Please use this identifier to cite or link to this item: http://hdl.handle.net/1822/11217

TitleInsights into Candida world : the extracellular milieu
Author(s)Martins, Margarida Isabel Barros Coelho
Advisor(s)Oliveira, Rosário
Henriques, Mariana
Issue date23-Nov-2010
Abstract(s)Over the last years fungi have emerged as a major cause of human disease. Candida albicans is the most common cause of opportunistic mycoses, albeit Non-Candida albicans Candida (NCAC) species, namely Candida dubliniensis, Candida glabrata, Candida krusei, Candida parapsilosis, and Candida tropicalis, are emerging as pathogens. Candida species factors that might influence the pathogenesis of infection include the ability to: undergo a reversible conversion between yeast and filaments (only for C. albicans and C. dubliniensis), form biofilms, and secrete proteins and chemicals, such as E,E-farnesol (farnesol), into the extracellular medium. This work aimed to bring insights into Candida species world, following two main routes: first, the analyses of compounds released by Candida species into the extracellular medium focusing on extracellular DNA (eDNA) and alcohol compounds, and second, the identification of Candida species in clinical samples. Concerning eDNA, here we show that it is a component of C. albicans biofilm extracellular matrix (ECM). In addition, based on the degradation of eDNA by deoxyribonuclease (DNase) and addition of exogenous DNA at different stages of biofilm development, it was possible to define that eDNA contributes to the maintenance and stability of C. albicans mature biofilms. Furthermore, the study of the impact of the combined use of DNase and antifungals against C. albicans biofilms revealed that DNase increases the effectiveness of the antifungal agents amphotericin B and caspofungin. Our results present evidence that eDNA is a key element of the ECM in C. albicans, contributing to biofilm integrity and antifungal resistance. With respect to extracellular alcohols, the results presented herein show that C. albicans extracellular alcohol farnesol impairs C. glabrata, C. krusei, and C. tropicalis planktonic growth by interfering with cell viability and/or cell cycle, depending on the species. In addition, headspacesolid- phase microextraction and gas chromatography-mass spectrometry were used for profiling Candida extracellular alcohols. Those procedures allowed the identification and quantification of isoamyl alcohol, 2-phenylethanol (phenylethanol), 1-dodecanol, E-nerolidol (nerolidol), and farnesol in C. albicans and C. dubliniensis planktonic and biofilm cells supernatants. With the exception of nerolidol, these compounds were also identified in C. parapsilosis and C. tropicalis planktonic supernatants. Based on the quantifications performed in this work, the effect of the exogenous addition of commercial formulations of the alcohols was tested in vitro and in vivo. physiological levels of these alcohols were capable of inhibiting C. albicans and C. dubliniensis yeast to filament conversion, under filamentation inducing conditions, when used alone or in combination (simulating a 96-h culture supernatant). Furthermore, the evaluation of the impact of the individual addition of the extracellular alcohols against Candida species biofilm cells mitochondrial activity and biofilm biomass showed that these compounds differentially affect biofilm development. Of note, physiological levels of isoamyl alcohol, phenylethanol, and nerolidol interfered with C. parapsilosis and C. tropicalis biofilm development. In vivo, using the well established murine model of hematogenously disseminated candidiasis, it was shown that a solution simulating the composition of alcohols present in a C. albicans culture supernatant is able to increase mice survival, decreasing fungal burden, and filamentation in the kidney. Overall, these results show that extracellular compounds produced by Candida species regulate their virulence traits. Finally, we evaluated the oral Candida carriage in patients attending a dental clinic in Braga, Portugal. This study showed that although C. albicans is the Candida species most frequently isolated from the oral cavity in this population, NCAC species also contribute to the fungal burden. Notably, the majority of these NCAC species was co-isolated with C. albicans, evidencing the complex control of Candida species diversity and distribution in a natural environment. Taken together, these studies represent a very important contribution to our understanding of the composition of the extracellular milieu of Candida species and its relationship with the regulation of Candida biology, and open the possibility of the development of new treatment and/ or diagnostic strategies to combat candidiasis.
Ao longo dos últimos anos os fungos têm emergido como uma das principais causas de doenças humanas. Candida albicans constitui a principal causa de micoses oportunistas, embora outras espécies de Candida não albicans (CNA), como Candida dubliniensis, Candida glabrata, Candida krusei, Candida parapsilosis e Candida tropicalis estejam a emergir como patogénicos. São vários os factores que podem influenciar a patogénese, nomeadamente, a capacidade de transitar reversivelmente entre as formas de levedura e filamento (especificamente C. albicans e C. dubliniensis), a capacidade de formar biofilme e ainda a de libertar proteínas e outras classes de compostos, como o E,E-farnesol (farnesol), no meio extracelular. Esta tese teve como objectivo principal o estudo do universo das espécies de Candida seguindo, nesse sentido, duas linhas de investigação. Primeiro, efectuando a avaliação dos compostos libertados por espécies de Candida para o meio extracelular, especificamente DNA extracelular (DNAe) e compostos alcoólicos. Segundo, procedendo à identificação de espécies de Candida em amostras clínicas. Relativamente aos estudos que visaram o DNAe, os nossos resultados mostraram que este é um componente da matriz extracelular de biofilmes formados por C. albicans. Adicionalmente, com base na degradação do DNAe pela enzima desoxiribonuclease e na adição de DNA exógeno em diferentes fases de desenvolvimento do biofilme, foi possível estabelecer que o DNAe contribui para a manutenção e estabilidade de biofilmes maduros de C. albicans. Complementarmente, o estudo do efeito da combinação da desoxiribonuclease com agentes antifúngicos em biofilmes de C. albicans revelou que a desoxiribonuclease aumenta a eficácia dos agentes antifúngicos anfotericina B e caspofungina. Os nossos resultados evidenciam que o DNAe é um elemento chave da matriz extracelular de biofilmes de C. albicans que contribui para a sua integridade e resistência a agentes antifúngicos. Em relação aos álcoois extracelulares, os resultados apresentados nesta tese revelaram que o farnesol, álcool extracelular produzido por C. albicans, afecta negativamente o crescimento de células planctónicas de C. glabrata, C. krusei e C. tropicalis, por interferir com a viabilidade e/ou ciclo celular, dependendo da espécie em estudo. Adicionalmente, a técnica de microextracção em fase sólida, em modo de espaço de cabeça acoplada a cromatografia de gás-espectrometria de massa foi utilizada para determinar o perfil dos álcoois extracelulares de espécies de Candida. Esta análise permitiu a identificação e quantificação dos álcoois: álcool isoamílico,
TypeDoctoral thesis
DescriptionTese de doutoramento em Engenharia Biomédica
URIhttp://hdl.handle.net/1822/11217
AccessOpen access
Appears in Collections:CEB - Teses de Doutoramento / PhD Theses
BUM - Teses de Doutoramento

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