Mafalda Cavalheiro

Class 2016

Grant PD/BD/116946/2016

PhD thesis

Deciphering Mechanisms of Pathogenesis in Candida glabrata: in the Crossroad Between Drug Resistance and Virulence

Supervisor

• Miguel C Teixeira, Professor @ IST/ULisboa, PT

Collaborators

• Etienne Dague, LAAS-CNRS, Toulouse, France

PhD Degree complete: Discussion on October 12, 2020

THESIS ABSTRACT

In this thesis, the study of molecular mechanisms simultaneously involved in at least two of the three main processes for the survival of C. glabrata in the human host: biofilm formation, virulence and antifungal resistance; was pursued. This work presents two new regulators of biofilm formation, CgEfg1 and CgTec1, which together control 40% of all the transcriptomic changes happening in this complex process. In addition, biofilm formation was found to depend on unexpected players, like CgDtr1 and CgTpo4, which are key effectors of virulence in thispathogenic yeast, CgDtr1 as an acetate exporter whose action fights oxidative and acetic acid stress, while CgTpo4 is essential to resist the action of antimicrobial peptides and polyamines. Biofilm formation was also found to rely on the expression of two cell peripheral proteins, CgEpa3 and CgPil2, an adhesin and an eisosome component unraveled during the study of in vitro evolution towards azole resistance of a C. glabrata susceptible clinical isolate. CgEpa3 and CgPil2 are thus two effectors in azole resistance, the first believed to contribute to increased yeast aggregation, decreasing C. glabrata exposure to azole drugs, while CgPil2 contributes for lipid homeostasis at the plasma membrane, influencing the presence of MDR transporters involved in both antifungal resistance and biofilm formation. Altogether, this thesis gives evidence of new molecular mechanisms with intricate functions in biofilm formation, virulence and antifungal resistance, indicating that new therapeutic targets should be chosen according to their importance in as many fundamental roles in C. glabrata as possible.

RESUMO DA TESE

Nesta tese, o estudo dos mecanismos moleculares simultaneamente envolvidos em pelo menos dois dos três principais processos de sobrevivência de C. glabrata no hospedeiro humano: formação de biofilme, virulência e resistência a antifúngicos; foi perseguido. Este trabalho apresenta dois novos reguladores de formação de biofilme, CgEfg1 e CgTec1, que juntos controlam 40% de todas as alterações transcriptómicas que acontecem neste processo complexo. Adicionalmente, descobriu-se que a formação de biofilme é dependente de jogadores inesperados, como CgDtr1 e CgTpo4, que são efectores-chave de virulência nesta levedura patogénica, CgDtr1 como um exportador de acetato cuja acção combate o stress oxidativo e o derivado do ácido acético, enquanto o CgTpo4 é essencial para resistir à acção de péptidos antimicrobianos e poliaminas. A formação de biofilme foi descoberta estar dependente da expressão de duas proteínas celulares periféricas, CgEpa3 e CgPil2, uma adesina e um componente de eisossomas desvendados durante o estudo da evolução in vitro direccionada à resistência a azóis de um isolado clínico susceptível de C. glabrata. CgEpa3 e CgPil2 são assim dois efectores na resistência a azóis, o primeiro acredita-se contribuir para o aumento de agregação entre células de levedura, diminuindo a exposição de C. glabrata às drogas azóis, enquanto CgPil2 contribui para a homeostase lipídica na membrana plasmática, influenciando a presença de transportadores MDR envolvidos na resistência a antifúngicos e na formação de biofilme. De um modo geral, esta tese dá evidências de novos mecanismos moleculares com funções intricadas na formação de biofilme, virulência e resistência a antifúngicos, indicando que novos alvos terapêuticos devem ser escolhidos de acordo com a sua importância no maior número possível de papéis fundamentais em C. glabrata.

PUBLICATIONS

Papers

Cavalheiro M, Romão D, Santos R, Mil-Homens D, Pais P, Costa C, Galocha M, Pereira D, Takahashi-Nakaguchi A, Chibana H, Fialho AM, Teixeira MC, “Role of CgTpo4 in polyamine and antimicrobial peptide resistance: determining virulence in Candida glabrata”. International Journal of Molecular Sciences. 22, (1376), 2021.

Klemm S, Baum M, Qiu H, Nan Z, Cavalheiro M, Teixeira MC, Tendero C, Gapeeva A, Adelung R, Dague E, Castelain M, Formosa-Dague C, “Development of Polythiourethane/ZnO-based anti-fouling materials and evaluation of the adhesion of Staphylococcus aureus and Candida glabrata using single-cell force spectroscopy”, Nanomaterials. 11(2), 2021.

Pais P, Califórnia R, Galocha M, Viana R, Ola M, Cavalheiro M, Takahashi-Nakaguchi A, Chibana H, Butler G, Teixeira MC, “Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability”, Antimicrobial Agents Chemother. 64(9), 2020.

Santos, R., Cavalheiro, M., Costa, C., Takahashi-Nakaguchi, A., Okamoto, M., Chibana, H., Teixeira, M.C., “Screening the Drug:H+ Antiporter family for a role in biofilm formation in Candida glabrata”, Frontiers in Cellular and Infection Microbiology, 10: 29, 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.

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.

Cavalheiro, M., Pais, P., Galocha, M., Teixeira, M.C., “Host-pathogen interactions mediated by MDR transporters in fungi: as pleiotropic as it gets!”, Genes, 9: 332, 2018.

Cavalheiro, M., Teixeira, M.C., “Candida biofilms: threats, challenges and promising strategies”, Frontiers in Medicine, 5: 28, 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.

Teixeira, M.C., Monteiro, P., Palma, M., Costa, 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 inSaccharomyces cerevisiae”. Nucleic Acids Res. gkx842, 2017. (DOI: 10.1093/nar/gkx842)

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”. Front. Cell. Infect. Microbiol. 6:131, 2016 (DOI: 10.3389/fcimb.2016.00131)

Costa, C., Ribeiro, J., Miranda, I.M., Silva-Dias, A.I., Cavalheiro, M., Costa-de-Oliveira, S., Rodrigues, A.G., Teixeira, M.C., “Clotrimazole drug resistance in Candida glabrata clinical isolates correlates with increased expression of the drug:H+ antiporters CgAqr1, CgTpo1_1, CgTpo3 and CgQdr2″, Front. Microbiol. 7:526, 2016.

Costa C, Ponte A, Pais, P, Santos R, Cavalheiro M, Takashi Y, Chibana H, Teixeira MC, “New mechanisms of flucytosine resistance in C. glabrata unveiled by a chemogenomics analysis in S. cerevisiae”, PLoS ONE, 10(8):e0135110, 2015.

Book Chapters

Cavalheiro M, Teixeira MC (2018) In the Crossroad Between Drug Resistance and Virulence in Fungal Pathogens, In: Skoneczny M. (eds) Stress Response Mechanisms in Fungi. Springer, Cham.

Cavalheiro M, Costa C, Pais P, Teixeira MC (2016) Acquisition of resistance to azole antifungal drugs in Candida species: imidazoles versus triazoles, In: Ketoconazole: Clinical Pharmacokinetics, Uses and Potential Side Effects. Nova Science Publishers, Inc., NY, USA.

Oral Communications

Cavalheiro M, Costa C, Wang C, Silva-Dias A, Miranda IM, Silva RM, Rodrigues AG, Butler G, Teixeira MC, Transcriptomic Analysis of the in vitro Evolution of a Susceptible Candida glabrata Clinical Isolate towards Azole Resistance”, Annual Meeting of the Portuguese Society of Genetics, 14-15 June 2018, Porto, Portugal.

Poster Communications

Cavalheiro M, Costa C, Silva-Dias A, Miranda IM, Chibana H, Rodrigues AG, Butler G, Teixeira MC, “New insights into the clinical acquisition of azole resistance: witnessing Candida glabrata evolution”, 29th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 13-16 April 2019, Amsterdam, The Netherlands

Cavalheiro M, Costa C, Wang C, Silva-Dias A, Miranda IM, Rodrigues AG, Butler G, Teixeira MC, “Determining the molecular mechanisms behind the in vitro evolution of a susceptible Candida glabrata clinical isolate towards azole resistance”, 14th ASM Conference on Candida and Candidiasis,April 15-19, 2018, Providence, RI, USA

Cavalheiro M, Santos R, Romão D, Mil-Homens D, Pais P, Costa C, Viana R, Santos S, Fialho AM, Teixeira MC, “Determining virulence by protecting the cell from host induced stresses: the unexpected role of C. glabrata multidrug transporters”, MicroBiotec2017, December 7-8, 2017, Porto, Portugal (best poster presentation award)

Cavalheiro M, Santos R, Romão D, Mil-Homens D, Pais P, Costa C, Galocha M, Fialho AM, Teixeira MC, “A new role for C. glabrata drug:H+ antiporters: in the crossroad between drug resistance and virulence”, 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

Cavalheiro M, Costa C, Wang C, Miranda IM, Silva-Dias A, Rodrigues AG, Butler G, Teixeira MC, Unveiling the mechanisms of azole drug resistance acquisition in a clinical isolate: a transcriptomics approach, PYFF6 – 6th Conference on Physiology of Yeasts and Filamentous Fungi, 11 -14 July 2016, Lisbon, Portugal.

Awards

Travel Grant Award given by the Microbiology Society, at Candida and Candidiasis 2020 conference, Monte Real, Canada. (postponed due to covid-19)

Travel Grant Award given by the American Society for Microbiology, at 14th ASM Conference on Candida and Candidiasis, 15-19 April 2018, Providence, RI, EUA.

Best Poster Communication in the Symposium Cellular Microbiology and Pathogenesis given by the Portuguese Society of Microbiology, at MICROBIOTEC’17, 7-9 December 2017, Porto, Portugal.

DOCTORAL PROGRAM (36 ECTS)

• General Doctoral Training (6 ECTS)

• Advanced Experimental Techniques and methodologies (6 ECTS)

• Bioentrepreneurship (6 ECTS)

• Outreach and Teaching Skills (6 ECTS)

• Structural Biology (6 ECTS)

• Gene Therapy (6 ECTS)

Advanced courses: Genome Assembly and Annotation Course, by Elixir, 23-27 October 2017, Instituto Gulbenkian Ciência, Oeiras, Portugal

Current Position/Instutution

Junior R&D Project Manager at A4F – Algae for Future, Department of Research, Development and Innovation, Estrada do Paço do Lumiar, Campus do Lumiar, Ed E-R/C, 1649-038, Lisboa, Portugal