson Correlation 1 N * Correlation is significant at the 0.05 level (2-tailed). 42 44 Fig. 1: Biofilm characterization flowchart. WSP: Quantification by phenol-sulfuric acid method. ASP: Quantification by phenol-sulfuric acid method. Biofilm in 2 mL of 0.89% NaCl Supernatant (Soluble content of the ECM) Pellet washed twice with water 100 µL for plating Pellet resuspended with water (Insoluble content of the ECM and cells) Centrifugation at 5,500 g (10 min/4ºC) Protein: Quantification by Bradford method. eDNA: Isolation by phenol- chloroform method. Matrix protein Quantification by Bradford method. 100 µL for dry weight 43 45 Figure 2. Visual macroscopic appearance and overall C. albicans biofilm structure by VPSEM. The WT strain present typical thick biofilm architecture in visual appearance (a) with the presence of abundant ECM (b and c), exemplified by the arrows (c). The Δ/Δ efg1 mutant shows sparse thin biofilm growth patterns (d) and an ECM (e, f) morphologically distinct from the WT (b, c) and Δ/Δ tec1 strains (h, i). The Δ/Δ tec1 mutant present defects in visual appearance (g) compared to the WT strain (a), however, its ECM is morphologically similar to the WT (h, i, see arrows). 44 46 Figure 3. C. albicans biofilms structures and corresponding quantitative data from CLSM assays. (a) Representative three-dimensional and orthogonal images of the structural organization of the 48h biofilms of C. albicans wild-type (1) and mutant strains- Δ/Δ efg1 (2) and Δ/Δ tec1(3) (green color denotes labeling with SYTO9 for live yeast cells). Mean and SD of biovolume (b) and average biofilm thickness (c) of C. albicans wild-type and mutant strains (Δ/Δ efg1 and Δ/Δ tec1) determined by COMSTAT2 analysis. The average biovolume and biofilm thickness were calculated from five independent samples from each strain. Values followed by the same letter are not significantly different (P>0.05), as determined by an analysis of variance for all pairs using Tukey’s test. The profile of the distribution of C. albicans WT and mutant strains (Δ/Δ efg1 and Δ/Δ tec1) in the biofilms is represented in (d). 45 47 Supplemental material Figure S1: Growth curves of C. albicans strains SN 425 (WT), CJN 2302 (Δ/Δ efg1) and CJN 2330 (Δ/Δ tec1). Planktonic cultures were performed in YNB medium supplemented with 100 mM of glucose and incubated at 37º C. The optical density (OD at 540 nm) and the population (Log10 CFU mL-1) were determined over time. 4,5 5 5,5 6 6,5 7 7,5 0 0,2 0,4 0,6 0,8 1 0 2 4 6 8 10 12 14 Lo g 1 0 C FU m L-1 O D 5 4 0 n m Hours Δ/Δ tec 1 OD Log 4,5 5 5,5 6 6,5 7 7,5 0 0,2 0,4 0,6 0,8 1 0 2 4 6 8 10 12 14 Lo g 1 0 C FU m L-1 O D 5 4 0 n m Hours WT OD Log 4,5 5 5,5 6 6,5 7 7,5 0 0,2 0,4 0,6 0,8 1 0 2 4 6 8 10 12 14 Lo g 1 0 C FU m L-1 O D 5 4 0 n m Hours Δ/Δ efg 1 OD Log 46 48 3.2 Publicação 2 Fluconazole impacts the extracellular matrix of fluconazole-susceptible and resistant Candida albicans and Candida glabrata biofilms. Beatriz Helena Dias Panariello, Marlise I. Klein, Ewerton Garcia de Oliveira Mima, Ana Cláudia Pavarina* Department of Dental Materials and Prosthodontics, São Paulo State University (Unesp), School of Dentistry, Araraquara, São Paulo, Brazil. *Corresponding Author: Dr. Ana Cláudia Pavarina e-mail: pavarina@foar.unesp.br Phone: +55 16 33016544 Fax: +55 16 33016406 Address: Rua Humaitá, 1680, 14801-903, Araraquara, São Paulo, Brazil. *Artigo formatado de acordo com o periódico JOURNAL OF ORAL MICROBIOLOGY 47 49 ABSTRACT Background: Fluconazole (FLZ) is a drug commonly used for the treatment of Candida infections. However, β-glucans in the extracellular matrices (ECM) hinder FLZ penetration into Candida biofilms, while extracellular DNA (eDNA) collaborates with biofilm’s architecture and resistance. Methods: This study characterized biofilms of fluconazole-sensitive (S) and -resistant (R) Candida albicans and Candida glabrata in the presence or absence of FLZ focusing the ECM traits. Biofilms of C. albicans ATCC 90028 (CaS), C. albicans ATCC 96901 (CaR), C. glabrata ATCC 2001 (CgS) and C. glabrata ATCC 200918 (CgR) were grown with RPMI medium with or without biofilm FLZ sub-minimum inhibitory concentrations (MIC) per strain (37°C/48 h). Biofilms were assessed by CFU/mL, biomass and ECM components (alkali- soluble polysaccharides-ASP, water-soluble polysaccharides-WSP, eDNA and proteins). Scanning Electron Microscopy (SEM) was also performed. Data were analyzed by two-way ANOVA tests (p ≤ 0.05). Results: In biofilms, FLZ reduced the CFU/mL of all strains (p=0.000), except for CaS (p=0.937). However, the ASP amounts in CaS were significantly reduced by FLZ (p=0.034), while the drug had no effect on the ASP amounts of the other strains (p>0.05). Total biomasses and WSP were significantly reduced by FLZ in the ECM of all microorganisms (p=0.000), but eDNA and proteins amounts were not influenced by the presence of FLZ nor by the type of the strain (p>0.05). FLZ affected the cell morphology and biofilm structure by hindering hyphae formation in CaS and CaR biofilms, by decreasing the number of cells in CgS and CgR biofilms and by yielding sparsely spaced cell agglomerates on the substrate. Conclusion: FLZ hindered the accumulation of WSPs and reduced the biomasses of the biofilms by decreasing hyphae prolongations and the number of cells. KEYWORDS: Biofilm, Candida albicans, Candida glabrata, Extracellular matrix, Fluconazole, Fluconazole-resistant. 48 50 Introduction Candida spp. are commensal fungi of the oral cavity of healthy individuals that can become opportunistic pathogens in some situations, for example, when there are changes in immune system, metabolic dysfunction or in high-age population [1-7]. Moreover, growing usage of broad-spectrum antibiotics, cytotoxic chemotherapies and transplantation also intensifies the risk for infections by these opportunistic fungi [7]. These infections are known as candidiasis. Candida albicans is the main specie associated to this disease, while Candida glabrata is the most predominant non-albicans specie that has been linked to the development of oral infections [1-6]. C. glabrata is considered a pathobiont pathogen [7-8], mainly due to its innate resistance to azoles [9-10]. Candida infections are frequently related to the establishment of biofilms. Biofilms are composed by microbial cells that are attached to a substrate and surrounded by an extracellular matrix (ECM) [11]. The ECM is composed of secreted microbial and host-derived substances and cells lysis [11], and collaborates to the conservation of the biofilms architecture and to the preservation of stable interactions between cell-cell, cell-surface, and the environment [12]. Although polysaccharides and protein are the most widely studied substances in biofilms ECMs, other molecules, such as nucleic acids, are important to their function [11]. Treatments for oral infections caused by Candida use topical [13] and systemic [14] antifungal medication, such as Fluconazole (FLZ). Systemic antifungal medication is usually prescribed for individuals with compromised overall health and in the episodes of recurrent infections [14]. An important aspect to be considered in therapy with topical or systemic antifungal refers to the resistance that Candida species can present to these drugs. Resistance to antifungal agents can be defined as the persistence or progression of an infection after application of antimicrobial treatment [15-16]. Some studies have observed the emergence of resistant microorganisms during long-term or prophylactic treatment [17-18]. Intrinsic or 49 51 primary resistance occurs when a microorganism has low susceptibility to a medication, prior to its exposure to the agent. Some Candida species possess intrinsic resistance to antifungal drugs, especially to fluconazole [7, 10]. In contrast, the secondary resistance is one that can be developed by the microorganism after long periods of exposure to antifungal drugs [15]. Thus, a major concern with Candida spp. biofilms is that their cells may have reduced susceptibility against azoles and polyenes, due to development of resistance [19-20]. The resistance of Candida biofilms is multifactorial and involves the stimulation of drug efflux pumps, the cells physiological state and the protection exerted by the ECM via β-glucans, that bind to FLZ and amphotericin B [21], avoiding the diffusion of these antifungals through biofilms [22]. C. albicans biofilms that grew under constant flow produced a higher quantity of ECM than those grown statically and presented higher resistance to amphotericin B, showing that the matrix can expressively influence on drug resistance in Candida [23]. In the same study, the authors detected that the ECM of C. albicans GDH 2346 (NCYC 1467) had carbohydrate, protein, hexosamine, phosphorus and uronic acid. Nevertheless, the main constituent of the ECM C. albicans was glucose (32%) [23]. Besides β-glucan, it has been revealed that the extracellular DNA (eDNA) is also a relevant constituent of the ECM that guarantees the structure integrity of C. albicans biofilm [24]. Furthermore, nuclear magnetic resonance (NMR) investigation verified interaction of ECM aggregate with fluconazole, demonstrating a role in drug resistance [25]. The difficulties in determining the key ECM components have been a challenge to the understanding of how the ECM interferes in Candida drug resistance [26]. Thus, better knowledge of the assemblage and functional properties of the matrix and FLZ influence on it will enable the design of more effective therapies to control Candida pathogenesis and biofilm development Here we characterized the biofilms of fluconazole-susceptible and -resistant C. 50 52 albicans and C. glabrata strains in the presence or absence FLZ to evaluate the drug interference in the ECM of these microorganisms. Material and Methods Microorganisms Four American Type Culture Collection (ATCC) Candida species were used in this study. C. albicans ATCC 90028 (fluconazole-susceptible; CaS) was originally isolated from blood in Iowa, USA. C. albicans ATCC 96901 (fluconazole-resistant; CaR) was isolated from the mouth of an HIV-positive patient in Omaha, NE, USA. C. glabrata ATCC 2001 (fluconazole-susceptible; CgS) was isolated from feces in Wayne, PA, USA and C. glabrata ATCC 200918 (fluconazole-resistant; CgR) was isolated from human tongue in Santa Rosa, CA, USA. Growth curves To ensure the reproducibility of the biofilms model, growth curves were constructed based on optical density (OD) at 540 nm wavelength. Growth curves were performed on three different occasions with three replicates. The number of colony forming units (CFU) were determined at four-time points (Figure 1). The microorganisms kept at -80ºC were seeded onto Petri dishes with SDA (Sabourand dextrose agar- DIFCO, Detroit, Michigan, USA) culture medium supplemented with chloramphenicol (50 mg/L) [27] and incubated at 37°C for 48 h. Then, 5 colonies of each microorganism were added separately to tubes containing YNB medium (Yeast Nitrogen Base- DIFCO, Detroit, Michigan, USA) supplemented with 100 mM of glucose [27] and these pre- inoculums were incubated at 37°C for 16 h. Afterwards, the pre-inoculums were diluted with fresh YNB medium plus 100 mM glucose (1:20 dilution for C. albicans strains and 1:10 for C. glabrata strains) to form the inoculums, and the OD540 nm and CFU were determined every 2 h 51 53 in a total of 16h for each strain. Biofilms formation was performed with cells at mid-log growth phase of each microorganism (Figure 1). Susceptibility testing and fluconazole concentrations for biofilms formation The CLSI M27-A3 broth microdilution susceptibility method [28] was performed to examine the minimal inhibitory concentrations (MIC) of FLZ against planktonic fluconazole susceptible and –resistant C. albicans and C. glabrata strains. Fluconazole powder (F8929, Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile ultrapure water [29]. As a negative control, a 100 µL of 2× concentrated RPMI 1640 buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic (MOPS) plus a 100 µL of sterile ultrapure water was used (no cells and antifungal agent). As a positive control, only cell suspensions were tested without the antifungal agent. Serial two-fold dilutions of fluconazole (range: 0.125 to 512 μg/ml) in RPMI 1640 medium buffered to pH 7.0 with 0.165 MOPS buffer were inoculated in 96-well plates with each microorganism suspension adjusted to achieve a final inoculum concentration of 0.5x103 to 2.5x103 cells/mL based on the growth curves (Figure 1). The plates were incubated at 37°C and observed for the presence or absence of growth at 24 h. In addition to visual endpoint readings, the optical density of each plate well was measured at 562 nm after 24 h of incubation. MICs in spectrophotometer were based on the density of the growth control and were considered the lowest drug concentrations that resulted in minimum 90% decrease in growth related to the drug-free growth control [30-31]. As cells in biofilms are more resistant to drugs [32], the concentrations of 5X and 10X were tested in biofilms [33]. The 5X MIC concentration was chosen because 10X MIC almost completely inhibited the formation of biofilm (data not shown), and this was not the goal of the study since the inhibition of biofilm does not allow the evaluation of FLZ effects on biofilms’ ECMs. Thus, 5X MIC concentrations were used for biofilm formation. 52 54 Biofilm formation and processing Biofilms formation and processing for Log10 CFU/mL, total biomass, insoluble biomass, proteins (from the insoluble part of the biofilm and from the supernatant), ASP, WSP and eDNA were performed according to the methodology described by Panariello et al. (2017) [34]. Briefly, biofilms of CaS, CaR, CgS and CgR were formed for 48 h in RPMI buffered with MOPS (pH 7.0). Biofilms (48 h) were washed twice with 0.89% NaCl and detached by individually scratching the wells with a pipette tip and 2 mL 0.89% NaCl. Then, biofilms were separated for Log10 (CFU/mL) determination (0.1 mL) and for total biomass determination [35] (0.1 mL). After that, biofilms were dynamically vortexed, centrifuged (5,500 x g/10 min/4ºC) and the supernatant containing the soluble part of the matrix was separated from the pellet, which contains the insoluble part of the matrix. The supernatant was divided for the quantification of WSPs (1 mL) [36], eDNA (0.650 mL) [37] and matrix protein in the soluble portion (0.150 mL) [38]. The pellet was washed twice and resuspended in sterile ultrapure water, then, it was separated for the quantification of insoluble biomass (0.8 mL), protein from the insoluble portion (0.05 mL) [38] and ASPs (0.95 mL) [36]. Scanning electron microscopy (SEM) Biofilms were grown over polystyrene pieces obtained from the bottom of 24-well plates [39]. Before its use, they were sterilized in a microwave for 3 min at 650 W [40] and dried at flow chamber with UV light for 30 minutes. After 48 h of biofilms growth, the media was removed, and the wells were washed twice with 1 mL of sterile 0.89% NaCl. Next, the biofilm samples were fixed with 2.5% glutaraldehyde (60 min/ room temperature), washed twice with sterile 0.89% NaCl, and dehydrated. The dehydration process was performed with series of washes with ethanol 70% and 90% for 60 min each, followed by 5 washes of 30 min with absolute ethanol. The samples were stored in a desiccator with silica for 7 days to guarantee moisture- free samples. Then, the samples were fixed in aluminum stubs, sputter coated with gold, and 53 55 observed with a JEOL JSM-6610LV Scanning Electron Microscope, using magnifications of 160, 600 and 1200. Statistical analyses Normal distribution of data was verified by Shapiro-Wilk test and homogeneity of variance was checked by Levene test (α = 0.05). The quantitative data of CFU/mL, biomass, proteins from the insoluble portion and ECM components were statistically analyzed by two- way analysis of variance (ANOVA) considering the presence or absence of FLZ and the different strains (CaS, CaR, CgS, CgS). When the postulation of normality was not encountered, data were ranked and non-parametric analysis (ANOVA on ranks) was applied (α=0.05). For multiple comparisons, Tukey post hoc test was applied for homoscedastic data and Games-Howell post hoc test for heteroscedastic data (α=0.05). Analyses were done in the software SPSS (IBM® SPSS® Statistics, version 20, Chicago, IL, USA). Results Susceptibility testing and fluconazole concentrations for biofilms formation The MIC was performed in duplicate at three different occasion. The MIC90 for planktonic cells were: CaS= 16 µg/mL; CaR= 256 µg/mL; CgS= 8 µg/mL and CgR= 256 µg/mL. The MIC90 concentration found for planktonic cells of CaS; however, is not typical for sensitive strains. As cited before, cells in biofilms are more resistant than cells in planktonic cultures, thus, for biofilm formation, 5X MIC was applied: CaS= 80 µg/mL; CaR= 1280 µg/mL; CgS= 40 µg/mL and CgR= 1280 µg/mL. Biofilm and ECM characterization Log10 (CFU/mL) presented normal distribution and homoscedasticity (Levene test: p=0.110), thus it was analyzed by two-way ANOVA with “FLZ” and “strains” as main effects. There was a significant interaction between the factors “FLZ” and “strains” (p=0.000), so one-way ANOVA followed by Tukey post hoc test was performed. FLZ 54 56 significantly reduced (p=0.000) the CFU/mL of CaR (reduction of 0.88 logs), CgS (reduction of 0.71 logs) and CgR (reduction of 0.70 logs). On the other hand, the statistics pointed that the reduction of 0.67 logs caused by FLZ on CaS biofilms was not statistically significantly (p=0.937). Moreover, CaS presented the lowest CFU/mL of all the strains in the absence of FLZ (p=0.000). Mean and standard deviations of Log10 (CFU/mL) are represented in Figure 2. The total biomass data did not show normal distribution; therefore, data were ranked. The non-parametric analysis showed that the factor “FLZ” caused significant interference in the results (p=0.000), reducing the biomasses of all the strains (Figure 3). The insoluble biomass data did not show normal distribution; thus, data were ranked, and non-parametric analysis was performed. An interaction between the factors “FLZ” and “strains” was observed (p=0.000). Levene’s test showed that data were heteroscedastic (p=0.06), so one-way ANOVA on ranks with Welch correction and Games-Howell post hoc test was applied. The insoluble biomass of CaS (p=0.038), CaR (p=0.000) and CgR (p=0.000) were significantly reduced by the presence of FLZ. On the other hand, CgS showed a higher insoluble biomass in the presence of FLZ when compared to CgS in the absence of FLZ (p=0.000). Mean and standard deviations of insoluble biomasses are represented in Figure 4. Protein data from the insoluble portion met the criteria of normality, therefore two-way ANOVA was performed, showing that there were no interactions of factors “FLZ” and “strain” in the results obtained (p>0.224). Thus, no post hoc tests were performed. Consequently, protein amounts from the insoluble portion were not affected by FLZ and the quantities of this component are not influenced by the type of the strain (Figure 5). For WSP data the assumption of normality was not found, thus data were ranked. The non-parametric analysis showed that the only factor that influenced the quantity of WSP was “FLZ” (p=0.000), reducing this component in the ECM of all the evaluated strains (Figure 6). 55 57 ASP data did not meet the assumptions of normality; therefore, data were ranked. Levene’s test pointed to the homogeneity of variances (p=0.294). As the non-parametric test showed that there was a significant interaction between the factors “FLZ” and “strains” (p=0.002), data were submitted to one-way ANOVA on ranks with Tukey post hoc test. Statistics demonstrated that ASP amount was significantly reduced by FLZ on CaS (p=0.034). However, for the other strains, FLZ did not alter ASP amounts (p>0.200). Moreover, CgS produces significantly smaller amounts of ASP than the other evaluated strains (p=0.008). Mean and standard deviations of ASP are represented in Figure 7. eDNA data did not show normal distribution, so data were ranked. The non-parametric analysis revealed that there were no interactions of any factors in eDNA data (p>0.006), thus, no post hoc tests were performed. Therefore, eDNA amounts were not affected by FLZ and the type of the strain does not interfere with the amounts of eDNA (Figure 8). Matrix protein data did not meet the assumptions of normality and data were ranked. The non-parametric analysis showed that the “strain” was the only factor that interacted with the results obtained (p= 0.000). Levene’s test showed that matrix protein data are homoscedastic (p=0.176), therefore one-way ANOVA on ranks with Tukey post hoc test was applied for multiple comparisons. However, the analysis showed no statistical differences in the amount of matrix proteins between the strains (p>0.05) (Figure 9). Biofilm structure SEM was performed to examine the overall biofilm structure of different microorganisms in the presence and in the absence of FLZ. Especial attention was given to the cells morphology and spatial organization on substrate, as well as on ECM covering and/or linking cells in these biofilms. Figure 10 shows the SEM images of CaS (Figure 10 A, B, C) and CaS+FLZ (Figure 10 D, E, F), demonstrating that FLZ reduced the size of hyphae. A closer 56 58 look at the structures shows that without FLZ cells are embedded in the ECM, as shown by the arrow (Figure 10 C). Figure 11 shows the SEM images of biofilms formed by CaR (Figure 11 A, B and C) and CaR+FLZ (Figure 11 D, E and F). The CaR biofilm possesses large amounts of yeast cells and elongated hyphae structures (Figure 11 A, B, C). Figure 11 C shows the union of cells within biofilms through their extracellular matrices, as exemplified by the arrow. FLZ reduced the size and number of hyphae, remaining mostly cells with yeast morphology (Figure 11 C, D, E). Figure 12 shows the SEM images of biofilms formed by CgS (Figure 12 A and B) and CgS+FLZ (Figure 12 C and D). CgS biofilm is composed by yeast cells (Figure 12 A) linked by ECM (Figure 12 B). The presence of FLZ reduced the number of cells (Figure 12 C) and the ECM (Figure 12 D). Figure 13 shows the SEM images of biofilms formed by CgR (Figure 13 A and B) and CgR+FLZ (Figure 13 C and D). CgR biofilm presents a high quantity yeast cells (Figure 13A) and clusters interconnected by ECM (Figure 13 B). The presence of FLZ reduced the number of cells (Figure 13 C) and the ECM (Figure 13 D). Discussion Fluconazole is often a chosen treatment for Candida infections because of its low cost and availability for oral administration [41]. This drug prevents the biosynthesis of ergosterol through the cytochrome P450 enzyme 14-α demethylase, which catalyzes the conversion of lanosterol to ergosterol [42]. The reduction of ergosterol changes the fluidity of the membrane and the activity of numerous membrane-bound enzymes, hindering the fungal growth and replication [42]. However, fluconazole-resistance is rising in Candida species [41, 44-45], requiring a better understanding of the action of this drug in Candida biofilms. Due to the mechanism of action of FLZ, variations in the architecture of the ECM may happen during 57 59 Candida biofilm formation in the presence of this medication [33, 46]. Here we characterized the biofilms formed by fluconazole-susceptible and -resistant C. albicans and C. glabrata strains in the presence or absence FLZ to evaluate the drug interference in the ECM of these microorganisms. Surprisingly, no significant reduction in CaS counts was observed after exposure to 5x MIC doses of FLZ (Figure 2), and the images showed that FLZ reduced the size of hyphae in strain (Figures 10 C, D, E). However, the growth of CaR biofilms in the presence or absence of FLZ showed that the drug significantly reduced its population (Log10 CFU/mL) (Figure 2). In addition, SEM images showed that FLZ reduced hyphae formation in CaR (Figures 11 D, E, F). This result corroborates to a previous study that observed that FLZ has a direct inhibitory effect on hyphal formation [47]. Because FLZ interferes with the ergosterol pathway and ergosterol is necessary for hyphae formation, the presence of FLZ inhibits the transition from yeast to hypha [47], even in a culture media that promotes C. albicans filamentous morphology. Thus, taking into consideration the hyphae is the invasive form of C. albicans [48], the reduction of its size in both CaS and CaR is an important result to the reduction of the virulence of this strain. Likely to what happened to C. albicans strains, FLZ significantly reduced the CFU/mL in CgS and CgR (Figure 2). These results were confirmed by the SEM images of CgS (Figures 12 D and E) and CgR (Figures 13 D and E), which showed fewer cells and cell agglomerates are more sparsely distributed on the substrate. It has been reported that C. glabrata has intrinsic decreased susceptibility to FLZ and another classes of azoles antifungals [41, 49-50], nevertheless, the results of the present study showed that biofilm formation with 40 µg/mL of FLZ for CgS and 1280 µg/mL of FLZ for CgR can reduce the cell viability of these microorganisms. Therefore, the log reduction values for all strains is very close (CaS: reduction of 0.67 logs; CaR: reduction of 0.88 logs; CgS: reduction of 0.71 logs and CgR: reduction of 0.70 logs). 58 60 An important finding of this study was that FLZ acted on the ECM of all the microorg