Mónica
Galocha
...
DP_AEM student
Institute for Bioengineering and Biosciences (IBB)
Doctoral Program in Biotechnology and Biosciences (Técnico)
2018-2022
Miguel Cacho Teixeira
Mónica
0
0

Phd Thesis

Genome-wide evolution towards antifungal drug resistance in Candida glabrata: towards the identification of new, clinically relevant, players.

Mónica da Luz Galocha PD/BD/143027/2018

 

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, 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%) [1]. Candidiasis is the most common fungal disease in the world [2], being Candida species responsible for 12.7% of all catheter-associated urinary infections and 14.6% of central line–associated bloodstream infections [3], corresponding to the major cause of systemic fungal infections [4]. Candida glabrata, together with Candida albicans, is responsible for ~400,000 life-threatening infections per year [5]. 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.

 

Objectives

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.

 

Detailed description

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.

 

Tasks Timeline

• 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)

 

References

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2. Brown, G.D.; Denning, D.W.; Gow, N.A.R.; Levitz, S.M.; Netea, M.G.; White, T.C. Hidden Killers: Human Fungal Infections. Sci. Transl. Med. 2012, 4, 165rv13-165rv13, doi:10.1126/scitranslmed.3004404.

3. Suleyman, G.; Alangaden, G.J. Nosocomial Fungal Infections: Epidemiology, Infection Control, and Prevention. Infect. Dis. Clin. North Am. 2016, 30, 1023–1052, doi:10.1016/j.idc.2016.07.008.

4. Fausto, A.; Rodrigues, M.L.; Coelho, C. The still underestimated problem of fungal diseases worldwide. Front. Microbiol. 2019, 10, 1–5, doi:10.3389/fmicb.2019.00214.

5. Cavalheiro, M.; Pais, P.; Galocha, M.; Teixeira, M. Host-Pathogen Interactions Mediated by MDR Transporters in Fungi: As Pleiotropic as it Gets! Genes (Basel). 2018, 9, 332, doi:10.3390/genes9070332.

6. 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. Int. J. Mol. Sci. 2019, 20.

7. Pais, P.; Galocha, M.; Teixeira, M.C. Genome-Wide Response to Drugs and Stress in the Pathogenic Yeast Candida glabrata. In Progress in Molecular and Subcellular Biology 58,; Springer International Publishing, 2019; Vol. 58, pp. 155–193 ISBN 978-3-030-13034-3.

8. 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. Microb. Cell 2019, 6, 142–159, doi:10.15698/mic2019.03.670.

9. Ben-Ami, R.; Zimmerman, O.; Finn, T.; Amit, S.; Novikov, A.; Wertheimer, N.; Lurie-Weinberger, M.; Berman, J. Heteroresistance to fluconazole is a continuously distributed phenotype among candida glabrata clinical strains associated with in vivo persistence. MBio 2016, 7, doi:10.1128/mBio.00655-16.

10. Arastehfar, A.; Daneshnia, F.; Zomorodian, K.; Najafzadeh, M.J.; Khodavaisy, S.; Zarrinfar, H.; Hagen, F.; Shahrabadi, Z.Z.; Lackner, M.; Mirhendi, H.; et al. Low level of antifungal resistance in iranian isolates of candida glabrata recovered from blood samples in a multicenter study from 2015 to 2018 and potential prognostic values of genotyping and sequencing of PDR1. Antimicrob. Agents Chemother. 2019, 63, doi:10.1128/AAC.02503-18.

11. Sanguinetti, M.; Posteraro, B.; Fiori, B.; Ranno, S.; Torelli, R.; Fadda, G. Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance. Antimicrob. Agents Chemother. 2005, 49, 668–79, doi:10.1128/AAC.49.2.668-679.2005.

12. Sanglard, D.; Ischer, F.; Calabrese, D.; Majcherczyk, P.A.; Bille, J. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob. Agents Chemother. 1999, 43, 2753–65.

13. Akins, R.A. An update on antifungal targets and mechanisms of resistance in Candida albicans. 2005, 285–318, doi:10.1080/13693780500138971.

14. Costa, C.; Ribeiro, J.; Miranda, I.M.; Silva-Dias, A.; 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. 2016, 7, 1–11, doi:10.3389/fmicb.2016.00526.

15. Costa, C.; Dias, P.J.; Sá-Correia, I.; Teixeira, M.C. MFS multidrug transporters in pathogenic fungi: Do they have real clinical impact?; Frontiers Research Foundation, 2014; Vol. 5 MAY, pp. 1–8;.

16. Costa, C.; Pires, C.; Cabrito, T.R.; Renaudin, A.; Ohno, M.; Chibana, H.; Sá-Correia, I.;

Teixeira, M.C. Candida glabrata drug:H+ antiporter CgQdr2 confers imidazole drug resistance, being activated by transcription factor CgPdr1. Antimicrob. Agents Chemother. 2013, 57, 3159–67, doi:10.1128/AAC.00811-12.

17. 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. Mol. Cell. Proteomics 2016, 15, 57–72, doi:10.1074/mcp.M114.045344.

18. Costa, C.; Henriques, A.; Pires, C.; Nunes, J.; Ohno, M.; Chibana, H.; Sá-Correia, I.; Teixeira, M.C. The dual role of candida glabrata drug:H+ antiporter CgAqr1 (ORF CAGL0J09944g) in antifungal drug and acetic acid resistance. Front. Microbiol. 2013, 4, 170, doi:10.3389/fmicb.2013.00170.

19. Costa, C.; Nunes, J.; Henriques, A.; Mira, N.P.; Nakayama, H.; Chibana, H.; Teixeira, M.C. Candida glabrata drug:H+ antiporter CgTpo3 (ORF CAGL0I10384G): Role in azole drug resistance and polyamine homeostasis. J. Antimicrob. Chemother. 2014, 69, 1767–1776, doi:10.1093/jac/dku044.

20. 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. Front. Microbiol. 2016, 7, 2045, doi:10.3389/fmicb.2016.02045.

21. Vermitsky, J.-P.; Earhart, K.D.; Smith, W.L.; Homayouni, R.; Edlind, T.D.; Rogers, P.D. Pdr1 regulates multidrug resistance in Candida glabrata: gene disruption and genome-wide expression studies. Mol. Microbiol. 2006, 61, 704–722, doi:10.1111/j.1365-2958.2006.05235.x.

22. Ferrari, S.; Ischer, F.; Calabrese, D.; Posteraro, B.; Sanguinetti, M.; Fadda, G.; Rohde, B.; Bauser, C.; Bader, O.; Sanglard, D. Gain of Function Mutations in CgPDR1 of Candida glabrata Not Only Mediate Antifungal Resistance but Also Enhance Virulence. PLoS Pathog. 2009, 5, e1000268, doi:10.1371/journal.ppat.1000268.

23. Caudle, K.E.; Barker, K.S.; Wiederhold, N.P.; Xu, L.; Homayouni, R.; Rogers, P.D. Genomewide expression profile analysis of the Candida glabrata Pdr1 regulon. Eukaryot. Cell 2011, 10, 373–83, doi:10.1128/EC.00073-10.

24. Vermitsky, J.-P.; Edlind, T.D. Azole resistance in Candida glabrata: coordinate upregulation of multidrug transporters and evidence for a Pdr1-like transcription factor. Antimicrob. Agents Chemother. 2004, 48, 3773–81, doi:10.1128/AAC.48.10.3773-3781.2004.

Publication and Meetings

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. 

Galocha, M., Costa, I. V., Teixeira, M. C. (2020). Carrier-Mediated Drug Uptake in Fungal Pathogens. Genes, 11(11), 1324.

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 reports10(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 chemotherapy64(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 research48(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.

8th Workshop of the PhD Program in Applied and Environmental Microbiology (DP_AEM) in University of Minho, Braga from 10-13 February 2020

Seminar of the PhD Program in Applied and Environmental Microbiology (DP_AEM) at 10 February 2020, Virtual meeting.

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 

Awards

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.