Class 2015
PhD Grant: SFRD/BD/113631/2015
PhD thesis
Unveiling the complex regulatory network governing antifungal drug resistance in Candida glabrata: from new regulators to new effectors
Supervisor
• Miguel Teixeira, Professor @ IST/ULisboa, PT
Collaborators
• Geraldine Butler, Professor @ University College Dublin, IR
PhD Degree complete: Discussion on September 27, 2019; Pass with Distinction and Honour
THESIS ABSTRACT
The acquisition of drug resistance has been implicated in the failure of antifungal therapy, especially against infections caused by Candida glabrata. It is thus crucial to understand the molecular basis of this phenomenon, in order to guide the design of more suitable therapeutic strategies. With this overall goal in mind, the work described in this thesis aims to contribute to extend knowledge on the role of transcription regulatory networks in antifungal resistance in C. glabrata.
The PathoYeastract database (www.pathoyeastract.org) was built, comprising a series of tools to predict and analyze transcription regulatory associations in the four major Candida pathogenic species. All published data on Transcription Factor (TF) regulatory associations, and DNA binding motifs was curated and stored in the database. Moreover, tools for the comprehensive analysis on gene or genome-wide transcriptional responses were developed, which further enable the prediction of regulatory networks and cross-species comparison based on orthologous associations described for other yeast species.
Additionally, the role of three transcription factors in antifungal resistance was evaluated. Although the role of the TF Pdr1 in fluconazole resistance had been well established, that is not the case for other antifungals, especially the effect of Pdr1 at the protein expression levels. Based on the identification of CgPdr1 as a determinant of resistance to imidazoles and 5-flucytosine, the effect of CgPdr1 expression on membrane protein levels in response to clotrimazole and 5-flucytosine was studied, using iTRAQ-MS-based proteomics. The concentration of 37 or 32 membrane proteins was found to change upon clotrimazole or 5-flucytosine stress, respectively, Pdr1 controlling about 50% of those changes. Based on the obtained results, the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2 were found to mediate clotrimazole resistance, while CgFlr1 and CgFlr2 were found to contribute to 5-flucytosine resistance, in both cases by decreasing intracellular drug accumulation. Cell wall remodeling was also found to occur in response to both drugs, in the case of clotrimazole through the specific participation of the CgGas1 cell wall protein.
It is frequently the occurrence of Gain-of-Function (GOF) mutations in CgPdr1, leading to its activation, that results in constitutive expression of drug efflux pumps and, consequently, azole drug resistance. However, several studies reported the identification of C. glabrata clinical isolates that display azole resistance, but no CgPdr1 GOF mutations, suggesting the existence of additional unknown mechanisms of antifungal resistance in this yeast. As a result of a screening for new TFs that might be involved in antifungal drug resistance, the TFs CgRpn4 and CgMar1 were found as additional determinants of fluconazole resistance in C. glabrata. Their role in this phenomenon, assessed through RNA-sequencing, was found to include, at least, the control of plasma membrane lipid metabolism. CgRpn4 was found to be required for the activation of ergosterol biosynthesis genes, including direct activation of the azole target CgERG11. CgMar1 was found to regulate sphingolipid metabolism, particularly the activation of the sphingolipid flippase CgRsb1 and the incorporation of sphingosine in the plasma membrane. Obtained results support the notion that CgRpn4 and CgMar1 participate in the control of ergosterol and sphingolipid levels during fluconazole stress, respectively, contributing to decrease plasma membrane permeability and the consequent fluconazole accumulation in C. glabrata cells.
Altogether, this thesis extends our understanding of how transcription regulation is orchestrated in Candida species, by providing a new computational tool for its analysis, while presenting two novel regulatory networks that contribute to azole resistance in C. glabrata.
RESUMO DA TESE
A aquisição de resistência a drogas tem sido associada ao insucesso da terapia antifúngica, especialmente em infeções causadas por Candida glabrata. Por esta razão, é crucial perceber as bases moleculares deste fenómeno, de forma a guiar o desenvolvimento de estratégias terapêuticas mais adequadas. Com este objetivo, o trabalho descrito nesta tese tem o intuito de contribuir para o conhecimento do papel de redes de regulação transcricional na resistência a antifúngicos em Candida glabrata.
A base de dados PathoYeastract (www.pathoyeastract.org) for desenvolvida, incluindo um conjunto de ferramentas para analisar e prever associações regulatórias nas quatro espécies de Candida mais prevalentes. Toda a informação publicada relativa a associações regulatórias de Fatores de Transcrição (FT) e motivos de ligação ao DNA foi recolhida e incorporada na base de dados. Foram desenvolvidas ferramentas para a análise integrada de respostas transcricionais ao nível de genes ou genomas completos, permitindo a previsão de redes regulatórias e comparações inter-espécies baseadas em associações ortólogas descritas noutras espécies de leveduras.
O papel de três fatores de transcrição na mediação da resistência a antifúngicos foi estudado. Embora a função do FT CgPdr1 na resistência a fluconazol esteja bem estudada, o mesmo não é o caso para outro antifúngicos, especialmente o efeito do FT CgPdr1 ao nível da expressão proteica. Com base na identificação do FT CgPdr1 como sendo um determinante de resistência a imidazóis e a 5-flucitosina, o efeito da sua expressão na resposta ao nível de proteínas de membrana em resposta a clotrimazol e 5-flucitosina foi estudado, usando para tal proteómica baseada em iTRAQ-MS. Verificou-se que a concentração de 37 ou 32 proteínas de membrana encontra-se alterada durante tratamento com clotrimazole ou 5-flucitosina, respetivamente, e que o FT Pdr1 controla cerca de 50% dessas alterações. Com base nestes resultados, verificou-se que os antiportadores droga:H+ CgTpo1_1 e CgTpo1_2 conferem resistência a clotrimazol, enquanto que os antiportadores droga:H+ CgFlr1 e CgFlr2 conferem resistência a 5-flucitosina; em ambos os casos actuam por diminuir a acumulação intracelular de antifúngico. A remodelação da parede celular também ocorre em resposta a ambos os antifúngicos, sendo que no caso do clotrimazole o mesmo se deve, pelo menos, à presença da proteína de parede CgGas1.
A ocorrência de mutações de ganho de função no FT CgPdr1, levando à sua ativação, é frequentemente a causa da expressão constitutiva de bombas de efluxo, resultando na resistência a antifúngicos da família dos azóis. No entanto, vários estudos referem a descoberta de isolados de C. glabrata resistentes a azóis que não possuem mutações de ganho de função no FT CgPdr1, o que sugere a existência de mecanismos adicionais de resistência a azóis que são desconhecidos nesta levedura. Como resultado de uma triagem para encontrar novos FTs que possam estar envolvidos em resistência a antifúngicos, os FTs CgRpn4 e CgMar1 foram identificados como determinantes de resistência a fluconazol em C. glabrata. A sua função neste fenómeno foi avaliada através da técnica de sequenciação de RNA, tendo sido associada ao controlo do metabolismo de lípidos da membrana plasmática. O FT CgRpn4 regula a ativação de genes de síntese de ergosterol, incluindo ativação direta do gene CgERG11, que constitui o alvo dos azóis. O FT CgMar1 regula metabolismo de esfingolípidos, nomeadamente a ativação do translocador de esfingolípidos CgRsb1 e a incorporação de esfingosina na membrana plasmática. Os resultados obtidos mostram que os FTs CgRpn4 e CgMar1, respetivamente, participam no controlo dos níveis de ergosterol e esfingolípidos durante tratamento com fluconazol, contribuindo assim para diminuir a permeabilidade de membrana e consequentemente a acumulação do antifúngico em células de C. glabrata.
De modo geral, esta tese contribui para o aumento do conhecimento quanto à regulação transcricional em C. glabrata e como a mesma está organizada, através do desenvolvimento de ferramentas computacionais de análise e apresentando duas novas redes de regulação que contribuem para resistência a azóis em C. glabrata.
PUBLICATIONS
Papers
Pais, P., California, R., Galocha, M., Viana, R., Ola, M., Cavalheiro, M., Takahashi-Nakaguchi, A., Chibana, H., Butler, G., Teixeira, M.C., “Candida glabrata transcription factor Rpn4 mediates fluconazole resistance through regulation of ergosterol biosynthesis and plasma membrane permeability”, Antimicrobial Agents and Chemotherapy, 64: e00554-20, 2020.
Monteiro, P.T., Oliveira, J., Pais, P., Antunes, M., Palma, M., Cavalheiro, M., Galocha, M., Godinho, C.P., Martins, L.C., Bourbon, N., Mota, M.N., Ribeiro, R.A., Viana, R., Sá-Correia, I., Teixeira, M.C., “YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts”, Nucleic Acids Research, 48: D642-D649, 2020.
Pais, P., Galocha, M., Miranda, I.M., Rodrigues, A.G., Teixeira, M.C., 2019, “Draft genome sequences from three clinical isolates of the pathogenic yeast Candida glabrata”, Microbial Resource Announcements, 8(35). pii: e00278-19, 2019.
Galocha, M., Pais, P., Cavalheiro, M., Pereira, D., Viana, R., Teixeira, M.C., “Divergent approaches to virulence in C. albicans and C. glabrata: two sides of the same coin”, International Journal of Molecular Sciences, 20(9). pii: E2345, 2019.
Pais, P., Galocha, M., Viana, R., Cavalheiro, M., Pereira, D., Teixeira, M.C., “Microevolution of the pathogenic yeasts Candida albicans and Candida glabrata during antifungal therapy and host infection”, Microbial Cell, 6(3): 142 – 159, 2019.
Cavalheiro, M., Costa, C., Silva-Dias, A., Miranda, I.M., Wang, C., Pais, P., Pinto, S.N., Mil-Homens, D., Sato-Okamoto, M., Takahashi-Nakaguchi, A., Silva, R.M., Mira, N.P., Fialho, A.M., Chibana, H., Rodrigues, A.G., Butler, G., Teixeira, M.C., “Unveiling the mechanisms of in vitro evolution towards fluconazole resistance of a Candida glabrata clinical isolate: a transcriptomics approach”, Antimicrobial Agents and Chemotherapy, 63: e00995-18, 2019.
Teixeira, M.C., Monteiro, P.T., Palma, M., Catarina, C., Godinho, C.P., Pais, P., Cavalheiro, M., Antunes, M., Lemos, A., Pedreira, T., Sá-Correia, I., YEASTRACT, an upgraded database for the analysis of transcription regulatory networks in Saccharomyces cerevisiae. Nucleic Acids Research, 46: D348-D353, 2018.
Romão, D., Cavalheiro, M., Mil-Homens, D., Santos, R., Pais, P., Costa, C., Takahashi-Nakaguchi, A., Fialho, A.M., Hiroji Chibana, H., Teixeira, M.C., “A new determinant of Candida glabrata virulence: the acetate exporter CgDtr1″, Frontiers in Cellular and Infection Microbiology, 7: 473, 2017.
Santos, R., Costa, C., Mil-Homens, D., Romão, D., de Carvalho, C.C.R., Pais, P., Mira, N.P., Fialho, A.M., Teixeira, M.C., “The multidrug resistance transporters CgTpo1_1 and CgTpo1_2 play a role in virulence and biofilm formation in the human pathogen Candida glabrata”, Cellular Microbiology, 19(5): e12686, 2017.
Monteiro, P.T., Pais, P., Costa, C., Manna, S., Sá-Correia, I., Teixeira, M.C., “The PathoYeastract database: an information system for the analysis of gene and genomic transcription regulation in pathogenic yeasts”, Nucleic Acids Research, 45(D1): D597-D603, 2017.
Pais, P., Pires, C., Costa, C., Okamoto, M., Chibana, H., Teixeira, M.C., “Membrane proteomics analysis of the Candida glabrata response to 5-flucytosine: unveiling the role and regulation of the drug efflux transporters CgFlr1 and CgFlr2”, Frontiers in Microbiology, 7:2045, 2016.
Pais, P., Costa, C., Cavalheiro, M., Romão, D., Teixeira, M.C., “Transcriptional control of drug resistance, virulence and immune system evasion in pathogenic fungi: a cross-species comparison”, Frontiers in Cellular and Infection Microbiology, 6: 131, 2016.
Pais, P., Costa, C., Pires, C., Shimizu, K., Chibana, H., Teixeira, M.C., “Membrane proteome-wide response to the antifungal drug clotrimazole in Candida glabrata: role of the transcription factor CgPdr1 and the Drug:H+ Antiporters CgTpo1_1 and CgTpo1_2”, Molecular and Cellular Proteomics, 15(1):57-72, 2016
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Oral Communications
Pais P, Galocha M, Califórnia R, Pimenta A, Mil-Homens D, Fialho AM, Butler G, Teixeira MC, “Functional characterization of the transcription factor CgMrr1 suggests new players governing azole drug resistance in Candida glabrata”, 14th ASM Conference on Candida and Candidiasis, April 15-19, 2018, Providence, RI, USA
Pais P, Galocha M, Califórnia R, Butler G, Teixeira MC, “Beyond MDR transporter-mediated azole resistance in Candida glabrata: functional characterization of the transcription factors CgRpn4 and CgMrr1”, XXI Jornadas de Biologia de Leveduras, 8-9 June 2018, Braga, Portugal
Pais P, Califórnia R, Galocha M, Chibana H, Butler G, Teixeira MC, “Candida glabrata transcription factor Rpn4 regulates fluconazole resistance by transcriptional control of ergosterol biosynthesis and plasma membrane stability”, 29th European Congress of Clinical Microbiology and Infectious Diseases – ECCMID, 13-16 April 2019, Amsterdam, The Netherlands
Pais P, Galocha M, Califórnia C, Viana R, Ola M, Okamoto M, Monteiro PT, Chibana H, Butler G, Teixeira MC, “Tackling azole antifungal resistance in Candida glabrata: in the crossroad between three complex regulatory pathways”, MicroBiotec2019, December 5-7, 2019, Coimbra, Portugal
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Poster Communications
P. Pais, C. Costa, C. Pires, R. Santos, M. Cavalheiro, M.C. Teixeira, “New mechanisms of 5-flucytosine resistance in the human pathogen Candida glabrata: unveiled through chemogenomics and proteomics approaches”, MicroBiotec2015, December 10-12, 2015, Évora, Portugal
P. Pais, C. Costa, C. Pires, D. Romão, M. Cavalheiro, M.C. Teixeira. “Membrane proteomics approaches unveil new mechanisms of 5-flucytosine and clotrimazole antifungal resistance in the human pathogen Candida glabrata”, 6th Conference on Physiology of Yeast and Filamentous Fungi (PYFF 6), Lisboa, Portugal
Pais P, Galocha M, Cavalheiro M, Costa C, Califórnia R, Leitão C, Pereira D, Butler G, Teixeira MC, “Deciphering transcription regulation of antifungal drug resistance in the pathogenic yeast Candida glabrata”, MicroBiotec2017, December 7-8, 2017, Porto, Portugal
Pais P, Costa C, Pires C, Cavalheiro M, Teixeira MC, “Using pathoproteomics to unveil new players in antifungal drug resistance in the human pathogenic yeast Candida glabrata”, HFP2017 – Advanced Lecture Course in Molecular Mechanisms of Host-Pathogen Interactions and Virulence in Human Fungal Pathogens, May 13-19, 2017, La Colle sur Loup, France
Pais P, Galocha M, Monteiro PT, Teixeira MC, “The PathoYeastract database: a tool for the study of transcription regulation in Candida species”, 14th ASM Conference on Candida and Candidiasis, April 15-19, 2018, Providence, RI, USA
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DOCTORAL PROGRAM (36 ECTS)
Curricular Units:
• General Doctoral Training (6 ECTS)
• Advanced Experimental Techniques and methodologies (6 ECTS)
• Bioentrepreneurship (6 ECTS)
• Advanced Topics in Bioengineering and Biosciences (6 ECTS)
• Outreach and Teaching Skills (6 ECTS)
• Computational Biology (6 ECTS)
Advanced courses: Genome Assembly and Annotation Course, by Elixir, October23-27 2017, Instituto Gulbenkian Ciência, Oeiras, Portugal
Current Positions:
12/2021-present: PhD PPC Strategist at Supreme Optimization
09/2021-present: Freelance Scientific Writer at CRISPR Medicine News
01/2020-06/2020: Post-doc fellow (BIODATA) @ Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
09/2019-12/2019: Researcher (BIODATA) @ Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal