Class 2017
PhD Grant: PD/BD/135139/2017
PhD thesis
Deepening the role of CgHaa1- and CgPdr1- pathways in stress resilience and pathogenesis of Candida glabrata to foster new antifungal treatments
Supervisor
• Nuno P. Mira , Professor @ IST/ULisboa, PT
Collaborators
• Frederic Devaux, Professor @ Université Pierre et Marie Curie, FR
• Ken Haynes, University of Exeter, UK
PhD Degree complete: Discussion on June, 2022
THESIS ABSTRACT
The emergence of Candida glabrata strains resistant to currently used antifungals is one of the more relevant factors determining the outcome of patients suffering from infections caused by this species pushing the need of identifying suitable alternative drugs and new targets different from those targeted by current antifungals. Transcription factors involved in response to environmental stress (including those that respond to the presence of antifungals in the environment) present themselves as an interesting set of possible targets considering their essential role in mounting appropriate tolerance responses. Also of importance, is the fact that the inhibition of their activity can result in a wide impact in the cell since it can affect, simultaneously, the expression of multiple genes, including those that could be required for survival in the presence of the stressor. This thesis is focused on detailing molecular aspects of two pathways controlled by two transcription factors, CgPdr1, a pivotal player in response to drugs across different Yeast species, and CgHaa1, demonstrated to serve as a determinant of tolerance to acetic acid in some species, including C. glabrata. It is expected that the detailing of the molecular mechanisms by which these CgPdr1- and CgHaa1- dependent pathways function, can help to foster their possible use as targets in the development of new anti-Candida treatments. Using comparative transcriptomic and genomic analyses of azole susceptible and resistant strains, it was possible to demonstrate that the acquisition of mutations in the coding sequence of CgPDR1 resulting in hyper-activation of this regulator is the most commonly found resistance mechanism, being possible to identify new variants that were not previously described: CgPdr1K274Q, CgPdr1I392M and CgPdr1I803T. To further understand how the impact of these three novel hyper-activating mutations affect the biochemical activity of CgPdr1, their impact in the tridimensional structure of the protein was studied (using in silico modelling) along with possible changes in the set of promoters and interactors directly bound in vivo. Altogether the results obtained in this thesis confirm that hyper-active CgPdr1K274Q variant recognizes in vivo the same set of promoters recognized by the wild-type CgPdr1, although they show enhanced capacity to induce expression of a set of target genes. It was possible to identify a set of proteins interacting with wild-type and with the K274Q variant of CgPdr1, but further studies will be required to understand whether these differences in the interaction to both variants are responsible for the detected different capabilities of the two proteins to induce target gene expression. The dissection of the CgHaa1-dependent regulatory network was also dissected in this thesis as well as a possible involvement of this system in biofilm formation and pathogenesis of this species. Using ChIP-Seq it was possible to demonstrate that CgHaa1 directly binds to at least 22 genes in vivo, a few of them (e.g. CgTPO3, CgYRO2, CgYGP1) with orthologs in S. cerevisiae being previously shown to confer protection against acetic acid. Only 20% of the direct regulon of CgHaa1 identified is conserved from that of S. cerevisiae, which indicates a noteworthy divergence of this transcriptional networks in both species. The evolution of the direct Haa1 network in C. glabrata may be the result of the recognition of a new cis-element identified in silico, that binding by the transcription factor requires further confirmation. The results obtained also demonstrate that CgHaa1 is required for maximal adherence and biofilm formation of C. glabrata on both biotic and abiotic surfaces.
RESUMO DA TESE
O aparecimento de estirpes de Candida glabrata resistentes aos antifúngicos atualmente utilizados é um dos factores mais relevantes que determinam o desfecho dos doentes que sofrem de infecções causadas por estas leveduras, tornando-se necessário identificar fármacos alternativos e que afectem outros alvos diferentes dos visados pelos antifúngicos actuais. Os factores de transcrição envolvidos na resposta ao stress ambiental (incluindo os que respondem à presença de antifúngicos) são um conjunto interessante de possíveis alvos, tendo em conta o seu papel em determinar uma resposta adequada. Esta tese centra-se no escrutínio de aspectos moleculares de duas vias controladas por dois factores de transcrição, CgPdr1, um ator fundamental na resposta a fármacos em diferentes espécies de leveduras, e CgHaa1, que se demonstrou ser um determinante da tolerância ao ácido acético em algumas espécies, incluindo C. glabrata. Espera-se que a descrição pormenorizada dos mecanismos moleculares pelos quais estas vias dependentes de CgPdr1- e CgHaa1- funcionam, possa ajudar a promover a sua possível utilização como alvos no desenvolvimento de novos tratamentos anti-Candida. Utilizando análises comparativas de transcriptómica e genómica de estirpes susceptíveis e resistentes aos azóis, foi possível demonstrar que a aquisição de mutações na sequência de codificação de CgPDR1, resultando na hiperactivação deste regulador, é o mecanismo de resistência mais frequentemente encontrado, sendo possível identificar novas variantes que não foram previamente descritas: CgPdr1K274Q, CgPdr1I392M e CgPdr1I803T. Para compreender melhor como é que o impacto destas três novas mutações hiper-activadoras afecta a atividade bioquímica da CgPdr1, foi estudado o seu impacto na estrutura tridimensional da proteína (utilizando modelação in silico), juntamente com possíveis alterações no conjunto de promotores e interactivos diretamente ligados in vivo. No conjunto, os resultados obtidos nesta tese confirmam que a variante hiperactiva CgPdr1K274Q reconhece in vivo o mesmo conjunto de promotores reconhecidos pela CgPdr1 de tipo selvagem, embora mostre uma maior capacidade de induzir a expressão de um conjunto de genes alvo. Foi possível identificar um conjunto de proteínas que interagem com o tipo selvagem e com a variante K274Q de CgPdr1, mas serão necessários mais estudos para perceber se estas diferenças na interação com as duas variantes são responsáveis pelas diferentes capacidades detectadas das duas proteínas para induzir a expressão de genes alvo.
A dissecção da rede de regulação dependente de CgHaa1 foi também dissecada nesta tese, bem como um possível envolvimento deste sistema na formação de biofilme e patogénese desta espécie. Utilizando ChIP-Seq foi possível demonstrar que CgHaa1 se liga diretamente a pelo menos 22 genes in vivo, alguns deles (e.g. CgTPO3, CgYRO2, CgYGP1) com ortólogos em S. cerevisiae que previamente demonstraram conferir proteção contra o ácido acético. Apenas 20% do regulador direto de CgHaa1 identificado é conservado em relação ao de S. cerevisiae, o que indica uma divergência notável destas redes transcricionais em ambas as espécies. A evolução da rede direta de Haa1 em C. glabrata pode ser o resultado do reconhecimento de um novo cis-elemento identificado in silico, cuja ligação pelo fator de transcrição requer confirmação adicional. Os resultados obtidos também demonstram que CgHaa1 é necessário para a máxima aderência e formação de biofilme de C. glabrata em superfícies bióticas e abióticas.
PUBLICATIONS
Papers
Salazar SB, Pinheiro MJF, Novais D, Soares AR, Lopes MM, Ferreira T, Rodrigues V, Fernandes F and Mira NP*, Disclosing azole resistance mechanisms in resistant C. glabrata strains encoding wild-type or gain-of-function CgPDR1alleles through comparative genomics and transcriptomics, G3: Genes, Genomes and Genetics, 12:7,10.1093/g3journal/jkac110; (2022)
Salazar, S.B., Simões, R.S., Pedro, N.A., Pinheiro, M.J., Carvalho, N.F.N.N., Mira, N.P., An overview on conventional and non-conventional therapeutic approaches for the treatment of candidiasis and underlying resistance mechanisms in clinical strains, Journal of Fungi (Basel). 10;6(1), 2020.
Salazar, S.B., Wang, C., Musterkotter, M., Okamoto, M., Takahashi-Nakagchi, A., Chibana, H., Lopes, M., Güldener, U., Butler, G. and Mira, N., “Comparative genomic and transcriptomic analyses unveil novel features of azole resistance and adaptation to the human host in Candida glabrata”, FEMS Yeast Research 1;18(1), 2018.
Oral Communications
Salazar, S.B., Valez, N., Sotti-Novais, D., Simões, R., Souza, J. A., Faustino, M. J., Faustino, C., Mendonça, C., Lopes, M. M., Vitória Rodrigues, V., Mira, N. P. “Unveiling azole resistance mechanisms in Candida glabrata clinical isolates encoding wild-type or gain-of-function CgPdr1 alleles”, Candida and Candidiasis, 21-27 March 2021, vitual event
Salazar, S.B., Pedreira, T., Chibana, H., Lopes, M.M., Güldene, U., Mira, N.P., Genomic Adaptative Mechanisms mediating Azole Resistance and Adaptation to the Human Host in Candida glabrata, with emphasis on the role of CgPDR1 regulon”, XXI Jornadas de Biologia de Leveduras, 8-9 June 2018, Braga, Portugal
Salazar, S.B., Bernardo, R., Cunha, D.V., Wang, C., Chibana, H., Silva, S., Pereira, L., Sá-Correia, I., Azeredo, J., Butler, G., Mira, N.P., “Genetic adaptive mechanisms mediating response ans tolerance to acetic acid stress in the human pathogen Candida glabrata: role of the CgHaa1-dependent signalling pathway”, Microbal Stress Meeting: Systems to Molecules and Back, 23-25 April 2018, Kinsale, Ireland (Best talk by an Early Career Researcher sponsored by the journal Genes)
Poster Communications
Maria Joana Pinheiro, Sara B. Salazar, Carolina Soares, Vitória Rodrigues, Fábio Fernandes, Nuno P. Mira, Disclosing azole resistance mechanisms in Candida glabrata clinical strains through OMICS analyses: beyond CgPdr1, MicroBiotec23, Covilhã, 7-9 Dec 2023
Salazar, S.B., Valez, N.,Pinheiro, M.J., Pedro, N.A., Lourenço, A., Mira, N. P. “Modulation of C. glabrata response and resistance to azoles in the presence of acetic and lactic acids at low pH”. Candida and Candidiasis, 21-27 March 2021, vitual event
Salazar, S., Silva, S., Mil-Homens, D., Pimenta, A.I., Chibana, H., Azeredo, J., Fialho, A.M., Mira, N.P., The Candida glabrata CgHaa1-dependent system is required for biofilm formation, adhesion to epithelial cells and maximal virulence against Galleria mellonela”, Advanced Lecture Course on Human Fungal Pathogens, 18-24 May 2019, Nice, France
Salazar, S.B., Pedreira, T., Henriques, R., Wang, C., Musterkotter, M., Okamoto, M., Takahashi-Nakaguchi, A., Chibana, H., Lopes, M.M., Güldener, U., Butler, G., Mira, N.P., “Comparative genomic and transcriptomic analysis unveil novel features of azole resistance and adaptation to the human host in Candida glabrata”, MicroBiotec2017 December 7-8, 2017, Porto, Portugal
DOCTORAL PROGRAM (36 ECTS)
Curricular units:
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Advanced Topics in bioengineering and Biological Sciences (6 ECTS)
• Gene therapy (6 ECTS)
Advanced courses:
• 8th Advanced Lecture Course on Human Fungal Pathogens, by FEMS, 18-24 May 2019, La Colle sur Loup, Nice, France
• Teaching STEM Students – Transformation Guide for teaching Assistants 1st Ed., by Academic Development Office, 10-12 September 2018, Instituto Superior Técnico, Lisbon, Portugal
• Analysis of High-Throughput Sequencing Data, by European Bioinformatics Institute (EMBL-EBI), 07-10 November 2017, Welcome Genome Campus, Cambridge, UK
Current position/address
Post-doctoral researcher at IGC; Invited Assistant Professor at Polytechnic Institute of Setúbal