Mónica da Luz Galocha PD/BD/143027/2018
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, to guide the design of more suitable therapeutic strategies.
This PhD project aims to unveil new mechanisms of acquired drug resistance in C. glabrata clinical isolates. First, we will identify new players involved in the drug resistant phenotype of clinical isolates. With that aim, a collection of C. glabrata clinical isolates will be characterized regarding their ability to resist antifungal drug action. In parallel, a directed evolution approach will be used to evolve in vitro C. glabrata susceptible clinical isolates towards drug resistance. In vivo and in vitro induced genomic alterations will be identified through whole-genome sequencing followed by SNP profiling, leading to the identification of yet unknown players in drug resistance phenomenon that might constitute new important drug targets. The relevance of the identified new players underlying C. glabrata acquired drug resistance will be studied in depth, characterizing the molecular basis of the observed phenotypes.
Ultimately, we aim to identify new drug targets to improve existing therapeutic strategies to tackle C. glabrata increasing infections.
Keywords: Candida glabrata, clinical isolates, drug resistance mechanisms, directed evolution, genome-wide changes, RNA-sequencing.
State of the Art
Candida species are accountable for mucosal and invasive candidiasis, the later associated with high mortality rates (40-60%) . Candidiasis is the most common fungal disease in the world , being Candida species responsible for 12.7% of all catheter-associated urinary infections and 14.6% of central line–associated bloodstream infections , corresponding to the major cause of systemic fungal infections . Candida glabrata, together with Candida albicans, is responsible for ~400,000 life-threatening infections per year . Given the increase of the immunocompromised population, the number of infections by these pathogens will continue to increase. Current therapeutic options are on the verge of becoming obsolete, as several Candida species exhibit intrinsic or acquired resistance to most antifungal drugs. Particularly, the increasing prevalence of C. glabrata as a human pathogen has been mostly associated with its rapid acquisition of antifungal resistance [6–8]. Although a number of antifungal resistance mechanisms have been deciphered over the years, several resistant clinical isolates do not to display any of the known mechanisms of resistance [9,10]. The main mechanism of acquired azole resistance is the up-regulation of ABC (ATP-binding Cassette) drug transporters, mainly CgCdr1 [11–13], and, to a lower extent, of the MFS (Major Facilitator Superfamily) drug:H+ antiporters [5,14–20], which catalyse the efflux of azoles preventing their intracellular accumulation. Generally, the constitutive up-regulation of the ABC efflux pumps is due to the emergence of gain-of-function (GOF) mutations in the CgPDR1 gene, encoding the key regulator of multidrug resistance in C. glabrata [21,22]. In contrast to what is observed in C. albicans, several studies suggest that mutations in the drug target encoding genes ERG11 is not a significant mechanism of acquired azole resistance in C. glabrata clinical isolates [11,23,24]. Although CgPDR1 GOF mutations are considered the major mechanism of clinical acquisition of azole resistance, several resistant clinical isolates do not to display this or any other of the known mechanisms of azole resistance [9,10], suggesting the presence of yet unknown alternative paths to acquired azole resistance in C. glabrata. Given that drug resistance imposes great concern as it has been increasing in the clinical setting and that some of the known resistance mechanisms were only observed in lab mutated strains, understanding the molecular basis of acquired drug resistance in clinical isolates is crucial to guide the design of more suitable therapeutic strategies.
This PhD project aims to address the development of drug resistance in C. glabrata clinical isolates by understanding the molecular basis of the resistant phenotype. To do so, a combination of large-scale genome-wide approaches will be used to unveil genomic changes that might provide insights into the mechanisms of acquired drug resistance as a way to design suitable strategies to deal with the arising problem of C. glabrata resistant clinical isolates, for which there is no guaranteed therapy. The selected transdisciplinary approach, steaming from genomics and bioinformatics to molecular biology, microbial physiology and clinical epidemiology, aims at identifying new mechanisms networks governing the ability of C. glabrata cells to act as successful pathogens, by surpassing antifungal drug therapy.
A second innovative aspect of this project is its focus on providing personalized medicine, from the perspective of the bug. Based on sequencing a high number of genomes of clinical isolates, it is our goal to associate phenotypic characteristics to specific genomic profiles and/or specific biomarker expression and SNPs. This will enable the use of these molecular traits to discriminate drug resistance profiles, in order to advise the best possible therapy. This innovative perspective will also be extended to other pathogenesis-relevant phenotypes, guiding the surgical design of the most suitable therapeutic strategy.
Finally, this project will provide novel targets to be used alone or in combination therapy, and prevent the development of antifungal resistance, while maintaining the effectiveness of the currently used drugs.
The main goal of this proposal is to improve current therapeutic options for the treatment of Candida infections, based on deep understanding of the molecular mechanisms underlying drug resistance development in Candida glabrata clinical setting. The expected implications of the execution of this project include improved health care and quality of life for the millions of people infected with superficial candidiasis and improved chances of survival to the hundreds of thousands that develop systemic candidiasis (in line with Goal 3 of the UN2030 Agenda for sustainable development).
First, a directed evolution approach will be used to in vitro induce susceptible clinical isolates towards drug resistance, by prolonged exposure to a determined antifungal. Additionally, a collection of C. glabrata clinical isolates, obtained from collaborative hospitals, will be scrutinized regarding their ability to resist antifungal drugs. Drug resistance-related mutations will be identified using whole-genome sequencing allied with SNP profiling, providing insights into new mechanisms underlying acquired drug resistance in the clinical setting. Genes identified as probable players in a yet unknown drug resistance pathway will be functionally characterized, ultimately leading to the identification of new potential drug targets.
It is expected that this PhD project will contribute for the advancement of scientific knowledge on this topic but also to provide promising drug targets for the future development of antifungal drugs and/or improving the currently available therapies to fight C. glabrata infections. Ultimately, this research plan is focused on enhancing the conditions for treatment of these infections and improve human health and quality of life.
Current therapeutic options for the increasing Candida infections are on the verge of becoming obsolete, as several Candida species exhibit intrinsic or acquired resistance to most of the currently used classes of antifungal drugs. C. glabrata has risen dramatically in frequency as a significant cause of blood stream infection (BSI) since the introduction of azole drugs in the
1980s. The increase in the prophylactic use of azoles for high-risk individuals undoubtedly contributed to the increasing development of C. glabrata resistance to these antifungal drugs, which are significantly effective in eradicating infections caused by other Candida species. These drugs have been used for decades as the standard treatment against candidosis and are still the only oral treatment option. Instead of developing new drugs to overcome antifungal resistance, a process which is both very costly and time-consuming, therapeutic strategies can be improved by enhancing the efficacy of existing drugs, such as azoles. Therefore, it is worth identifying the genes and cellular pathways involved in the development of drug resistance. C. glabrata presents high levels of intrinsic and acquired resistance to azole antifungals, especially due to overexpression of multidrug resistance transporters activated by the transcription factor Pdr1. Although GOF mutations in the multidrug regulator Pdr1 constitutes the main mechanism of acquired azole resistance in C. glabrata clinical isolates, several resistant isolates do not display any of the known resistance mechanisms. Additionally, some of the resistance mechanisms that we know (i.e., upregulation and/or alterations of the drug target Erg11) were only seen in lab manipulated strains, which raises the question: are they clinically relevant?
This PhD proposal aims to understand which mechanisms of drug resistance, beyond Pdr1, are underlying the resistant phenotype observed in C. glabrata azole resistant isolates. To tackle this, a directed evolution approach will be used to in vitro induce susceptible clinical isolates towards drug resistance by prolonged exposure to posaconazole, which is a new generation triazole with broad-spectrum activity. Additionally, a collection of C. glabrata clinical isolates, obtained from collaborative hospitals, will be scrutinized regarding their ability to resist antifungal drugs.
The first task will focus on a genome-wide analysis of selected C. glabrata clinical isolates, exhibiting extremely high and extremely low MIC values against azole drugs. Drug resistance-related mutations will be identified using whole-genome sequencing allied with SNP profiling, providing insights into new mechanisms underlying acquired drug resistance in the clinical setting. Non-relevant SNPs (i.e., synonymous SNPs, strain-related SNPs and SNPs present in susceptible isolates) will be discarded. This analysis will render a group of mutations and/or genes of interest that might underlying, at least partially, of the observed resistant phenotype.
The second task will be the phenotypic screening of the selected possible resistance-related genes. To do so, different antifungal susceptibility assays will be performed, as well as subcellular localization assays.
The third task will be the evaluation of the impact of the identified novel players in C. glabrata acquired drug resistance by assessing the functional differences of wild-type vs multiple deletion mutants, devoid of said players. This work will be performed in collaboration with the lab of Prof. Patrick Van Dijck, from the VIB-KU Leuven Center for Microbiology.
Finally, the fourth task will consist in the extensive functional characterization of the new effectors in drug resistance pathways, identified in the previous tasks, in order to unveil new mechanisms of acquired resistance in C. glabrata clinical isolates.
Overall, this research plan will contribute to find answers to important questions regarding the genome-wide evolution towards antifungal drug resistance of C. glabrata in the clinical setting, but also gather clues leading to the identification of new drug targets, thus contributing for the future development of novel therapeutic strategies to fight C. glabrata associated infections.
• 1st Year
o Curricular units of the Doctoral Program (2 semesters)
o 1st task – Genome-wide analysis of Candida glabrata clinical isolates (6 months)
o 2nd task – Phenotypic screening of selected relevant resistance-related genes (6 months)
• 2nd and 3rd Year
o 3rd task - Evaluation of the impact of the identified novel players in C. glabrata acquired drug resistance: assessing the functional differences of wild-type vs multiple deletion mutants, devoid of said players (in collaboration with the lab of Prof. Patrick Van Dijck, from the VIB-KU Leuven Center for Microbiology - https://cfm.vib.be/labs/van-dijck-lab) (6 months)
o 4th task - Functional characterization of the new effectors in drug resistance pathways, identified in the previous tasks (since 2nd year through half of 4th year)
• 4th Year
o 4th task - Functional characterization of the new effectors in drug resistance pathways, identified in the previous tasks (since 2nd year through half of 4th year)
o Write PhD thesis (6 months)
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Cavalheiro, M., Romão, D., Santos, R., Mil-Homens, D., Pais, P., Costa, C., Galocha, M., Pereira, D., Takahashi-Nakaguchi, A., Chibana, H., Fialho, A.M., Teixeira, M.C. (2021). Role of CgTpo4 in Polyamine and Antimicrobial Peptide Resistance: Determining Virulence in Candida glabrata. Int J Mol Sci, 22(3): 1376.
Viana, R., Pais, P., Cavalheiro, M., Galocha, M., Teixeira, M. C. (2021). Host-Induced Stress Response in Human Pathogenic Fungi. In book: Reference Module in Life Science.
Monteiro, P. T., Pedreira, T., Galocha, M., Teixeira, M. C., & Chaouiya, C. (2020). Assessing regulatory features of the current transcriptional network of Saccharomyces cerevisiae. Scientific reports, 10(1), 1-11.
Viana, R., Dias, O., Bastos, J., Lagoa, D. R. S., Galocha, M., Rocha, I., & Teixeira, M. C. (2019). Genome-scale metabolic model of the human pathogen C. albicans: aiming the identification of promising new drug targets.
Pais, P., Califórnia, R., Galocha, M., Viana, R., Ola, M., Cavalheiro, M., ... & Teixeira, M. C. (2020). Candida glabrata transcription factor Rpn4 mediates fluconazole resistance through regulation of ergosterol biosynthesis and plasma membrane permeability. Antimicrobial agents and chemotherapy, 64(9), e00554-20.
Monteiro, P. T., Oliveira, J., Pais, P., Antunes, M., Palma, M., Cavalheiro, M., Galocha, M., Godinho, C., Martins, L.C., Bourbon, N., Mota, M. N., Ribeiro, R. A., Viana, R., Sá-Correia, I., Teixeira, M. C. (2020). YEASTRACT+: a portal for cross-species comparative genomics of transcription regulation in yeasts. Nucleic acids research, 48(D1), D642-D649.
Pais, P., Galocha, M., Miranda, I.M., Rodrigues, A.G., Teixeira, M.C.. Draft genome sequences from three clinical isolates of the pathogenic yeast Candida glabrata. Microbial Resource Announcements accepted for publication.
Galocha, M., Pais, P., Cavalheiro, M., Pereira, D., Viana, R., & Teixeira, M. C. (2019). Divergent Approaches to Virulence in C. albicans and C. glabrata: Two Sides of the Same Coin. International journal of molecular sciences, 20(9), 2345.
Pais, P., Galocha, M., & Teixeira, M. C. (2019). Genome-Wide Response to Drugs and Stress in the Pathogenic Yeast Candida glabrata. In Yeasts in Biotechnology and Human Health (pp. 155-193). Springer, Cham.
Pais, P., Galocha, M., Viana, R., Cavalheiro, M., Pereira, D., & Teixeira, M. C. (2019). Microevolution of the pathogenic yeasts Candida albicans and Candida glabrata during antifungal therapy and host infection. Microbial Cell, 6(3), 142.
Cavalheiro, M., Pais, P., Galocha, M., & Teixeira, M. (2018). Host-pathogen interactions mediated by MDR transporters in fungi: as pleiotropic as it gets!. Genes, 9(7), 332.
7th Workshop of the Doctoral Program in Applied and Environmental Microbiology (DP_AEM); September 24-27, 2018, Braga.
HFP2019 – Advanced Lecture Course in Molecular Mechanisms of Host-Pathogen Interactions and Virulence in Human Fungal Pathogens, 18-24 May 2019, La Colle sur Loup, France - Galocha, M., Silva-Dias, Ana., Pais, P., Cavalheiro, M., Miranda, I. M., Chibana, H., Rodrigues, A. G., Teixeira, M. C., Candida glabrata evolution towards posaconazole resistance irrespective of PDR1 GOF mutations, Poster communication
Portuguese Society of Genetics Annual Meeting, 14-15 June 2018, Porto, Portugal. - Pais P, Galocha M, Monteiro PT, Teixeira MC, PathoYeastract: a free web information system designed to fully exploit transcription regulation data in pathogenic yeasts, Poster communication
Best PhD Poster Presentation Award - Poster communication at HFP2019 – Advanced Lecture Course in Molecular Mechanisms of Host-Pathogen Interactions and Virulence in Human Fungal Pathogens, 18-24 May 2019, La Colle sur Loup, France.