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Candida albicans is the main causative agent of candidiasis and one of the most frequent causes of nosocomial infections worldwide. In order to establish an infection, this pathogen supports effective stress responses to counter host defenses and adapts to changes in the availability of important nutrients, such as alternative carbon sources. These stress responses have clear implications on the composition and structure of Candida cell wall. Therefore, we studied the impact of lactate, a physiologically relevant carbon source, on the activity of C. albicans RLM1 transcriptional factor. RLM1 is involved in the cell wall integrity pathway and plays an important role in regulating the flow of carbohydrates into cell wall biosynthesis pathways. The role of C. albicans RLM1 in response to lactate adaptation was assessed in respect to several virulence factors, such as the ability to grow under cell wall damaging agents, filament, adhere or form biofilm, as well as to immune recognition. The data showed that growth of C. albicans cells in the presence of lactate induces the secretion of tartaric acid, which has the potential to modulate the TCA cycle on both the yeast and the host cells. In addition, we found that adaptation of C. albicans cells to lactate reduces their internalization by immune cells and consequent % of killing, which could be correlated with a lower exposure of the cell wall -glucans. In addition, absence of RLM1 has a minor impact on internalization, compared with the wild-type and complemented strains, but it reduces the higher efficiency of lactate grown cells at damaging phagocytic cells and induces a high amount of IL-10, rendering these cells more tolerable to the immune system. The data suggests that RLM1 mediates cell wall remodeling during carbon adaptation, impacting their interaction with immune cells., This study was supported by the Portuguese National Funding Agency for Science, Research and Technology FCT. RA received FCT Ph.D. fellowship (PD/BD/113813/2015) and AC-B received FCT Ph.D. fellowship SFRH/BD/133513/2017. The work on CBMA was supported by FEDER through POFC-COMPETE and by FCT through strategic funding UID/BIA/04050/2013. The work on CEB was supported by Pest-OE/EQB/LA0023/2013, from FCT, "BioHealth - Biotechnology and Bioengineering approaches to improve health quality," Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER, and the project "Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB," Ref. FCOMP-01-0124-FEDER-027462., info:eu-repo/semantics/publishedVersion

Candida spp. often inhabit niches that are glucose-limited but rich in alternative carbon sources, such as lactate or acetate, an ability that contributes to cells’ virulence. In glucose-poor niches, Candida albicans cells express JEN1 and JEN2 genes encoding the carboxylic acids transporters Jen1 and Jen2, respectively, which have been reported to be important in the early stages of infection. In this work, we aimed at analysing biofilm formation and antifungal drug resistance of C. albicans cells grown either in the presence of glucose or lactic acid. Additionally, we tested the involvement of Jen1 and Jen2 on these processes. Our results show that biofilm formation and susceptibility to fluconazole depend on the carbon source used. Wild-type and jen1jen2 lactic acid-grown cells formed more biofilm biomass, with predominance of yeast cells, than the ones grown in glucose. In the presence of this sugar a hyphae network is observed only for wild-type cells. In the presence of lactic acid, a jen1jen2 mutant strain exhibited a more compact biofilm with higher resistance to fluconazole when compared to the wild type. In the case of planktonic cells, the phenotype was exactly the opposite; the double mutant strain was more susceptible to fluconazole in lactic acid containing media. These findings show that carboxylic acids transporters have an important role in biofilm formation and in the acquisition of resistance to antifungal drugs, supporting the view that adaptation of Candida cells to the carbon source present in host niches affects their pathogenicity.

Dissertação de mestrado em Molecular Genetics, In recent decades, fungal pathogens have become a global health problem. Candida albicans, a common opportunistic commensal microorganism, is the most frequent cause of systemic candidiasis and nosocomial bloodstream fungal infections. C. albicans can colonize various parts of the host under abnormal conditions, especially in immunocompromised patients. The main feature of C. albicans that has made it a serious pathogen is its ability to adapt to different host niches and utilize alternative carbon sources, like carboxylic acids, in glucose-poor settings, such as the colon and vagina. Yeast plasma membrane carboxylate transporters belonging to the AceTr Transporter family, known as Atos (Acetate Transporter Orthologs), seem to play a crucial role in the uptake of these substrates in C. albicans. Previous work identified the orthologs of the Saccharomyces cerevisiae Ato1 (Ady2) monocarboxylate transporter in several Candida species. In C. albicans, eight Ato orthologs were detected, whose functions are still unknown. The purpose of this thesis was to characterize the function and structure of selected Ato family members and Jen transporters in C. albicans by heterologous expression in S. cerevisiae. We constructed 5 plasmids by Gap repair containing selected ATO genes under the control of the GPD promoter. Additionally, using the CRISPR-Cas9 method, we generated C. albicans strains with GFP fusions at the 3’ terminal of ATO and JEN genes to investigate the expression and subcellular localization of the selected transporters. Fluorescence microscopy analyses revealed that CaAto1- GFP and CaAto2-GFP were localized at the plasma membrane in the presence of lactate and acetate, suggesting these transporters play an important role in the utilization of these substrates. Moreover, an in-silico analysis of Ato proteins was performed to determine their three dimensional structure and docking with substrates. We found that the NPAPLGL(M/S), and FLY regions are conserved in selected Atos. Additionally, molecular docking showed that all selected C. albicans Ato proteins have four acetate-binding sites. Also, a comparison of the predicted pore radius and 3D structure of CaAto proteins with ScAto1 suggests that CaAto1 and CaAto2 have the same pore size and structure as ScAto1, which may indicate their identical roles, whereas CaAto3, CaAto4, and CaAto5 were dissimilar and thus might present a different specificity., Nas últimas décadas, as infeções fúngicas tornaram-se um problema de saúde global. Candida albicans, um microrganismo comensal, mas também oportunista, é o agente infecioso responsável pela maioria das candidíases sistémicas e infeções fúngicas da corrente sanguínea. Esta levedura pode colonizar várias partes do corpo, especialmente em doentes imunocomprometidos. A levedura C. albicans adapta-se aos diferentes nichos existentes no hospedeiro e utiliza fontes de carbono alternativas, como ácidos carboxílicos, em ambientes escassos em glucose, como o cólon e a vagina. Os transportadores de carboxilatos da membrana plasmática de levedura pertencentes à família AceTr, conhecidos como Atos (Acetate Transporter Orthologs), parecem desempenhar um papel crucial na absorção destes substratos em C. albicans. Estudos prévios identificaram os ortólogos do transportador de acetato Ato1 (Ady2) de Saccharomyces cerevisiae em várias espécies do género Candida. Em C. albicans, foram detetados oito ortólogos, cujas funções ainda são desconhecidas. O objetivo desta tese foi a determinação da função e estrutura de transportadores de C. albicans pertencentes à família Ato e Jen, por expressão heteróloga em S. cerevisiae. Para o efeito, construímos 5 plasmídeos contendo os genes ATO selecionados sob o controlo do promotor GPD, usando a metodologia de Gap Repair. Adicionalmente, usando o método CRISPR-Cas9, gerámos estirpes de C. albicans contendo fusões GFP no terminal 3' dos genes ATO e JEN para determinar a expressão e a localização subcelular dos transportadores selecionados. Ensaios de microscopia de fluorescência revelaram que na presença de lactato e acetato, as proteínas CaAto1-GFP e CaAto2-GFP se localizaram na membrana plasmática, sugerindo que estes transportadores desempenham um papel importante na utilização desses substratos. Foi também realizada uma análise in-silico das proteínas Ato para determinar a sua estrutura tridimensional e a interação com substratos. Verificámos que os domínios NPAPLGL(M/S) e FLY são conservados nestas proteínas. As proteínas Ato de C. albicans aqui estudadas apresentam quatro locais putativos de ligação acetato. A previsão da estrutura 3D e do raio do poro da proteína evidenciaram que a CaAto1 e a CaAto2 têm o mesmo tamanho e estrutura de poros que a ScAto1, o que pode indicar que terão funções idênticas, enquanto que CaAto3, CaAto4 e CaAto5 ao terem estruturas distintas podem possuir uma especificidade diferente.