genomic analysis of a candida glabrata clinical isolate ... · genomic analysis of a candida...
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Genomic analysis of a Candida glabrata clinical isolate
resistant to antifungals unveils novel features of drug
resistance in this pathogenic yeast
Sara Barbosa Salazar
Thesis to obtain the Master of Science Degree in
Biotechnology
Supervisor: Prof. Dr. Nuno Gonçalo Pereira Mira
Examination Committee
Chairperson: Prof. Dr. Isabel Maria de Sá-Correia Leite de Almeida
Supervisor: Prof. Dr. Nuno Gonçalo Pereira Mira
Member of the Committee: Prof. Dr. Ana Paula Fernandes Monteiro Sampaio
Carvalho
June 2015
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Acknowledgements
At first, I would like to express my gratitude to my supervisor, Professor Nuno Mira, for
giving me the opportunity of becoming a part of this project. He always shown to be enthusiastic
about our work, which was essential for my motivation in this work. I specially would like to thank
him for believing in me and the work I developed and the patience put in this last stage of the
master thesis that were essential for the presentation of this work.
I would also like to express my sincere acknowledgements to Professor Isabel Sá-Correia
for hosting my stay at the BSRG laboratory and also for her scientific contribution for this work
which is imprinted in the experience that the group has acquired in the exploration of OMICS
approaches for the elucidation of molecular mechanisms of resistance to drugs in Yeasts. I would
also like to thank Professor Maria João Sousa from the University of Minho for the help in the
estimation of FFUL887 genome size by flow cytometry. My acknowledgements are also going to
Professor Geraldine Butler and CanWang for all the help with the transcriptomic analysis.
Acknowledgments to Professor Maria Manuel Lopes, Dr. Rosa Barros and Dr. Teresa Ferreira of
CHLC for help in facilitating access to the cohort of clinical isolates used in this study. I would also
like to thank financial support of project PanCandida – Towards the development of a pan-
genomic DNA chip for the early detection of invasive candidiasis caused by C. albicans and C.
glabrata, sponsored by the Gilead Génese Program; of Pfizer research program WI178570; and
of FCT (UID/BIO/04565/2013).
As final acknowledgments, I thank Catarina Costa for her patience, who helped in the
laboratory whenever I needed. I would also like to thank my fellow students Nicole Rodrigues, Zé
Tó Rodrigues, Diana Cunha, Catarina Prata, and Pedro Pais for their support, friendship and for
all the laughs that is most certainly the reason I have kept my sanity and sense of humor through
the difficulties I endured during the realization of this project. My thanks also go to my friends that
were part of all my academic studies for all the support through time and to make this experience
one of the most rewarding yet. A special thanks goes for the support of Pedro that always shared
my interest in science and to both him, Farinhas and Fara for their support outside the work
environment and for their unshakable friendship. Finally, I want to express my gratitude for my
parents who supported me unconditionally and specially to my mother that inspired me to be who
I am today.
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Abstract
An alarming increase in the incidence of infections caused by C. glabrata has been
reported in the last years, in part, due to the emergence of strains resistant to azoles. Although
some knowledge has been gathered on the elucidation of the molecular mechanisms of
resistance to antifungals in C. glabrata, little is known on the genetic adaptive responses that
occur at the genomic level. In this work the genome sequences of a C. glabrata clinical isolate
(named FFUL887) resistant to voriconazole, fluconazole and caspofungin was compared with the
genome of the reference strain CBS138, which was found to be susceptible to all the above-
referred antifungals. The genomic sequence determined for the FFUL887 isolate includes 12.29
Mb, corresponding to 99.1% of the total genome size estimated by flow cytometry. Around 80.000
genomic variations were identified, 10.000 of them corresponding to missense mutations
occurring in the coding sequence of 3.200 genes (60% of the predicted C. glabrata ORFeome).
Around 100 proteins previously associated with drug resistance in C. glabrata were found to
harbour mutations in the FFUL887 genome including the transcription factor CgPdr1, a key player
in the control of drug resistance in C. glabrata. Using a transcriptomic analysis it was found that
the FFUL887 isolates over-expresses several described targets of CgPdr1 including the drug
efflux pumps CgCDR1, CgPDH1 and CgQDR2, all previously demonstrated to contribute for
azole resistance in C. glabrata. These observations, together with phenotypic data, demonstrate
that the CgPdr1 encoded by FFUL887 has a new gain-of-function mutation.
Key words: Acquired antifungal resistance; Candida glabrata; Comparative genomics
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Resumo
Nos últimos um aumento alarmante de infecções por parte de Candida glabata, em parte
é relato, em parte, devido ao aparecimento de estirpes resistentes a azóis. Embora já algum
conhecimento tenha sido adquirido levando à elucidação dos mecanismos moleculares de
resistência a antifungicos em C. glabrata, pouco se sabe relacionado a adaptação genética a
nível genético. Neste trabalho o genoma de um isolado clínico de C. glabrata (denominado
FFUL887) resistente a voriconazole, fluconazole e tolerante a caspofungina, foi comparado com
o genoma da estirpe de referência CBS138, mais susceptivel às mesmas drogas. A sequência
do genoma obtida do isolado FFUL887 inclui 12.29 Mb, correspondendo a 99.1% do tamanho
total do genoma estimado por citometria de fluxo. Cerca de 80.000 variações foram identificadas
no genoma, 10.000 correspondendo a mutações sem sentido ocorrentes em sequências
codificantes de cerca de 3.200 genes (60% do ORFeome previsto de C. glabrata). Cerca de 100
proteinas previamente associadas à resistência de drogas em C. glabrata, contêm mutações no
genoma de FFUL887 incluindo o factor de transcrição CgPDR1, um regulador chave no controlo
de resistência a drogas em C. glabrata. Com o uso de análise transcriptomica foi possível
confirmar que o isolado FFUL887 sobreexpressa alvos descritos do factor de transcriçao CgPdr1,
incluindo transportadores responsáveis pelo efluxo de drogas CgCDR1, CgPDH1 and CgQDR2,
transportadores estes previamente descritos como contribuintes para a resistência a azóis em C.
glabrata. Estas observações, em conjunto com os resultados fenotípicos, sugerem o CgPDR1
codificado por FFUL887 tem uma nova mutação de ganho-de-função.
Key words: Resistencia adquirida a antifungicos; Candida glabrata; genómica comparativa
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Table of Contents
Acknowledgements ........................................................................................................................ ii
Abstract ......................................................................................................................................... iii
Resumo ......................................................................................................................................... iv
Table of Contents .......................................................................................................................... v
List of Figures ............................................................................................................................... vii
List of Tables ................................................................................................................................. xi
Abbreviations ............................................................................................................................... xiii
1. Introduction ............................................................................................................................ 1
1.3 Antifungal drugs and their biological targets ....................................................................... 4
1.3.1. Overview.......................................................................................................................... 4
1.3.2 Azoles ........................................................................................................................... 5
1.3.3. Echinocandins ................................................................................................................. 5
1.3.4 Candida glabrata resistance to azoles and echinocandins .......................................... 6
1.4. Molecular mechanisms underlying C. glabrata resistance to azoles ................................. 7
1.4.1 Modifications related to ergosterol biosynthesis and import ........................................ 7
1.4.2. Role of multi-drug efflux pumps................................................................................... 9
1.4.3. The pleiotropic drug resistance transcription factor CgPdr1 ....................................... 9
1.4 4. Mitochondrial function ............................................................................................... 12
1.5. Molecular mechanisms underlying C. glabrata resistance to echinocandins .................. 12
1.5.1. Modifications in glucan synthase............................................................................... 12
1.6. Contribution of genome-wide studies for the comprehension of antifungal drug resistance
in C. glabrata ........................................................................................................................... 13
2. Material and Methods .............................................................................................................. 15
2.1. Strains and growth media ................................................................................................. 15
2.2. Antifungal solutions .......................................................................................................... 15
2.4. Growth curves in the presence of fluconazole, voriconazole, anidulafungin and
caspofungin. ............................................................................................................................ 18
2.5. Genomic DNA extraction .................................................................................................. 18
vi
2.6. Genome Sequencing and Annotation .............................................................................. 18
2.7. Microarray profiling of FFUL887 and CBS138 strains ..................................................... 19
2.8. Assessment of gene expression based on real time RT-PCR ......................................... 20
2.9. Quantification of biofilm formation in the presence of the antifungals ............................. 21
2.10. Adherence to biotic and abiotic surfaces ....................................................................... 21
2.11. Caspofungin time-kill assays .......................................................................................... 21
3. Results and Discussion ........................................................................................................... 22
3.1 Profiling of antifungal resistance within a cohort of clinical C. glabrata isolates ............... 22
3.2. Phenotypic characterization of the FFUL887 isolate in the presence of voriconazole,
anidulafungin, caspofungin or fluconazole. ............................................................................. 25
3.3 Genome Sequencing and Annotation ............................................................................... 30
3.4 Functional distribution of genes harboring mutations in the FFUL887 strain .................... 35
3.5 Mutations occurring in genes associated with antifungal resistance in C. glabrata .......... 39
3.5.1. Genes involved in azole and in echinocandin resistance ......................................... 41
3.5.2 Genes involved in azole resistance: emphasis on the transcription factor CgPdr1 ... 43
References .................................................................................................................................. 53
Annex A ....................................................................................................................................... 60
Annex B ....................................................................................................................................... 61
Annex C ....................................................................................................................................... 62
Annex D ....................................................................................................................................... 63
Appendix E .................................................................................................................................. 65
Annex F ....................................................................................................................................... 70
Annex G ....................................................................................................................................... 71
Annex H ....................................................................................................................................... 72
Annex I ........................................................................................................................................ 75
Annex J ........................................................................................................................................ 78
vii
List of Figures Figure 1 - Worldwide and Portugal prevalence of Candida spp. isolates collected from patients with
diagnosed Candida infections. Epidemiological studies chosen for this representation used a higher
dataset. Worldwide data was retrieved from Castanheira et al. 2014 and Portugal data from Paulo, C.
et al., 2009. Other epidemiological studies from Portugal are further explored in this section, were the
majority show a similar emergence of NCAC……………….……………………………………………....3
Figure 2 - Antifungal drug classes and their targets. A. Azoles inhibit fungal growth by interfering with
the synthesis of ergosterol and causing the accumulation of the toxic sterol diols intermediates in the
membrane which thereby results in membrane stress. B. Flucytosine is processed in the cell originating
compounds, 5-fluorouridine monophosphate and 5-fluorodeoxyuridine monophosphate that inhibit
DNA synthesis and RNA synthesis. C. Polyenes bind to ergosterol in the fungal cell membrane forming
membrane-spanning channels that cause leakage of cellular components and osmotic cellular lysis.
D. Echinocandins inhibit (1,3)-β-D-glucan synthase, resulting in cell wall stress and a loss of cell wall
integrity. The image was altered from Cowen, L., 2008 ……………………………………………………4
Figure 3 - Chemical structure of azoles fluconazole and voriconazole. Image was altered from Mast,
N. et al 2013 [1] ………………………………………………………..........................................................5
Figure 4 - Chemical structure of anidulafungin and caspofungin. Image was altered from Emri, T.,
2013…...………………………………………………………….................................................................6
Figure 5 - The C. glabrata mevalonate and ergosterol biosynthetic pathways. The enzyme Erg11
targeted by azoles is highlighted in the grey box. Genes highlighted in bold correspond to those whose
deletion increased susceptibility of C. glabrata to azoles. Underlined sterols are accumulated upon
inhibition of Erg11 function in C. glabrata. ............................................................................................8
Figure 6 - The functional class genes transcriptional regulated by CgPdr1. The most common genes
upregulated in PDR1 GOF transcriptomes and adhesion genes known to be regulated by CgPdr1 are
also summarized. Information was retrieved from the transcriptome studies from Claude, K., et al.,
2011, Tsai, H., et al., 2010 and Vermitsky, J., et al., 2006. ................................................................10
Figure 7 - Described C. glabrata PDR1 gain-of-function mutations, according to the information
available in Ferrari, S., et al., 2009, Tsai, H., et al., 2010, Berilla, N. and Subik, J., 2010. ………......11
Figure 8 - General mechanism describing the activation of Pdr1/Pdr3 in response to drugs within a
fungal cell. Drug can binds to the XBD domain of the pleiotropic transcription factors Pdr1/Pdr3. Acting
as a nuclear receptor, Pdr1/Pdr3-drug complex can them associate to the KIX domain of the Gal11p
subunit of the Mediator complex and recruit RNA polymerase II to the promoter region of PDR genes
initiating transcription. Image was retrieved from Goffeau, A., 2008 [2]. ….........................................11
viii
Figure 9 - Schematic representation of the procedure used to prepare the 96-multiwell plates used to
determine MIC of voriconazole, anidulafungin and caspofungin for the different C. glabrata isolates. B:
blank; GC: control. Image altered from EUCAST discussion document E.Dis 7.1. In the case of
fluconazole the same methodology was applied with the difference that the initial stock concentration
was of 12 800. .....................................................................................................................................16
Figure 10 - Summary of the results obtained in this study regarding profiling of susceptibility and
resistance of C. glabrata clinical isolates to fluconazole (A) and voriconazole (B). In brackets it is
indicated the number of isolates that was found to be susceptible or resistant to the azoles tested. In
panel C the dataset of isolates resistant to voriconazole and fluconazole was compared, highlighting
the existence of 7 isolates that are resistant to both drugs. ………………………………………………23
Figure 11 - Distribution of MIC values of fluconazole and voriconazole obtained for the cohort of C.
glabrata clinical isolates tested in this work (panels A and B) or reported by EUCAST (panels C and
D). The dashed line indicates the resistance breakpoint for fluconazole or the epidemiological cut-off
value for voriconazole, above which the isolates are considered resistant to these drugs…………....24
Figure 12 - Distribution of the MIC values of anidulafungin and caspofungin obtained for the cohort of
C. glabrata clinical isolates tested in this work (panel A and B) and reported by EUCAST (panel C).
The dashed line indicates the resistance breakpoint for anidulafungin, above which the isolates are
considered resistant to the drug. …………………………………………………………………………….25
Figure 13 - Growth curves of CBS138 (⃝) and FFUL887 () strains in RPMI 1640 2% glucose (A),
or in this same medium supplemented with fluconazole (16 mg/L and 32 mg/L, panels B and C) or
voriconazole (1 mg/L or 0.5 mg/L panels D and E). Growth was followed based on the increase in
OD595nm of the culture during 42h. The growth curves shown are representative of three independent
experiments that gave rise to the same growth pattern…………………………………………………….27
Figure 14. Growth curves of CBS138 (⃝) and FFUL887 () strains in RPMI 1640 2% glucose (A), or
in this same medium supplemented with anidulafungin (0.03 mg/L and 0.06 mg/L, panels B and C) and
caspofungin (0.125 mg/L and 0.25 mg/L, panels D and E). Growth was followed based on the increase
in OD595nm of the culture during 42h. The growth curves shown are representative of three independent
experiments that gave rise to the same growth pattern…………………………………………………….27
Figure 15 - Comparison of the growth of CBS138 and FFUL887 isolates in the presence of 4 mg/L of
ketoconazole and 1 mg/L of clotrimazole. The growth shown is representative of two independent
experiments in which each isolate was assayed twice…………………………………………………….28
Figure 16 - Measure of biofilm production after 24h of growth in RPMI 1640 2% G growth medium or
in this same growth medium supplemented with the indicated concentrations of fluconazole,
voriconazole, caspofungin and anidulafungin. In each panel the isolates are ordered according to their
resistance to the different antifungals. The results shown are representative of two independent
experiments in which each isolate was assayed twice……………………………………………………29
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Figure 17 - Resistance of FFUL887 and CBS138 cells to killing induced by 0.25 mg/L of caspofungin.
Cells of the two strains were cultivated in RPMI 1640 2% G growth medium in control supplemented
with 0.25 mg/L caspofungin ( and , respectively) or 0 mg/L ( and, respectively) for 12h during
which cell viability of the two cultures was at designated times. The viability results shown are
representative of three independent experiments………………………………………………………….30
Figure 18 – “Population snapshot” of FFUL887 and CBS138 against the entire C. glabrata MLST
database. Each dot represents a ST group. Blue dots represent founding genomes. Both CBS138 and
FFUL887 are singletons (strain with a unique ST value of the entire database)……………………….32
Figure 19 - Percentage of genes having non-synonymous SNPs mutations in the FFUL887 strain,
when compared to the reference strain CBS138…………………………………………………………..33
Figure 20 - Distribution of the non-synonymous mutations per the 3200 genes whose coding sequence
differed in the FFUL887 and in the CBS138 genomes. The genes were clustered according to their
chromosomal localization…………………………………………………………………………………….34
Figure 21 - Adherence of FFUL887 and CBS138 cells to biotic and abiotic surfaces. Cells of the two
strains were cultivated in polystyrene 96-microwell plates or in these same plates pre-coated with
fibronectin (10 µg/ml) or vitronectin (10 µg/ml) for 4 and 8h. After incubation, the amount of biomass
present was quantified using the crystal violet staining method. ……………………………………….35
Figure 22 - Functional clustering of the proteins found to have different amino acid sequences in
FFUL887 and in CBS138 strains, according with MIPS functional catalogue. Enriched functional
classes (p-value below 0.01) are indicated with *………………………………………………………….36
Figure 23. Functional clustering of proteins found to have truncated sequences in FFUL887 compared
to CBS138 strains, according with MIPS functional catalogue ……………………………………………37
Figure 24 - Modifications in the amino acid sequence of the glucan synthase genes CgFks1 and
CgFks2 encoded by FFUL887, when compared to their counter-partners encoded by the CBS138
strain. Domains from both proteins were predicted by Pfam Domain [3]. Grey boxes represent
transmembrane domains predicted by TMHMM SERVER [4]. …………………….…………..………40
Figure 25 - Partial alignment of C. glabrata CgFks2 protein sequence of CBS138 and FFUL887 to the
paralogue CgFks1, and orthologue sequences of S. cerevisiae S288c, C. albicans SC5314, C.
parapsilosis CDC317 and C. tropicalis MYA-3404 using ClustalW2 [5]. Mutation found in CgFks2 of
FFUL887 are highlighted. ………………………………………………………………………………..….40
Figure 26 - Alterations in the amino acid sequence of the protein domains of intervenient of the HACS,
CgCch1 and CgMid1 encoded by FFUL887 when compared to their counter-partners encoded by the
CBS138 strain. The domains from CgMid1 were predicted based on the homology with S. cerevisiae
Mid1 retrieved from Iida. H., et al., 1994 The S4 and P domains of CgCch1 were predicted based on
the homology with S. cerevisiae Cch1 retrieved from Paidhungat, M. and Garret, S., 1997 and frizzled
x
cysteine-rich domain was predicted by Phyre2. Grey boxes represent transmembrane domains
predicted by TMHMM SERVER. Peptide signals were predicted by SignalP …………………….…….42
Figure 27 - Alterations in the amino acid sequence of the proteins involved in azole resistance encoded
by FFUL887, when compared to their counter-partners encoded by the CBS138 strain. Neutral
mutations are shown in grey. The domains shown for Pdh1 were retrieved from Miyazaki, H., et al.,
1998, Upc2A and HST1 domains were predicted based on the homologue domains from S. cerevisiae
described by Davies, B., 2005 and Kadosh, D. and Struhl, K., 1998 using ClustalW2. Grey boxes
represent transmembrane domains, as predicted by the TMHMM algorithm……………………………45
Figure 28 - Alterations in the amino acid sequence of the CgPdr1 transcription factor encoded by
FFUL887, when compared to its counter-partners encoded by the CBS138 strain. Neutral mutations
are represented in grey. CgPdr1 domains were retrieved from Tsai, H., 2010 ……..………………….46
Figure 29 - MIC values for fluconazole and voriconazole evaluated by the EUCAST dilution method
for the DSY562, SFY114, SFY115 and FFUL887 strains…………………………………………………47
Figure 30 - Comparison of the transcript levels of CgPDR1, CgCDR1, CgPDH1 and CgQDR2 genes
in FFUL887 and in the CBS138 strains, as revealed by real time RT-PCR. …………………………48
Figure 31 - Modifications in the amino acid sequence of the CgPkc1 transcription factor encoded by
FFUL887, when compared to its counter-partners encoded by the CBS138 strain. Pkc1domains were
predicted by Pfam Domain. The regulatory and activation domains from Rlm1, the inhibitory/Swi6 and
Slt2 interaction domain from Swi4 were predicted based on the homologue domains from S. cerevisiae
described by Watanabe, Y., et al. 1997 and Siegmud, R. and Nasmyth, K., 1996 using
ClustalW2………………………………………………………………………………………………………51
Figure 32 - Measured MIC values for the antifungals fluconazole and voriconazole of the cohort clinical
isolates used in this study and the reference strain CBS138 (white). Resistant isolates are market in
black, while isolates with sensitive/intermediate resistance are marked in grey. ………………………62
Figure 33 - Measured MIC values for the antifungals anidulafungin and caspofungin of the cohort
clinical isolates used in this study and the reference strain CBS138 (white). Resistant isolates are
market in black, while isolates with sensitive/intermediate resistance are marked in grey……………63
xi
List of Tables
Table 1 - Intrinsic susceptibility patterns of “wild-type” populations of Candida species, according with
EUCAST. In this case the phenotype of the wild-type populations is defined based on the MIC values
obtained for the majority of the isolates. S- Susceptibility; I - Intermediate and R – Resistance. Table
altered from Arendrup, M., 2013 …..…………………………………………………………………………7
Table 2 - Multi-drug efflux pumps from the ABC and MFS family described to be involved in azole
resistance in C. glabrata. ……………………………………………………………………………………..9
Table 3. Summary of described mutations found in the coding sequence of CgFKS1 and CgFKS2
genes and that contribute for enhanced resistance to echinocandins in C. glabrata. The amino acid
sequence of the “hot spot regions” of CgFks1 and CgFks2 proteins is underlined. Δ indicates a non-
sense mutation. ……………………………………………………………………………………………….13
Table 4 - Concentration range of antifungal agents used on this study…………………………………17
Table 5 - MIC values of the different antifungals used during this work corresponding to resistance
breakpoints, as indicated by EUCAST. In the case of voriconazole EUCAST has not defined an exact
breakpoint but strains are consider resistance when exhibiting a MIC higher than 1 mg/L, the EUCAST
epidemiological cut-off (ECOFF). Due to significant inter-laboratory variation in MIC values, ranges for
caspofungin EUCAST breakpoints have not yet been established………………………………………17
Table 6 - Parameters used in the CLC Genomics Workbench operations. ……………………………19
Table 7 - Primer sequences used to perform RT-PCR……………………………………………………20
Table 8 - MIC values for fluconazole, voriconazole, anidulafungin and caspofungin, as determined by
the EUCAST recommended microdilution method………………………………………………………...25
Table 9 - Results of FFUL887 genome sequence, assembly and variations detection, in comparison
with the genome of the reference strain CBS138. These results were obtained using the software CLC
Genomics Workbench………………………………………………………………………………………...31
Table 10 – Characterization of the MLST allelic profile of CBS138 and FFUL887 strains using
cglabrata.mlst.net……………………………………………………………………………………………..32
Table 11 - Distribution in number of the missense mutations per genes of FFUL887………………….33
Table 12 - Proteins affected by premature STOP codons at 20% of its sequence either originated by
a nonsense mutation or frameshift mutations. The nomenclature used for variation report is taken from
www.hgvs.org/mutnomen/...................................................................................................................40
xii
Table 13. Genes described to mediate resistance to azoles and echinocandins in C. glabrata that were
found to harbour mutant variations in the FFUL887 isolate. The nomenclature used for variation report
is taken from www.hgvs.org/mutnomen/..............................................................................................41
Table 14. Genes described to mediate azole resistance and/or associated to multidrug resistance in
C. glabrata that were found to harbour mutant variations in the FFUL887 isolate. The nomenclature
used for variation report is taken from www.hgvs.org/mutnomen/.......................................................44
Table 15. Expression of CgPDR1 documented target genes in C. glabrata FFUL887 and in CBS138
strains, as revealed by microarray analysis. The set of documented CgPdr1 targets is accounting to
the results of Claude, K., et al., 2011, Tsai, F., et al., 2010 and Vermitsky, J. et al., 2006 …………...49
Table 16. Genes described to mediate resistance to echinocandins in C. glabrata found to harbour
mutant variations in the FFUL887 isolate. The nomenclature used for variation report is taken from
www.hgvs.org/mutnomen/...................................................................................................................51
Table 17 - List of isolates tested and the local where the sample was collected. To some cases a MIC
of fluconazole was tested previously on the hospital. The washed sample corresponds to
bronchoalveolar lavage fluids. Isolates signalized with an asterisk were isolated from patients with
AIDS that followed fluconazole treatment…………………………………………………………………..61
Table 18 - Modification in putative and known adhesion-like proteins found in FFUL887. The
nomenclature used for variation report is taken from www.hgvs.org/mutnomen/. Premature truncation
of the protein and if or not GPI-anchor signal is eliminated or introduced are also summarized……..64
Table 19 - Genes affected by premature STOP codon as result of frameshift and nonsense mutations
found in FFUL887. The nomenclature used for variation report is taken from www.hgvs.org/mutnomen/.
Premature truncation of the protein and if or not GPI-anchor signal is eliminated or introduced are also
summarized……………………………………………………………………………………………………66
Table 20 - Proteins with size increased has result of frameshift mutations found in FFUL887. The
nomenclature used for variation report is taken from www.hgvs.org/mutnomen/. ……………………..71
Table 21 - Genes described to mediate fluconazole and/or voriconazole and caspofungin resistance
in C. glabrata that were found to harbour mutant variations in the FFUL887 isolate. The nomenclature
used for variation report is taken from www.hgvs.org/mutnomen/.......................................................72
Table 22 - Genes described to mediate fluconazole and/or voriconazole resistance in C. glabrata that
were found to harbour mutant variations in the FFUL887 isolate. The nomenclature used for variation
report is taken from www.hgvs.org/mutnomen/...................................................................................73
Table 23 - Genes described to mediate caspofungin resistance in C. glabrata that were found to
harbour mutant variations in the FFUL887 isolate. The nomenclature used for variation report is taken
from www.hgvs.org/mutnomen/...........................................................................................................76
Table 24 - Expression of genes described to mediate fluconazole and/or voriconazole or caspofungin
resistance in C. glabrata in FFUL887 and in CBS138 strains, as revealed by microarray analysis…. 79
xiii
Abreviations ABC ATP-Binding Cassette
BSA Bovine Serum Albumin
C. albicans Candida albicans
C. glabrata Candida glabrata
C. guilliermondii Candida guilliermondii
C. krusei Candida krusei
C. parapsilosis Candida parapsilosis
C. tropicalis Candida tropicalis
cDNA Complementary Deoxyribonucleic Acid
CFU Colony Forming Units
CO infections Community-onset Infections
ddH20 Double Distilled Water
DMSO Dimethyl Sulfoxide
DNA Deoxyribonucleic Acid
DNAse Deoxyribonuclease
dNTP Deoxynucleotide Triphosphates
ECOFF Epidemiological Cut-off Value
EDTA Ethylenediaminetetraacetic Acid
EPA family Epithelial Adhesion Family
GOF Gain-of-function
GPI Glycosylphosphatidylinositol
HOG High Osmolarity Glycerol
MAPK Mitogen-Activated Protein Kinase
MDR Multi-drug Resistance
MFS Major Facilitator (MF) Superfamily
MIC Minimum Inhibitory Concentration
NCAC Non-Candida albicans Candida Species
NGS Next-Generation Sequencing
OD600nm Optical Density At 600 Nm
ORF Open Reading Frame
PDR Pleiotropic Drug Resistance
PDRE Pdr Response Elements
PGM Personal Genome Machine
RNA Ribonucleic Acid
RNase Ribonuclease
RPM Rotations per Minute
RPMI Roswell Park Memorial Institute Medium
RT-PCR Reverse Transcription Polymerase Chain Reaction
S. cerevisiae Saccharomyces cerevisiae
SBF SCB Binding Factor
SCB Swi4-Swi6 Cell Cycle Box
SNP Single-nucleotide Polymorphism
TRIS Tris(Hydroxymethyl)Aminomethane
YPD Yeast Peptone Dextrose
1
1. Introduction
1.1 Introduction to the theme of the thesis
Despite some genome-wide approaches had been used, especially in the latter years, to
characterize global responses of C. glabrata isolates resistant to azoles [6-8], there is still scarce
information on the adaptive responses that occur at the level of the genome sequence. In fact,
with the exception of one study described further in the introduction section that focused
acquisition of resistance to caspofungin [9], only a very limited number of studies had been
performed examining the genomic alterations of azole-resistant isolates. Through a phenotypic
screening performed according to the EUCAST recommended methodologies [10] one C. glabrata
isolate, named FFUL887, was identified as being resistant to both fluconazole and voriconazole.
This isolate also exhibited increased tolerance to caspofungin, when compared to the reference
strain CBS138 and to the remaining cohort of isolates tested. The demonstration of increased
resistance to both azoles and caspofungin was considered a particularly interesting trait since in
most cases there is a small overlap between isolates that are resistant to these two classes of
drugs [11, 12]. Having that in mind, and also a possible elucidation of the mechanisms involved in
the adaptation of C. glabrata to the human urinary tract (considering that FFUL887 was retrieved
from an urine sample), the genome sequence of this isolate was obtained and compared with the
sequence of the reference strain CBS138. Mechanistic insights underlying the increased
resistance of FFUL887 to antifungals were further dissected exploring the results of the genomics
approach and also results of a transcriptomic analysis. Emphasis has been put on the responses
controlled by the transcription factor CgPdr1, a well-described determinant of antifungal
resistance in C. glabrata clinical isolates [13-17].
1.2 Emergence of Candida glabrata and antifungal resistance
The incidence of infections caused by Fungi has risen in the past decades mainly due to
a significant increase in the size of the immunocompromised population, resulting from the
increase in life-expectancy and the utilization of aggressive therapeutic treatments that prolong
patient’s stays in hospitals, sometimes in a fragile condition[18-20]. Fungal infections range from
superficial rushes affecting the mucosas to life-threatening systemic infections in which the fungi
cross the blood stream and may colonize any major internal organ [18]. Almost 10% of the
bloodstream infections reported today are attributed to fungi, these having associated high rates
of mortality and contributing to increase hospital stays, which on the overall leads to a massive
economical burden for public healthcare systems [21-23]. Invasive infections caused by species of
the Candida genus, generally known as candidemia or invasive candidiasis, are responsible for
more fatalities than any other systemic mycosis having an associated mortality of around 40%
[21]. Around 2/3 of the invasive candidemias reported are thought to result from healthcare
2
associated-infections, the main risk factors for this being the use of central venous catheters, long
term parenteral nutrition, surgery and transfer of the yeasts via nursing staff handling [18-20, 24]. The
remaining 1/3 of the infections are considered community-onset infections (CO), these being
developed by patients that exhibit clinical manifestations of the disease prior hospitalization.
Frequently, patients with CO-candidemia have a history of prior hospitalization or of abusive use
of antimicrobials and/or home-based therapies that have an immunosuppressive effect [24, 25].
Among Candida spp., C. albicans is the more common causative agent of invasive and
superficial fungal infections; however, in the recent years the number of infections caused by non-
albicans Candida species (NCAC) has been raising significantly [18]. C. glabrata is now the second
major cause worldwide of invasive fungal infections, followed by C. parapsilosis and C. tropicalis
[26] (Figure 1). In Portugal, as in Europe, C. albicans is the most frequent species within isolates
recovered from patients with candidiasis infections [26, 27], with C. glabrata generally ranking in
second [26, 27] (Figure 1). A similar distribution was observed in epidemiological studies performed
in healthcare facilities located in Portugal where C. glabrata was found to account for around 10%
of the candidemia cases [27-29]. In another epidemiological study performed with isolates collected
from Hospital de S. Marcos, located in Braga, and in an healthcare facility also located in Braga,
the prevalence of C. glabrata was lower as this species only accounted for 4% of the candidemia
cases, ranking after C. albicans (79%) and C. tropicalis (5.6%) [30]. An epidemiological study
performed at Hospital São João, in Porto, reported C. glabrata as the fourth most prevalent
Candida species recovered from patients with candidemia, after C. albicans, C. parapsilosis and
C. tropicalis [22]. Despite the differences, it is evident that infections caused by NCAC are
increasing in Portugal, as in Europe (Figure 1). C. glabrata is most common in patients above 16
years old, its prevalence increasing progressively with the age of the patient [27]. The mortality of
C. glabrata-associated candidemia is around 40%, mortality rates that are comparable with those
reported for C. albicans [31, 32]. However, in cases of delayed diagnosis the outcomes of patients
with candidemia caused by C. glabrata are more severe than those observed for patients
colonized with C. albicans [31].
3
Figure 1 - Worldwide and Portugal prevalence of Candida spp. isolates collected from patients with
diagnosed Candida infections. Epidemiological studies chosen for this representation used a higher
dataset. Worldwide data was retrieved from Castanheira, M. et al. 2014 [26] and Portugal data from
Paulo, C. et al., 2009 [27]. Other epidemiological studies from Portugal are further explored in this
section, were the majority show a similar emergence of NCAC.
One of the reasons underlying this emergence of infections caused by NCAC and, in
particular, by C. glabrata, relates with the high resistance of this yeast species to fluconazole, the
frontline drug used for both active and prophylactic treatments of candidiasis. In this context, the
massive use of fluconazole in the clinical practice led to the selection of the more tolerant species,
C. glabrata among them [18]. More recently, the widespread use of agricultural fungicides
structurally similar to azoles used in the clinical practice was also demonstrated to be on the basis
of the emergence of resistant strains, including those belonging to the C. glabrata species [33]. An
important distinctive trait of C. glabrata is the observation that isolates resistant to antifungals are
recovered from patients diagnosed with candidaemia of hospital- and of community- origin,
something that is not observed for other Candida spp. [25]. C. glabrata was also found to acquire
resistance to antifungals at a higher rate than any other Candida spp. [34, 35]. To overcome this
problem of azole-resistance, drugs alternative to fluconazole had been developed including new
azoles (e.g. voriconazole) and echinocandins [36]. Nevertheless, the number of strains resistant to
these drugs is increasing [37] thereby rendering clear that a thorough understanding of the
mechanisms underlying the development of acquired resistance to azoles and echinocandins is
urgent. In particular, the identification of new biological targets is essential.
4
1.3 Antifungal drugs and their biological targets
1.3.1. Overview
Since both fungal and host cells are eukaryotic the set of therapeutic targets that could
be used in the development of antifungals is limited. Figure 2 shows the classes of antifungal
drugs currently available in the market, as well as their known biological targets. Azoles and
flucytosine exert a fungistatic effect against Candida spp., while echinocandins and polyenes are
fungicidal [38]. The levels of resistance against azoles and flucytosine are considerably higher than
those registered against echinocandins, this being attributed to the static effect exerted by these
drugs [39]. Polyenes (e.g. amphotericin B) target the fungal plasma membrane taking advantage
of the different sterols composition of the membrane of fungi and mammalian cells, while
flucytosine (also known as 5-fluorocytosine) inhibits the activity of thymidylate synthetase thereby
affecting DNA synthesis and ultimately inhibiting cell division. Since cytosine deaminase, the
enzyme that processes flucytosine to its toxic form, is not expressed by human cells, 5-flucytosine
acts specifically against fungal cells [40, 41].
Figure 2 - Antifungal drug classes and their targets. A. Azoles inhibit fungal growth by interfering with
the synthesis of ergosterol and causing the accumulation of the toxic sterol diols intermediates in the
membrane which thereby results in membrane stress. B. Flucytosine is processed in the cell originating
compounds, 5-fluorouridine monophosphate and 5-fluorodeoxyuridine monophosphate, that inhibit
DNA synthesis and RNA synthesis. C. Polyenes bind to ergosterol in the fungal cell membrane forming
membrane-spanning channels that cause leakage of cellular components and osmotic cellular lysis.
D. Echinocandins inhibit (1,3)-β-D-glucan synthase, resulting in cell wall stress and a loss of cell wall
integrity. The image was altered from Cowen, L., 2008 [40].
5
This thesis has explored resistance traits of C. glabrata against azoles and echinocandins
and for that reason the following sections are dedicated to a more detailed description on the
toxicity mechanisms of these drugs and on the underlying resistance responses.
1.3.2 Azoles
Azoles are used since the late 1960’s and until nowadays this is the class of antifungals
comprising a higher number of compounds. Azoles can be divided into two groups: imidazoles
(e.g. clotrimazole, miconazole, ketoconazole) and triazoles (e.g. fluconazole, itraconazole,
voriconazole) [42, 43], depending on the molecules having two or three nitrogen atoms in the five-
membered nitrogen heterocyclic ring. While imidazoles are mostly used to treat topical fungal
infections, triazoles can be administered orally or in an intravenous formulation thereby being
used to treat invasive infections. Triazoles also have a broader spectrum of action [44, 45]. The
chemical structure of voriconazole and fluconazole, two triazoles that will be focused on this
thesis, is represented in Figure 3. The mechanism of action of azoles involves binding to the iron
atom located in the heme group present in the active site of the P450 demethylase, one of the
enzymes of the ergosterol biosynthetic pathway [1]. This binding inhibits enzyme activity
consequently leading to the accumulation of toxic sterols in the membrane (Figure 2). Besides
this, the depletion of ergosterol from the membrane also leads to severe alterations in the plasma
membrane structure impairing its normal function as a selective barrier. Furthermore, ergosterol
was also found to play an important role in stimulation of fungal growth [40, 42]. It was recently
demonstrated that the entrance of azoles to the inside of C. albicans cells occurs by facilitated
diffusion, although up to now such transporter(s) have not been identified [46].
Figure 3 - Chemical structure of azoles fluconazole and voriconazole. Image was altered from Mast,
N. et al 2013 [1]
1.3.3. Echinocandins
Echinocandins are the newest antifungal drug class in the market and the emergence of
strains resistant to these drugs is recent [20]. Caspofungin was the first echinocandin approved by
FDA in 2002, followed by micafungin in 2005 and anidulafungin in 2006. Echinocandins are
lipopeptides, having a cyclic hexapeptide core N-linked to a varying fatty acyl side chain, as shown
in Figure 4. The use of echinocandins is limited to administration by intravenous infusion and
therefore to use in hospitalized patients [20]
6
Figure 4 - Chemical structure of anidulafungin and caspofungin. Image was altered from Emri, T.,
2013 [20].
These drugs were found to be particularly effective in the treatment of fungal infections
since a limited exposure to the antifungal enables extensive suppression of fungal growth thereby
decreasing the chance of the development of invasive disease in infected hosts [47, 48]. The
majority of strains resistant to azoles are susceptible to echinocandins [49], probably reflecting the
different mechanisms by which these drugs act in the yeast cell. The mechanism of action of
echinocandins involves inhibition of 1,3-β-D-glucan synthase which consequently perturbs cell
wall synthesis [20]. 1,3-β-D-glucan is the major component of the fungal cell wall (accounts for 30-
60% of the cell wall in Candida) assuring the structural integrity of this organelle [50, 51]. The
inhibition of β-1,3-D-glucan synthase impairs proper cell wall formation leading to osmotic
instability and ultimately promoting an apoptotic or necrotic cell death [20]. The mechanistic nature
of glucan synthase inhibition by echinocandins is still poorly understood, as it is the mechanism
of action of the 1,3-β-D-glucan enzyme itself [50]. Studies in C. albicans showed that at low
concentrations echinocandins may enter the cells through a high affinity facilitated-diffusion and
through a facilitated-diffusion carrier that seems to operate freely in both directions. In conditions
of saturation nonspecific drug uptake can also take place through diffusion across the bilayer of
the plasma membrane [52].
1.3.4 Candida glabrata resistance to azoles and echinocandins
In Table 1 it is summarized the susceptibility pattern of wild-type populations of different
Candida spp. rendering clear the high resistance of C. glabrata and C. krusei to azoles.
Exposure of naive clinical C. glabrata isolates to azoles led to the development of
resistance to itraconazole and voriconazole after 2 to 4 days [35]. This result showed that pre-
exposure of C. glabrata to azoles is not a prerequisite for the development of resistance indicating
that the cells are already innately tolerant to these antifungal agents [35]. Some of the molecular
mechanisms underlying this innate and acquired resistance of C. glabrata to azoles will be further
discussed in the next section. The incidence of resistance to anidulafungin and caspofungin in C.
glabrata is only of 1.5% [53], however, as the number of patients treated with these drugs increases
the probability of the emergence of resistance also increases. It is interesting to note that while
7
C. glabrata, C. guilliermondii and C. parapsilosis show an increased tolerance to anidulafungin,
C. krusei and C. parapsilosis are particularly tolerant to micafungin, based on the information
available at EUCAST (http://www.eucast.org).
Table 1 - Intrinsic susceptibility patterns of “wild-type” populations of Candida species, according with
EUCAST. In this case the phenotype of the wild-type populations is defined based on the MIC values
obtained for the majority of the isolates. S- Susceptibility; I - Intermediate and R – Resistance. Table
altered from Arendrup, M., 2013 [36].
Candida spp.
Am
ph
ote
ricin
Ech
ino
can
din
s
Flu
co
nazo
le
Itra
co
nazo
le
Vo
rico
nazo
le
Po
sa
co
nazo
le
5-f
lucyto
sin
e
C. albicans S S S S S S S
C. glabrata S S I-R* I-R* S-I-R* S-I-R* S
C. krusei S S R I-R S-I-R* S-I-R* R
C. parapsilosis S S-I S S S S S
C. tropicalis S S S S S S S
*The wild-type populations of C. glabrata and C. krusei are less susceptible to all azoles than C.
albicans and not regarded as optional targets for azoles.
1.4. Molecular mechanisms underlying C. glabrata resistance to azoles
1.4.1 Modifications related to ergosterol biosynthesis and import
Mutations in the enzyme P450 demethylase (encoded by the ERG11 gene), the target of
azoles, has been described as a key mechanism by which several Candida strains acquire
resistance to azoles including C. albicans, C. krusei or C. tropicalis [54-59]. However, in C. glabrata
this does not occur and most often azole-resistant isolates are found not to have mutations in
CgERG11 and when mutations are found these do not correlate with increased resistance of the
isolates to azoles [14, 60-62]. These observations show that C. glabrata has evolved other
mechanisms of resistance to azoles besides target modification. While in C. albicans and S.
cerevisiae upon inhibition of Erg11 by exposure to azoles results in accumulation of the toxic
sterol 14α-methyl-3,6-diol [63-65], in C. glabrata this does not occur being accumulated instead 14-
α-methyl sterols, which are non-toxic [61, 66, 67] (Figure 5). Consequently, a C. glabrata mutant
devoid of CgERG11 is not more susceptible to azoles than the wild-type strain [61]. On the other
hand, deletion of CgERG1, CgERG4 and CgERG6 genes increased C. glabrata susceptibility to
azoles indicating that the function of these enzymes in the ergosterol biosynthetic pathway is
required for maximal tolerance of this yeast species to azoles [68-70].
8
Figure 5 - The C. glabrata mevalonate and ergosterol biosynthetic pathways. The enzyme Erg11
targeted by azoles is highlighted in the grey box. Genes highlighted in bold correspond to those whose
deletion increased susceptibility of C. glabrata to azoles [61, 67, 69, 70]. Underlined sterols are accumulated
upon inhibition of Erg11 function in C. glabrata [61, 66].
The ability of C. glabrata to promote the uptake of sterols (including cholesterol or
ergosterol) from the growth medium is also thought to underlie the high resilience of this yeast
species to azoles [61]. In specific, it is thought that the these sterols taken from the growth medium
can be further metabolized into ergosterol or directly incorporated in the plasma membrane, these
mechanisms compensating for the reduction of ergosterol in the membrane that occurs upon
azole stress [61, 66, 71, 72]. Consistently, the deletion of the ABC-transporter CgAus1, and of the
mannoprotein CgTIR3, both involved in the uptake of sterols [72, 73], led to an increase in
susceptibility of C. glabrata to azoles, provided that the cells are cultivated in a medium
supplemented with sterols [73]. Furthermore, up-regulation of CgAUS1 is observed when C.
glabrata cells are cultivated in a medium containing sterols and inhibitory concentrations of
fluconazole or itraconazole [74]. These results suggest a possible cross-regulation between the
ergosterol biosynthesis and ergosterol transporters [61, 73, 74].
9
1.4.2. Role of multi-drug efflux pumps
An important mechanism underlying multidrug resistance in Fungi is drug extrusion by
multi-drug resistance (MDR) transporters of the ATP binding cassette (ABC) or of the Major
Facilitator (MF) Superfamilies (MFS) [75]. In C. glabrata at least 4 multi-drug efflux pumps of the
ABC Superfamily have been implicated in tolerance to azoles [8, 76, 77], CgCdr1, CgPdh1, CgPdr16
and CgSNQ2; CgCdr1 exerting the more significant protective effect [60, 77, 78]. Consistently, clinical
C. glabrata isolates resistant to azoles were found to exhibit higher expression levels of CgCDR1,
CgPDH1 and CgSNQ2, comparing with the expression levels registered in susceptible isolates
[60]. However, it is important to stress that “azole-resistant isolates” not exhibiting higher
expression of CgCDR1 or CgPDH1 genes have also been identified, indicating that there are
other mechanisms relevant for azole resistance in C. glabrata beyond up-regulation of these drug-
efflux pumps [78]. The role of MFS-MDR transporters in C. glabrata tolerance to azoles has been
much less studied and only recently the role of these proteins in drug resistance has been
examined [79]. The results obtained showed the important role of the CgTpo3, CgQdr2, CgAqr1,
and CgTpo1_1 (CAGL0G03927g), CgTpo1_2 (CAGL0E03674g) transporters in conferring
tolerance to azoles in C. glabrata [80-83], however, it remains to be established if these transporters
underlie the azole-resistance phenotype exhibited by clinical isolates. The MDR transporters so
far implicated in C. glabrata resistance to azoles are summarized in Table 2.
Table 2 - Multi-drug efflux pumps from the ABC and MFS family described to be involved in azole
resistance in C. glabrata.
Superfamily Gene name/ORF name Role in drug resistance
ABC Superfamily
CAGL0M01760g/CDR1 [8, 60, 77, 84] Confer imidazole and triazole
resistance
Involved in azole resistance in
clinical isolates
CAGL0F02717g/PDH1 [60, 77, 85]
CAGL0I04862g/SNQ2 [60, 76, 77]
CAGL0J07436g/PDR16 [8, 86] Confer imidazole and triazoles
resistance
MFS Superfamily
CAGL0G03927g/Tpo1_1 [82] Confer imidazole and triazole
resistance CAGL0E03674g/Tpo1_2 [82]
CAGL0I10384g/TPO3 [81] Confer imidazole and triazole
resistance CAGL0G08624g/QDR2 [80]
CAGL0J09944g/AQR1 [83] Involved in resistance to
imidazoles
1.4.3. The pleiotropic drug resistance transcription factor CgPdr1
The transcriptional regulation of drug-efflux pumps in C. glabrata, as in other yeasts, is
controlled by a well-organized and complex regulatory network known as the pleiotropic-drug
resistance network (PDR) which is under the control of the transcription factor CgPdr1 [87].
Although some knowledge has been gathered in the function of the PDR network in C. glabrata
this fungi-specific network has been much better studied in S. cerevisiae than in any other species
[88-94]. In the budding yeast the PDR network is controlled by two orthologous transcription factors:
10
ScPdr1 and ScPdr3 [90, 91]. Elimination of ScPDR1 and ScPDR3 was found to increase S.
cerevisiae susceptibility to a wide range of structurally unrelated xenobiotics ranging from
antifungals, antibiotics to anticancer drugs and pesticides [88]. In C. glabrata only an orthologue of
ScPDR1 is found and its elimination increased susceptibility of this yeast species to fluconazole,
voriconazole, itraconazole and ketoconazole [6-8, 87]. Extensive transcriptomic profiling has been
showing that CgPdr1 regulates essentially the same biological functions described to under
regulation of ScPdr1 and ScPdr3 [87, 89]. Besides regulating the expression of ABC- and MFS-
MDR pumps, CgPdr1 has also been found to regulate the expression of genes involved in
lipid/fatty acid/sterol metabolism, in transcriptional regulation, in stress response, among other
functions (Figure 6). The involvement of CgPdr1 in the regulation of these genes has been
demonstrated in laboratory strains but also in gain-of-function CgPdr1 mutants recovered from
azole-resistant clinical isolates [14, 16, 17]. In Figure 6 are summarized the functional class of genes
found to be regulated by CgPdr1 [14, 16, 17] either in lab and in GOF CgPdr1 mutants.
Figure 6 - The functional class genes transcriptional regulated by CgPdr1. The most common genes
upregulated in PDR1 GOF transcriptomes and adhesion genes known to be regulated by CgPdr1 are
also summarized. Information was retrieved from the transcriptome studies from Claude, K., et al.,
2011 [16], Tsai, H., et al., 2010 [14] and Vermitsky, J., et al., 2006 [17].
A wide range of GOF point mutations has been described to occur in the coding sequence
of CgPDR1, a summary of them being shown in Figure 7. A distinctive trait of CgPdr1 GOF
mutants is the fact that the activity of the transcription factor becomes constitutively high thereby
resulting in the constitutive expression of its target genes even in the absence of a xenobiotic
stimulus [13, 14, 16, 17]. Several studies have demonstrated that the distribution of GOF mutations in
the CgPDR1 gene are not associated with specific domains (as it can be seen in the Figure 7)
and these can give origin to different transcription profiles by altering the overall structure of the
transcriptional regulatory network of the strain [13, 14, 16]. Recently the mechanism underlying the
activation of CgPdr1 by ketoconazole has been disclosed [89]. The results obtained showed that
11
ketoconazole is able to directly bind to a domain of CgPdr1 protein located at the C-terminal, this
being known as the xenobiotic binding domain (XBD) (Figure 8). This mechanism was
hypothesized to serve as a generalized mechanism of CgPdr1 activation in response to drugs,
thereby turning this transcription factor able to act as a nucleor receptor: upon binding of the drug,
the complex Pdr1-drug binds to the KIX domain of the mediator complex subunit, CgGal11A,
which thereby promotes the up-regulation of RNA polymerase II activity resulting in expression of
Pdr1-target genes, including drug-efflux pump-encoding genes [89] (Figure 8).
Figure 7 - Described C. glabrata PDR1 gain-of-function mutations, according to the information
available in Ferrari, S., et al., 2009 [13], Tsai, H., et al., 2010 [14], Berilla, N. and Subik, J., 2010 [15].
Figure 8 - General mechanism describing the activation of Pdr1/Pdr3 in response to drugs within a
fungal cell. Drug can binds to the XBD domain of the pleiotropic transcription factors Pdr1/Pdr3. Acting
as a nuclear receptor, Pdr1/Pdr3-drug complex can them associate to the KIX domain of the Gal11p
subunit of the Mediator complex and recruit RNA polymerase II to the promoter region of PDR genes
initiating transcription. Image was retrieved from Goffeau, A., 2008 [2].
12
1.4 4. Mitochondrial function
Another mechanism of resistance to azoles that has been described in C. glabrata is the
loss of mitochondrial function, also known as the petite phenotype [8, 77, 95]. The petite phenotype
is caused by mutations in the mitochondrial DNA which result in the formation of petite cells that
are unable to undergo aerobic respiration and that are therefore unable to use ethanol or glycerol
as sole sources of carbon and energy. Exposure of C. glabrata cells to azoles has been found to
induce mtDNA damage, presumably by direct binding of these drugs to mtDNA or due to an over-
accumulation of ROS caused by the impairment of mitochondrial function [77, 95]. Notably, C.
glabrata petite mutants were found to exhibit increased expression of CgCDR1 and CgPDH1
genes, and the composition of the plasma membrane was also different from the one registered
in parental strain cells being observed an increase in free ergosterol content and a decrease in
sterol intermediates [95]. The transcriptional alterations registered in petite mutants were proposed
to result from cross-talk between mitochondrial function and control of pleiotropic-drug response
by CgPdr1, something that has also been observed to occur in S. cerevisiae [95]. It is however
important to stress that the role of petite mutant phenotype in contributing for enhanced resistance
to azoles is not yet fully understood since in some studies these strains were found to exhibit an
hyper-susceptibility phenotype [61, 95].
1.5. Molecular mechanisms underlying C. glabrata resistance to echinocandins
1.5.1. Modifications in glucan synthase
The vast majority of the reports describing the molecular mechanisms underlying
increased resistance of C. glabrata to echinocandins in isolates involves mutations on the glucan
synthase-encoding genes CgFKS1 and CgFKS2 [96-110], a higher number of mutations being
reported to occur in the coding sequence of CgFKS2 [111]. In C. glabrata, the catalytic subunit of
glucan synthase is composed by the redundant alternative subunits CgFks1 and CgFks2, with
CgFks2 being the most abundant subunit during vegetative growth correlated with a higher
number of known mutations affecting echinocandin resistance [101, 111]. CgFKS3 also encodes
another putative FKS protein, however, this protein does not appear to have a role in glucan
synthase action showing very low expression levels and was not reported to be involved in
echinocandin resistance [111]. The GTP-binding protein Rho1p is the regulatory subunit of the
glucan synthase complex being involved in the control of the activation of its function [112]. The
described mutations on CgFKS1 gene leading to increased resistance to echinocandins are
mainly found in two hot spots located between the regions 621 to 638 a.a., and a recently
described hotspot in the region 676 to 686 a.a. [113], while Fks2 gene hot spots are located
between the regions 655 to 672 a.a. and 1374-1381 a.a., as shown in Table 3. It is not yet known
if the mutations contribute to increase resistance to echinocandins by affecting binding of the
echinocandins to the proteins or by affecting the action of the drug in an indirect manner [114].
13
Table 3. Summary of described mutations found in the coding sequence of CgFKS1 and CgFKS2
genes and that contribute for enhanced resistance to echinocandins in C. glabrata [96-110]. The amino
acid sequence of the “hot spot regions” of CgFks1 and CgFks2 proteins is underlined. Δ indicates a
non-sense mutation.
Mutation distribution
Fks1
Hot Spot 1 (621-638) Hot Spot 3 (676-686) Other E S Y F F L I L S L R D P I R N L S C P G E I S G S T Y Δ
L D T Y L W Y I V V L
A1037T
Fks2
Hot spot 1 (655-672) Hot Spot 2 (1374-1381) Other E S Y F F L I L S L R D P I R I L S Δ Δ WF R G E F S P V F H V Y G T Y
D W I R R Y T L L
E78D F649V L707S G680S
T926P
The increase in chitin synthesis has also been described to be a part of C. albicans
response to echinocandins, this response correlating with increased survival of this yeast species
in mice treated with caspofungin [115-117]. Such response was not observed when C. glabrata cells
are challenged with inhibitory concentrations of echinocandins [116], although, the elimination of
genes encoding enzymes of the chitin biosynthetic pathway were found to increase susceptibility
to echinocandins [9].
1.6. Contribution of genome-wide studies for the comprehension of
antifungal drug resistance in C. glabrata
The development of more advanced genetic tools to be used in Candida spp., especially
in C. glabrata, has opened the door to further dissection of the molecular mechanisms of tolerance
to antifungals at a genomic scale, broadening the view into how these yeasts develop resistance.
The experimental approaches used are distinct ranging from the use of large scale phenotypic
screenings performed with individual deletion mutants [6-8] to extensive transcriptomic [14, 16, 17],
genomic [9] or proteomic profiling [118].
Using a library of C. glabrata mutants based on the use of Tn7 transposons Kaur, R., et
al., 2006 [8], have identified key players underlying resistance to fluconazole, being of notice the
uncovered role of proteins involved in Ca2+ signalling in contributing for tolerance to this azole [8].
Another biological function that emerged as being relevant for tolerance to fluconazole is the
activity of the mediator complex (CgSrb8, CgNut1, and CgRgr1), a regulator of RNA polymerase
II activity; this protective effect being attributed to the function that this complex may have in
augmenting the important genes required for maximal tolerance to fluconazole. Confirming the
previously described involvement of the petite phenotype in improving C. glabrata resistance to
fluconazole, in this study two mutants devoid of CgSUV3 and CgSHE9, two mtDNA-encoded
genes, were also identified as being more resistant to the azole than the parental strain [8]. More
14
recently, a large collection of deletion mutants, engineered using elimination cassettes, has also
been screened to search for C. glabrata determinants of resistance to fluconazole and also to
voriconazole and caspofungin [7]. The results obtained support the evidence of the important role
played by the calcineurin pathway in C. glabrata tolerance to azoles (and also to echinocandins),
since the deletion of the two subunits of calcineurin (encoded by CgCNA1 and CgCNB1 genes),
its regulators (encoded by CgRCN1 gene) and the known upstream factors (encoded by CgCZR1)
resulted in altered susceptibility to both types of antifungals. Proteins belonging to the PKC-,
HOG1 or TORC1- signalling pathways were also identified as determinants of C. glabrata
tolerance to antifungals [7].
To better understand the molecular mechanisms underlying acquisition of resistance to
echinocandins in C. glabrata inside the human host, a recent study has explored a comparative
genomic analysis to compare the genome of a set of sequential C. glabrata isolates retrieved from
a patient with recurring bloodstream candidaemia under caspofungin therapy [9]. In the first isolate
recovered that showed a modest increase of resistance to caspofungin, compared to the isolate
that was recovered in the infection, several non-synonymous mutations were found in the coding
sequence of CgGPH1, CgCDC6 and CgTCB1/TBC2 genes. These genes encode, respectively,
a glycogen phosphorylase, a protein involved in DNA replication initiation and a protein involved
in membrane trafficking. Consistently, reconstruction of the mutations identified in CgCDC6 in the
genome of a naive isolate (susceptible to echinocandins) led to the development of a resistance
phenotype to caspofungin [9]. Analysis of the genome sequence of a second isolate, which was
more resistant to caspofungin than the first resistant isolate recovered, revealed mutations in
coding sequence of CgDOT6, CgMRPL11 and CgSUI2 genes, which encode, respectively, a
subunit of the RPD3L histone deacetylase complex, a protein of the large mitochondrial ribosomal
subunit and an alpha subunit of the translation initiation factor eIF2. However, the increase in
resistance to caspofungin was not attributed to these mutations since the isolate also harboured
a mutation in CgFKS2 coding sequence, which was considered to be on the origin of the
resistance phenotype [9].
15
2. Material and Methods
2.1. Strains and growth media
A cohort of 58 C. glabrata clinical isolates was used in this work. These clinical isolates
were recovered from patients attending three major Hospitals of the Lisbon area through the years
2000 and 2008 (Annex A) and were kindly provided by Prof. Maria Manuel Lopes, Faculdade de
Farmácia da Universidade de Lisboa and Dr. Rosa Barros, Head of the Microbiology Laboratory
of “Centro Hospitalar de Lisboa Central”. The reference strain Candida glabrata CBS138 strain
was also used.
C. glabrata cells were batch-cultured at 30ºC, with orbital agitation of 250rpm in the rich
growth mediums Yeast Peptone Dextrose (YPD) or RPMI (from Roswell Park Memorial Institute
Medium). YPD contains, per liter, 20g glucose (Merck Millipore), 10g yeast extract (HiMedia
Laboratories, Mumbai, India) and 20g Peptone (HiMedia Laboratories). RPMI, contains, per liter,
20.8g RPMI-1640 synthetic medium (Sigma), 36g glucose (Merck Millipore), 0.3g of L-glutamine
(Sigmaa) and 0.165 mol/L of MOPS (3-(N-morpholino) propanesulfonic acid, Sigma).
Components of RPMI-1640 medium are discriminated at EUCAST E.Dis 7.1 [10]. The pH of the
media was adjusted to 7.0 with NaOH as base, as recommended by EUCAST. Different C.
glabrata isolates were maintained at -80ºC in YPD medium supplemented with 30% glycerol (v/v)
(Merck). All media were prepared in deionized water.
YPD medium was sterilized by autoclave for 15 minutes at 121ºC and 1 atm. RPMI
medium was filtered with a 0.22-µm pore size filter and preserved at 4ºC until further use.
2.2. Antifungal solutions
The stock solutions of the antifungals were prepared from the powder and using DMSO
(Dimethyl sulfoxide, Sigma) as the solvent. Fluconazole was purchased from Sigma, caspofungin
was kindly provided by Merck and voriconazole and anidulafungin were kindly provided by Pfizer
International Inc. within the scope of the project “Unraveling the mechanisms of resistance to
anidulafungin and voriconazole in C. glabrata through a genomics approach”. Anidulafungin,
caspofungin and voriconazole were stored in sealed containers at -20ºC with a desiccant and
fluconazole was stored at room temperature, according with the recommendation of the
manufacturers. The solutions of antifungal drugs used in the phenotypic screened were prepared
taking into account the potency of the lot of antifungal drug powder in use. The amount of powder
or diluents need to prepare a standard solution was calculated as follows:
Weight (g) =Volume (L)×Concentration (mg/L)
Potency (mg g)⁄ (1.1)
Volume (L) =Weight (g)×Potency (mg g)⁄
Concentration (mg/L) (1.2)
16
2.3. Quantification of MIC50 using the micro dilution method
To assess resistance levels of the C. glabrata clinical isolates or of the reference strain
CBS138 to azoles and echinocandins the micro-dilution method recommended by EUCAST [10]
was used, A schematic representation of the method used is shown in Figure 9. The range of
concentrations used for each antifungal drug is shown in Table 4.
Figure 9 - Schematic representation of the procedure used to prepare the 96-multiwell plates used to
determine MIC of voriconazole, anidulafungin and caspofungin for the different C. glabrata isolates. B:
blank; GC: control. Image altered from EUCAST discussion document E.Dis 7.1 [10]. In the case of
fluconazole the same methodology was applied with the difference that the initial stock concentration
was of 12 800.
To prepare the 96multiwell-plates required for the microdilution assay, 300µl of the stock
solution of each antifungal was transferred for a new tube and 1:2 dilutions (in DMSO) were
performed in a final volume of 300µl, yielding concentrations ranging from 12800 mg/L to 25 mg/L
for FLC and from 1600 mg/L to 3 mg/L for the other drugs (as shown in Figure 9). This solutions
were designated as diluted solutions 1 (Figure 9). Afterwards, a second set of diluted solutions
17
was prepared by adding 100µl of the dilutions solution 1 to 9.9mL of RPMI 2% glucose medium
2x concentrated (Figure 9). These diluted solutions were named as diluted solutions 2 (Figure 9).
100µl of these diluted solutions 2 were used to inoculate the 96-microwell plates (Figure 9). 100µL
of a diluted cell suspension was used to inoculate the 96 multi-well plates, yielding a the final test
range of concentration of 64 mg/L to 0.125 mg/L for FLC and 8 mg/L to 0.015 mg/L for other
drugs. These cell suspensions were prepared from a pre-culture that was cultivated for 18h in
5mL of YPD at 30ºC and 250rpm orbital agitation. The initial OD of the cultures was approximately
0.025 corresponding to around 1.25x105 CFU/mL which is within the range of 0.5x105 – 2.5x105
CFU/mL recommended by the EUCAST protocol [10]. In column 11 the cells were diluted in 100µl
sterile drug-free medium to assess their growth performance in the absence of the antifungal and
in column 12 only the sterile drug-free growth medium was added (diluted 1:2 in sterile water) to
serve as blank. After inoculation, the 96-multiwell plates were incubated without agitation at 37°C
for 24h. After that time, cells were resuspended and the recommended OD530nm of the cultures
was read in a microplate reader. The value of the blank was subtracted from readings of the rest
of the wells. Resistance of each isolate was assessed based on results of the analysis of two
independent experiments in which each isolate was assayed twice (which means that four MIC
values were determined for each drug and for each isolate). The minimum inhibitory concentration
(MIC50) value was calculated, comparing the ratio of the OD in each of the wells with the OD
attained in control conditions, and compared to the EUCAST (www.eucast.org) recommended
breakpoints for the antifungal agents (summarized in Table 5).
Table 4 - Concentration range of antifungal agents used on this study.
Table 5 - MIC values of the different antifungals used during this work corresponding to resistance
breakpoints, as indicated by EUCAST. In the case of voriconazole EUCAST has not defined an exact
breakpoint but strains are consider resistance when exhibiting a MIC higher than 1 mg/L, the EUCAST
epidemiological cut-off (ECOFF). Due to significant inter-laboratory variation in MIC values, ranges for
caspofungin EUCAST breakpoints have not yet been established.
Antifungal Agent MIC breakpoints (mg/L)
Susceptibility Resistance
Fluconazole ≤0.002 >32
Voriconazole ≤1 >1
Anidulafungin ≤0.06 > 0.06
Antifungal agent Test range
(mg/L)
Stock Solution
(mg/L)
Fluconazole 0.125 - 64 12800
Voriconazole 0.015 - 8 1600
Caspofungin 0.015 - 8 1600
Anidulafungin 0.015 - 8 1600
18
2.4. Growth curves in the presence of fluconazole, voriconazole,
anidulafungin and caspofungin.
Growth curves of C. glabrata reference strain CBS138 and of the FFUL887 isolate in the
presence of fluconazole, voriconazole, caspofungin and anidulafungin were performed using
essentially the same experimental setup described above for the micro dilution method. Growth,
taken as the increase in OD595nm of the culture, was monitored hourly during 48h. Each experiment
was performed in duplicate.
2.5. Genomic DNA extraction
FFUL887 and CBS138 cells were grown in YPD until an OD600nm above 3.0. Afterwards
cells centrifuged at 5000rpm, during 5min at 4ºC and the supernatant discarded. Pellet was
resuspended in 1ml Sorbitol 1M (Sigma) and EDTA (tetrasodium salt dehydrate, Sigma-Aldrich)
0.1M at pH 7.5 solution and transferred to an eppendorf. Afterwards, 10 mg/ml zymolease (Zymo
research) was added and the solution was incubated at 37˚C until protoplast formation. The
sheroplasts were centrifuged at 5000rpm for 5min, and the pellet resuspended in 1mL Tris-HCL
with pH 7.4 50 mM (Sigma-Aldrich), and EDTA 20 mM solution. After this step, 30µl of SDS 10%
was added to the mixture. After an incubation step of 30min at 65˚C, 250µl of Potassium Acetate
(5M, Merck) was added to induce protein precipitation, this being followed by a 1h incubation on
ice. Afterwards the solution was centrifuged at 10000rpm for 10min and the supernatant
transferred to 2 new eppendorfs. 1 volume of cold isopropanol was used to wash the pellet
followed by centrifugation at 5000rpm for 15min. Supernatant was discarded and the resulting
pellet was incubated in 1mL ethanol 70% during 5min, and wash with ethanol 70% twice. The
pellet was dried in speed vacuum and resuspended in 200µl TE (pH 7.4). The final step, addition
of 0.5µl of RNase (10 mg/ml) followed by 1h incubation at 37ºC followed. Mixture was centrifuged
at 10000rpm during 15min and the supernatant was preserved at 4ºC till further use.
2.6. Genome Sequencing and Annotation
The genome of FFUL887 isolate was obtained at the next-generation sequencing (NGS)
laboratory of Stab Vida, using Ion PGM sequencing technology. Two rounds of deep sequencing
were performed and the reads (5 920 417) obtained were analysed using the software CLC
Genomics Workbench. The reads obtained in the sequencing step were trimmed based on
quality, as detailed below in Table 6. The trimmed reads were assembled using ‘de novo
assembly’ and mapped ‘against the reference genome of CBS138. Variation detection was
performed from the mapped reads using both probabilistic and quality-based variant detection
tools and InDels and Structural Variants tool. The parameters used at the different mentioned
operations are summarized in Table 6.
19
Table 6 - Parameters used in the CLC Genomics Workbench operations.
CLC Genomics Workbench Operation
Parameters
Quality trimming
Ambiguous trim: yes; ambiguous limit: 2; quality trim: 0.05; Remove 5’/3’ terminal nucleotides: no; discard short/long reads: no, mismatch cost: 2; insertion cost: 3; deletion cost: 3; length fraction: 0.5; similarity fraction: 0.8.
De novo assembly
Word size: 20; bubble size: 50; minimum contig length: 200; perform scaffolding: yes.
Mapping reads to reference
Reference: Candida glabrata CBS138; mismatch cost: 2; insertion cost: 3; deletion cost: 3; length fraction: 0.5; similarity fraction: 0.8; non-specific match handling: map randomly.
Probability variant detection
Ignore non-specific matches: yes; Ignore broken pairs: yes; Minimum coverage: 10; Variant probability: 50; Required variant count: 2; Required presence in forward and reverse reads: yes; Ignore variants in non-specific matches: yes; Filter 0; 454/Ion Torrent homopolymer indels: yes; Maximum expected variations: 1; Genetic code = 1 strand. Filters applied: Average Quality: ≥20
Quality-based variant detection
Neighbourhood radius: 5; Maximum gap and mismatch count: 2; Minimum neighbourhood quality: 15; Minimum central quality; 20; Ignore non-specific matches: yes; Ignore broken pairs: yes; Minimum coverage: 10; Variant probability: 50; Maximum expected variations: 1; Required presence in forward and reverse reads: yes; Ignore variants in non-specific matches: yes; Filter 0; 454/Ion Torrent homopolymer indels: yes; Genetic code = 1 strand. Filters applied: Average Quality: ≥20
InDels and Structural Variants
P-Value threshold: 0;0001; Maximum number of mismatches: 3.
Filters applied: Variant ratio: ≥0.8; # Reads; ≥10; Sequence complexity: ≥0.2
2.7. Microarray profiling of FFUL887 and CBS138 strains
The transcriptome of FFUL887 and CBS138 cells was compared in mid-log phase during
growth in RPMI growth medium. For this the two strains were cultivated in 25mL of YPD at 30ºC
with orbital agitation (250rpm) until mid-exponential phase and then re-inoculated in 150mL of
RPMI at 30ºC and 250rpm. When the OD600nm of the cultures achieved 2 cells were harvested by
centrifugation (8000xg, 7min, 4.°C – Beckman J2.21 Centrifuge, rotor JA.10) and immediately
frozen at -80ºC until further use. RNA extraction was performed using RiboPure™
RNA Isolation Kit (Ambion, Life Technologies, California, USA). The resulting cell pellet was
resuspended in 480µl of lysis buffer, 48µl of SDS and 480µl of a mixture of
Phenol:Chloroform:IAA. The suspension was then added to new tubes containing
about 750μL cold Zirconia Beads. Cell disruption was performed by position the tubes horizontally
on the vortex and agitated at maximum speed for 10min. Separation of the aqueous phase,
containing the RNA, from the organic phase was obtained by centrifugation for 5min at 16.100xg
at room temp. 1.90mL of Binding Buffer and 1.25mL of 100% Ethanol was added and the total
volume was centrifuged through a filter cartridge. Filter cartridge was washed with 700µl of Wash
Solution 1 and washed two times with 500µl of Wash Solution 2/3 followed 1min centrifugation
and an extra minute to ensure the complete removal of Wash solution. Total RNA obtained was
20
eluted in two times with 50µl of Elution Solution, previously heated at 95°C. DNase treatment was
perform by adding to the 100µl-RNA sample 8 U of DNase I and 10µl of 10X DNase I Buffer. The
mixture was incubated at 37°C during 30min. After incubation period 10µL of DNase Inactivation
Reagent were added to the mixture, which was then vortexed and left for 5min at room
temperature. The purified RNA (in the supernatant fraction) was collected by centrifugation at
>10000rpm for 3min and supernatant fraction was added to fresh tubes. RNA extracted was
quantified and integrity verified using Bioanalyser. The microarray analysis was performed
according with the methodology described by Rossignol, T., et al., 2007 [119].
2.8. Assessment of gene expression based on real time RT-PCR
Some of the results obtained in the microarray analysis were further confirmed using real
time RT-PCR. Cell growth and extraction of total RNA was performed in the same manner as
described above for the microarray analysis. Conversion of total RNA into cDNA was performed
using 1µg of RNA, 2.2µl MgCl2 25mM, 1.0µl Buffer 10x, 2.0µl dNTPs 2.5mM, 0.5µl Random
Hexamers, 0.2µl RNAse inhibitor, 1.85µl ddH20 and 0.25µl reverse transcriptase. The reverse-
transcription step was performed in a C1000 Thermal Cycler (Bio-Rad, Hercules, USA) with the
following experimental setup: 10min at 25°C, 30min at 48°C, 5min at 95°C. The subsequent
quantitative PCR step was performed using 2.5µL of the cDNA previously produced, 12.5µL
MasterMix (SYBR® Select Master Mix, Applied Biosystems), 2.5µL Primer Forward (4pmol/mL),
2.5µL Primer Reverse (at a concentration of 4pmol/mL) and 5µL ddH20. The protocol used for
the PCR step was the following: 2min at 50°C, 10min at 95°C, 15s at 95°C, 1min at 60°C, using
40 cycles. The sequence of the primers used is shown in Table 7.
Table 7 - Primer sequences used to perform RT-PCR.
Primer
Identification
Primer Sequence
CgACT1
Forward 5’-AGAGCCGTCTTCCCTTCCAT-3’
Reverse 5’-TTGACCCATACCGACCATGA-3’
CgPDR1 Forward 5’-CGATTGCCAACCCGTTAGA-3’
Reverse 5’-GACGACCTTGGTGTAGGAGTCAT-3’
CgCDR1 Forward 5’-GCTTGCCCGCACATTGA-3’
Reverse 5’- CCTCAGGCAGAGTGTGTTCTTTC-3’
CgPDH1 Forward 5’-GCCATGGTACCTGCATCGAT-3’
Reverse 5’-CCGAGGAATAGCAAAACCAGTATAC-3’
CgQDR2 Forward 5’-TCACTGCATAGTTTCATATCGGACTA-3’
Reverse 5’-TGCCGATATGTTCCCAAGTGA-3’
21
2.9. Quantification of biofilm formation in the presence of the antifungals
The amount of biofilm produced by the different C. glabrata clinical isolates in the
presence of fluconazole, voriconazole, caspofungin or anidulafungin was performed using the
crystal violet staining method. For this strains were cultivated under the same experimental setup
described above for the micro dilution method. After 18h of growth in the presence or absence of
the antifungals the cells were washed in PBS 1x (NaCl 8 g/L, KCL 0.2 g/L, Na2HPO4 1.44 g/L and
KH2PO4 0.24 g/L) and fixed with 200µl of methanol for 15min. After drying, 200µl of crystal violet
(Merck) was added to each cell suspension for staining. After 15 minutes, the biofilm was washed
with deionized water, resuspended in ethanol and the OD570nm was measured in a microplate
reader.
2.10. Adherence to biotic and abiotic surfaces
Adhesion of FFUL887 and CBS138 strains to polystyrene, fibronectin and vitronectin of the
different clinical isolates was measured in 96-multiwell plates using the crystal violet staining
method. When vitronectin and fibronectin were used, a pre-coating step of the plate was required.
This coating step was performed by adding 200µl of fibronectin (Sigma) and vitronectin (Sigma)
10µg/ml diluted in sterile PBS and leaving the plate rest overnight at 4ºC. After 4h and 8h of
cultivation of the two strains in the plates containing RPMI medium, the plates were washed two
times with sterile PBS and non-specific binding sites were blocked with 200µl sterile PBS with 2%
of bovine serum albumin (BSA, Sigma) during 1h at room temperature. Two washing steps with
sterile PBS were subsequently performed after which crystal violet was added, according to the
methodology described in 2.3.
2.11. Caspofungin time-kill assays
The viability of FFUL887 and CBS138 when exposed to 0.25 mg/L of caspofungin was
quantified according to the method described by Klepser, M., et al., 1998 [120]. A pre-culture of the
two strains was performed in YPD. This suspension was used to inoculate 10 mL of fresh RPMI
growth medium (at an initial cell concentration of 105 CFU/mL) either or not supplemented with
caspofungin. The cultures were incubated at 30ºC and 250rpm for 12h. Cell viability, based on
the number of CFU’s formed onto the surface of YPD plates, was measured at designated times.
22
3. Results and Discussion
3.1 Profiling of antifungal resistance within a cohort of clinical C. glabrata
isolates
Around 60 C. glabrata clinical isolates were screened for their tolerance to several
antifungals used in the clinical practice. These isolates, kindly provided by Prof Maria Manuel
Lopes [121] from Faculdade de Farmácia da Universidade de Lisboa, were isolated from various
sites including blood, oral cavity, pharynx, bronchoalveolar lavage fluids, faeces and urinary tract.
Further information on the clinical isolates recovered are available in Annex A. The antifungal
drugs used in this screening were fluconazole, voriconazole, anidulafungin (all kindly provided by
Pfizer) and caspofungin (kindly provided by Merck). The assessment of the isolates
tolerance/resistance to these antifungals was performed using the micro-dilution assay
recommended by EUCAST (European Committee on Antimicrobial Susceptibility Testing) [10].
This experimental protocol aims to quantify the MIC50 value, that is, the concentration of the
antifungal drug that leads to a 50% decrease in growth of the isolate, when compared with the
growth registered in the absence of the drug. If an isolate exhibits a MIC50 value above the
designated “resistance breakpoint” MIC, which is defined by EUCAST, the isolate is considered
resistant. For voriconazole no specific resistance breakpoint is recommended by EUCAST due to
the lack of sufficient evidence correlating MIC values and clinical outcomes and therefore isolates
are considered resistant to this drug if the MIC50 value is above 1 mg/L, the epidemiological cut-
off value (ECOFF) [122]. For caspofungin no breakpoint has also been defined by EUCAST due to
significant discrepancies found in inter-laboratory assays. Throughout the screening the reference
strain C. glabrata CBS138 was used as a reference to control reproducibility of the results.
The results obtained concerning the MIC values of fluconazole and voriconazole obtained
for the different isolates are shown in Annex B and summarized in Figure 10A and 10B,
respectively. Under the experimental conditions used, the MIC value of the reference strain
CBS138 was of 16 mg/L for fluconazole and 0.25 mg/L for voriconazole. Nine isolates (FFUL98,
FFUL412, FFUL443, FFUL674, FFUL830, FFUL866, FFUL878, FFUL887, FFUL4012) were
found to be resistant to fluconazole, while eight were found to be resistant to voriconazole
(FFUL412, FFUL443, FFUL674, FFUL677, FFUL830, FFUL866, FFUL878, FFUL887). These
numbers correspond to an incidence of resistance of about 16% and 14% of the total number of
isolates tested. Seven isolates (FFUL412, FFUL443, FFUL674, FFUL830, FFUL866, FFUL878,
FFUL887) were resistant to both azoles (Figure 10C). Other studies profiling antifungal resistance
in C. glabrata have demonstrated the existence of a high degree of cross-resistance between
fluconazole and voriconazole, of around 80% [123, 124]. Notably, three of the isolates herein
uncovered as exhibiting cross-resistance to voriconazole and fluconazole, FFUL412, FFUL443
and FFUL674, were retrieved from patients undergoing fluconazole-based therapy (Annex A).
None of the C. glabrata isolates tested could be considered susceptible to fluconazole as the MIC
values were always above 0,002 mg/L (Annex B). Consequently, the majority of the isolates were
23
clustered in the intermediate resistant class (Figure 10A). The relatively high percentage of
isolates found to be intermediately or highly tolerant to fluconazole found within the cohort of
isolates tested is consistent with the well-described innate tolerance of C. glabrata species to
azoles [18]. Despite the number of isolates examined in this study is small, it is interesting to
observe that the resistance incidence herein obtained is close to the values reported in other
surveillance tests which range 10-15% [124-126]. The same is also true for voriconazole since the
percentage of resistant isolates obtained in this work is of 14%, with previous studies reporting
an incidence of resistance close to 10% [124]. No correlation between the isolation site and the
level of resistance of the isolate was found concerning all the drugs tested.
Figure 10 - Summary of the results obtained in this study regarding profiling of susceptibility and
resistance of C. glabrata clinical isolates to fluconazole (A) and voriconazole (B). In brackets it is
indicated the number of isolates that was found to be susceptible or resistant to the azoles tested. In
panel C the dataset of isolates resistant to voriconazole and fluconazole was compared, highlighting
the existence of 7 isolates that are resistant to both drugs.
The distribution of MIC values to fluconazole and voriconazole obtained for the overall
cohort of isolates tested in this study is shown in Figure 11 panel A and B, respectively. The
distribution obtained is similar to the one provided by EUCAST, the slight differences obtained
being considered to be within the experimental error associated to the dilution test method
(www.eucast.org). Such similarity is quite relevant considering that the distribution obtained by
EUCAST comprises a significantly higher number of isolates than those that are used in this study
(Figure 11 panel C and D).
24
Figure 11 - Distribution of MIC values of fluconazole and voriconazole obtained for the cohort of C.
glabrata clinical isolates tested in this work (panels A and B) or reported by EUCAST (panels C and
D). The dashed line indicates the resistance breakpoint for fluconazole or the epidemiological cut-off
value for voriconazole, above which the isolates are considered resistant to these drugs.
The results obtained concerning the anidulafungin and caspofungin MIC values obtained
for the different isolates are shown in Annex C and summarized in Figure 12A and 12B,
respectively. Under the experimental conditions used, the MIC value of the reference strain
CBS138 was of 0.06 mg/L for anidulafungin and 0.125 mg/L for caspofungin. None of the isolates
tested were able to grow in concentrations of anidulafungin above 0.06 mg/L, the defined
breakpoint concentration (Annex C). Consequently, none of the isolates tested could be
considered resistant to anidulafungin. Interestingly, the FFUL665, FFUL696 isolates and CBS138
reference strain were found to be considerably more tolerant to anidulafungin than the remaining
isolates as it exhibited some growth in the presence of 0.06 mg/L of the drug, while none of the
others did (Annex C). The distribution of anidulafungin MICs for the herein tested cohort of isolates
is similar to the one reported by EUCAST, as shown in Figure 12C. Although the identification of
isolates resistant to caspofungin is not possible due to the absence of a defined breakpoint and
MIC distributions, a closer look into growth of the isolates in the presence of this antifungal renders
clear that isolate FFUL887 is considerably more tolerant than the remaining isolates being able
to grow in the presence of 0.25 mg/L of this drug (Annex C). Notably, FFUL887 was one of the
isolates that was also found to be resistant to fluconazole and voriconazole. Consistent with the
results herein obtained, other epidemiological surveys have also reported very low levels of
resistance to echinocandins (1.5%) [53].
25
Figure 12 - Distribution of the MIC values of anidulafungin and caspofungin obtained for the cohort of
C. glabrata clinical isolates tested in this work (panel A and B) and reported by EUCAST (panel C).
The dashed line indicates the resistance breakpoint for anidulafungin, above which the isolates are
considered resistant to the drug.
3.2. Phenotypic characterization of the FFUL887 isolate in the presence of
voriconazole, anidulafungin, caspofungin or fluconazole.
The FFUL887 isolate was further selected to proceed in this work considering its
interesting traits of resistance to fluconazole, voriconazole and caspofungin, as assessed by the
above described phenotypic screening. In Table 8 the MIC values that were obtained for the
FFUL887 strain and for the reference strain CBS138 are summarized.
Table 8 - MIC values for fluconazole, voriconazole, anidulafungin and caspofungin, as determined by
the EUCAST recommended microdilution method.
Strains MIC (mg/L)
Fluconazole Voriconazole Caspofungin Anidulafungin
FFUL887 >64 2 0.5 0.03
CBS138 16 0.25 0.125 0.06
3.2.1 Growth kinetics
To confirm the results of the phenotypic screening above described and to assess growth
kinetics of the FFUL887 isolate in the presence of the different antifungals the growth curves in
liquid growth medium of cells of this isolate and of the CBS138 strains in the presence of the
antifungals was followed through time. For this, the cells of the two strains were cultivated under
26
the same experimental conditions as those used in the test microdilution method with the
difference that instead of having only one read of absorbance in the end of the test (after 24h of
inoculation), the absorbance values were measured hourly for 42h. Two concentrations of the
different drugs were tested: one corresponding to the breakpoint (32 mg/L for fluconazole and 1
mg/L for voriconazole) and one below the breakpoint (16 mg/L for fluconazole and 0.5 mg/L for
voriconazole) (Figure 13). A control curve, without any drug added to the growth medium, was
also performed for each of the strains (Figure 13A). In the absence of the antifungal drug the two
strains exhibited a similar growth pattern indicating a similar fitness in the absence of stress
(Figure 13A). When the medium was supplemented with azole drugs, the FFUL887 isolate was
confirmed to grow better than CBS138 cells to fluconazole and voriconazole, in line with the
results obtained in the test dilution method (Table 8). Resistance of FFUL887 cells to voriconazole
and fluconazole seems innate since no lag phase was observed when these cells were suddenly
exposed to the drugs, albeit the growth rate of adapted populations was lower (0.0334 h-1 for
growth in the presence of 64 mg/L fluconazole and 0.0331 h-1 for growth in the presence of 1
mg/L voriconazole compared to control, 0.0439 h-1).
Similarly, growth curves of FFUL887 and CBS138 in the presence of two concentrations
of anidulafungin and caspofungin was also performed (Figure 14). Confirming the results obtained
in the microtiter dilution assays, the CBS138 strain was found to be considerably more tolerant to
anidulafungin than the FFUL887 isolate (Figure 14, panel C). On the other hand, in the presence
of 0.125 mg/L of caspofungin the FFUL887 isolate was more than the CBS138 strain (0.052 h-1
compared to 0.025 h-1) (Figure 14, panel D). When the concentration of caspofungin increased to
0.25 mg/L no significant differences in the growth rate of the two strains were observed, although,
the final biomass of the FFUL887 culture was higher (Figure 14, panel E).
27
Figure 13 - Growth curves of CBS138 (⃝) and FFUL887 () strains in RPMI 1640 2% glucose (A),
or in this same medium supplemented with fluconazole (16 mg/L and 32 mg/L, panels B and C) or
voriconazole (1 mg/L or 0.5 mg/L, panels D and E). Growth was followed based on the increase in
OD595nm of the culture during 42h. The growth curves shown are representative of three independent
experiments that gave rise to the same growth pattern.
Figure 14. Growth curves of CBS138 (⃝) and FFUL887 () strains in RPMI 1640 2% glucose (A), or
in this same medium supplemented with anidulafungin (0.03 mg/L and 0.06 mg/L, panels B and C) and
caspofungin (0.125 mg/L and 0.25 mg/L, panels D and E). Growth was followed based on the increase
in OD595nm of the culture during 42h. The growth curves shown are representative of three independent
experiments that gave rise to the same growth pattern.
28
To assess if the azole-tolerance phenotype exhibited by the FFUL887 strain was
generalized for azoles or limited to azoles of the triazole family (where fluconazole or voriconazole
are included in), growth of this strain in the presence of ketoconazole and clotrimazole, two
imidazoles, was examined. The cells were cultivated under the same experimental conditions
described above in the microdilution assays using 4 mg/L ketoconazole and 1 mg/mL of
clotrimazole (Figure 15). These concentrations were selected because they corresponded to
those concentrations above which C. glabrata isolates are considered resistant to clotrimazole or
ketoconazole, as recommended by CLSI or EUCAST, respectively. The results obtained (Figure
15) clearly show that FFUL887 cells are susceptible to both ketoconazole and clotrimazole as the
OD of the cultures was below the one required to consider the isolates resistant to these drugs
(indicated in dashed lines in Figure 15). Despite this, FFUL887 cells were found to grew better
than CBS138 in the presence of the two imidazoles, this being more evident in the presence of
clotrimazole (Figure 15).
Figure 15 - Comparison of the growth of CBS138 and FFUL887 isolates in the presence of 4 mg/L of
ketoconazole and 1 mg/L of clotrimazole. The growth shown is representative of two independent
experiments in which each isolate was assayed twice.
3.2.2 Biofilm formation
Resistance of Candida glabrata to echinocandins and azoles has been well associated
with the ability of this yeast species to form biofilms, this contributing to reduce cellular exposure
to the drug and thereby resulting in increased resistance [127-130]. Given this, it was examined
whether the increased tolerance of FFUL887 cells to fluconazole and voriconazole could result
from an enhanced ability of these cells to form biofilms. Besides the FFUL887 isolate and the
reference strain CBS138, three other isolates were also used in these assays: FFUL674, resistant
to voriconazole and fluconazole (Annex B); and FFUL46 and FFUL48, which, like FFUL887, were
also found to be tolerant to caspofungin (Annex C). Biofilm formation was quantified using the
commonly used crystal violet staining method. The experimental conditions used were similar to
those used in the microdilution assay and the concentrations of the different drugs used
corresponded to those of the resistance breakpoint (Figure 16). In the absence of drugs FFUL674,
FFUL887 and CBS138 cells exhibited a significantly higher ability to form biofilms than FFUL46
29
and FFUL48 (Figure 16A). When exposed to any of the drugs tested the ability of all the strains
to form biofilms was reduced (Figure 16). No correlation between the ability to form biofilms and
the resistance of the strains to azoles was observed since the both FFUL674 and FFUL887
strains, which were the strains more resistant to fluconazole and voriconazole, produced amounts
of biofilm similar to those produced by susceptible strains (Figure 16). The FFUL887 strain was
found to be the strain producing higher amounts of biofilm in the presence of 0.25 mg/L of
caspofungin, however, this trend was not observed when higher concentrations of this
echinocandin were used (results not shown). No correlation between resistance to anidulafungin
and ability to form biofilms was also observed (Figure 16). Altogether, the results obtained indicate
that the tolerance phenotype of the FFUL887 to fluconazole, voriconazole and caspofungin is not
linked to an increased capacity to form biofilms.
Figure 16 - Measure of biofilm production after 24h of growth in RPMI 1640 2% G growth medium or
in this same growth medium supplemented with the indicated concentrations of fluconazole,
voriconazole, caspofungin and anidulafungin. In each panel the isolates are ordered according to their
resistance to the different antifungals. The results shown are representative of two independent
experiments in which each isolate was assayed twice.
3.2.3 Resistance to echinocandin-induced death
Echinocandins have a reported fungicidal action against C. glabrata [38]. Taking this into
consideration, cell viability of FFU887 and CBS138 cells was compared upon sudden exposure
to 0.25 mg/L of caspofungin, a concentration that was found to induce loss of cell viability [120].
30
Remarkably, FFUL887 cells were found to more resistant to the killing effect exerted by
caspofungin than the CBS138 strain (Figure 17).
Figure 17 - Resistance of FFUL887 and CBS138 cells to killing induced by 0.25 mg/L of caspofungin.
Cells of the two strains were cultivated in RPMI 1640 2% G growth medium in control supplemented
with 0.25 mg/L caspofungin ( and , respectively) or 0 mg/L ( and, respectively) for 12h during
which cell viability of the two cultures was at designated times. The viability results shown are
representative of three independent experiments.
3.3 Genome Sequencing and Annotation
To better understand the genetic adaptive responses underlying the increased resistance
of the FFUL887 isolate to voriconazole, fluconazole and caspofungin the genome sequence of
this isolate was obtained and then compared with the publicly available genome sequence of the
CBS138 strain. The genome sequence of the FFUL887 isolate was obtained after two rounds of
sequencing in a PGM sequencer from Life Technologies (Ion Torrent technology) in the NGS
laboratory of StabVida. After the two rounds of DNA sequencing, 5 920 417 reads (average size
of 199.75 bp) were obtained, this being subsequently assembled into 769 contigs (Table 9). The
total number of assembled bases, 12.29 Mb, corresponds to 99.1% of the estimated genome size
of the FFUL887 strain, 12.4 Mb, as determined by flow cytometry. For the identification of SNPs
and other mutations the reads obtained from the FFUL887 genome were mapped against the
publicly available and well-annotated genome sequence of the CBS138 strain, using the CLC
Genomics Workbench software. Identification of variants between the two strains was performed
using both probabilistic variant detection and quality-based detection, which differ in the manner
of identify polymorphisms. The probabilistic variant detection uses a probabilistic model,
combination of a Bayesian model and Maximum likelihood to calculate prior probabilities and error
31
probabilities. Parameters are first calculated on the mapped reads alone and then follows the
calculation of the likelihood of the observed allele being the same as the reference genome and
variants are called. This method originates less variants than a quality-base detection. Quality-
base variant calling tool uses a neighbourhood Quality Standard (NQS) algorithm, using a
combination of quality filters and user-specified thresholds for coverage and frequency to call
variants covered by aligned reads. This algorithm assumes that bases surrounded by perfectly
aligned, consistently high-quality sequence, are more accurate predicted than predicted by a
probabilistic algorithm [131-133]. While the previous methods only detect indels that are spanned by
reads, the tool of InDels and Structural Variation can detect larger insertions, deletions. This tool
relies exclusively on the information derived from unaligned ends of the reads in the mapping.
Only those SNPs that were identified both in the probabilistic and in the quality-based detections
were further considered. A total of 79 076 mutations (78 865 corresponding to SNPs and 211 to
InDels) had been identified in the genomes of CBS138 and FFUL887, 55% (corresponding to 35
438) of them being located in non-coding regions and 45% (corresponding to 43 573) in coding
regions. A total of 3198 gene coding sequences were found to differ between FFUL887 and
CBS138, 25 942 of these differences corresponding to synonymous mutations and 10 065 to non-
synonymous ones. The statistics of genome sequencing, assembly and variant call performed
are summarized in Table 9.
Table 9 - Results of FFUL887 genome sequence, assembly and variations detection, in comparison
with the genome of the reference strain CBS138. These results were obtained using the software CLC
Genomics Workbench.
Genome Sequencing Statistics
Reads
Total number of sequenced bases 1 182 Mb
Number of reads 5 928 417
Read average size 199.75 bp
Assembly
Total number of matched bases 1 160 Mb
Total of contigs 769
Average Contig length 15 984 bp
Contig length Sum 12 291 887 bp
Coverage 95.8x
Variant calling
Total 78 865
Synonymous SNPs Non-synonymous SNPs In non-coding regions
25 942 10 065 42 858
InDels variant calling
Coding region 58
In non-coding region 153
On the overall the data obtained turned clear that there are very significant differences at
the genomic level between CBS138 and the FFUL887 isolate. To further understand the genetic
relatedness of FFUL887 and CBS138 the genes used for the MLST scheme in C. glabrata (FKS2,
LEU2, NMT1, TRP1, UGP1 and URA3) were compared in order to define the sequence-type (ST)
of the two strains [134]. Such analysis showed that the FFUL887 has new alleles for the FKS2 and
MNT1 genes thereby turning impossible to define at this moment the ST of this isolate. Alleles
defined by both strains using the database cglabrata.mlst.net.
32
Table 10 – Characterization of the MLST allelic profile of CBS138 and FFUL887 strains using
cglabrata.mlst.net.
Allele Allelic profile
CBS138 887
FKS2 8 New allele
LEU2 3 New allele
NMT1 5 7
TRP1 5 7
UGP1 1 3
URA3 1 6
The allelic profile of CBS138 and FFUL887 was then run with eBRUST [135] against the
list of C. glabrata isolates profiles in the database at cglabrata.mlst.net (Figure 18). The BRUST
algorithm allows to display the relationships between closely related isolates of a specie or
population, in which an ancestral genotype, denominated founding genome, increases in
frequency in the population, and while doing so, begins to diversify to produce a cluster of closely-
related genotypes that are all descended from the founding genotype. The founding genotype is
identify by the analysis of mutually exclusive groups of related genotypes in the population,
information obtain by MLST allelic profile. The output is displayed as a radial diagram (“population
snapshot”), centred on the predicted founding genotype [135]. The results show that CBS138 and
FFUL887 are distant related isolates that don’t diverge from the same founding genotype (Figure
18).
Figure 18 – “Population snapshot” of FFUL887 and CBS138 against the entire C. glabrata MLST
database. Each dot represents a ST group. Blue dots represent founding genomes. Both CBS138 and
FFUL887 are singletons (strain with a unique ST value of the entire database).
33
It would be impossible to focus on all the identified mutations in this thesis and therefore
focus has been put on non-synonymous mutations found in coding sequences as these are those
more prone to directly impact the physiology of FFUL887 and CBS138 cells. Besides helping to
understand the genetic traits that could underlie the antifungal resistance phenotype of the
FFUL887 isolate, the results obtained might also help to better understand mechanisms of C.
glabrata adaptation to the mammalian urinary and GI tracts, considering that the FFUL887 isolate
was recovered from urine sample and CBS138 (Annex A). In Figure 19 it is shown the distribution
of non-synonymous mutations over the C. glabrata chromosomes. Such analysis reveals that the
polymorphisms found in the FFUL887 isolate are distributed evenly throughout all the 14
chromosomes that compose the C. glabrata genome, being observed a decrease in the frequency
of mutations in the mitochondrial genome (Figure 19). This observation is consistent with previous
studies reporting that mitochondrial genome has a lower frequency of mutation then nuclear
chromosomes [136, 137]. The distribution of non-synonymous mutations along the 3198 genes that
were found to be altered in the FFUL887 strain are shown in Figures 20 and in Table 11. The vast
majority of the genes harbored less than 5 non-synonymous variations, however, in some genes
this number increased up to more than 30 mutations, being of notice the number of mutations
found in the CAGL0C00253g (115), CAGL0J11968g (30) and CgPWP4 (53) genes, all encoding
proteins with a presumed cell wall adhesins; in CAGL0K12078g (50), encoding a protein with
unknown function; and in CAGL0C00231g (42), encoding a presumed nucleobase transporter
(Figure 20).
Figure 19 - Percentage of genes having non-synonymous mutations in the FFUL887 strain, when
compared to the reference strain CBS138.
Table 11 - Distribution in number of the non-synonymous mutations per genes of FFUL887.
Nº of missense mutation per gene ≤5 6-10 11-15 16-20 21-30 31-50 >50
Nº of genes affected 2760 340 65 17 12 2 2
34
Figure 20 - Distribution of the non-synonymous mutations per the 3200 genes whose coding sequence
differed in the FFUL887 and in the CBS138 genomes. The genes were clustered according to their
chromosomal localization.
A closer look into the biological function of the genes that harbored more than 16
mutations clearly revealed the enrichment of proteins involved in adhesion, as detailed in Annex
D. It is possible that these heavily mutated genes are those under a stronger selective pressure.
Consistent with this, modification in adhesion properties through a high mutational rate of adhesin-
encoding genes has been considered a primary mechanism of pathogenicity evolution in C.
glabrata [138]. 51% of adhesin-related proteins in C. glabrata [139] were found to have non-
synonymous mutations in FFUL887. Several of these proteins involved in adhesion belong to the
EPA family, which is one the most studied virulence factors studied in C. glabrata [138]. It was
interesting to observe that besides extensive number of SNPs other mutations were identified in
the set of adhesins encoded by the FFUL887 genome including disruptive mutations in the coding
sequences of CgAWP7, CgEPA12, CAGL0J02552g, CAGL0C00253g genes, these genetic
alterations being predicted to eliminate the glycosylphosphatidylinositol (GPI)-anchor sequence
that links the adhesin to the 1,6-β-glucan chain (Annex D). Another difference was the number of
35
repeated sequences found in the coding sequence of the adhesin genes CAGL0F08833g and
CAGL0M03773g encoded by FFUL887 and CBS138.
The significant number of differences found in the coding sequences of genes related
with adhesion in CBS138 and in FFUL887 suggested that these isolates could have different
adherence capacities. In that sense, the ability of CBS138 and FFUL887 cells to adhere to biotic
and abiotic surfaces was tested. Polystyrene was used as an abiotic surface, considering its wide
use in studies focusing adherence in C. glabrata. The extracellular matrix proteins fibronectin and
vitronectin were used as examples of biotic surfaces. The ability of FFUL887 and CBS138 cells
to adhere to the different surfaces was measured based on crystal violet staining method after 4h
and 8h of cultivation. Both strains adhered rapidly to polystyrene and vitronectin, with the
FFUL887 cells reaching higher biomass values, especially for vitronectin (Figure 21). Adherence
to fibronectin was considerably slower and reached higher values for the CBS138 strain (Figure
21). Altogether, the results confirm the suggestion that CBS138 and the FFUL887 isolate have
differences in their adherence properties, although further studies have to be performed in order
to see if the genetic differences found in the genes related with adhesion in the two strains are
related with the observed differences in adhesive capacity. It is interesting that FFUL887 strain
exhibited higher adhesion to vitronectin considering that this is an extracellular matrix protein
found in the urinary tract [140], the location where this isolate was retrieved from.
Figure 21 - Adherence of FFUL887 and CBS138 cells to biotic and abiotic surfaces. Cells of the two
strains were cultivated in polystyrene 96-microwell plates or in these same plates pre-coated with
fibronectin (10 µg/ml) or vitronectin (10 µg/ml) for 4 and 8h. After incubation, the amount of biomass
present was quantified using the crystal violet staining method.
3.4 Functional distribution of genes harboring mutations in the FFUL887
strain
The set of FFUL887 proteins that were found to harbor mutations, in comparison with the
CBS138 proteome, were clustered according to their biological function using the MIPS
Functional Catalogue Database tool [141]. The distribution obtained is shown in Figure 22.
A significant number of proteins having a biological function related with metabolism have
been found to have different sequences in FFUL887 and in CBS138 including proteins involved
in carbohydrate metabolism (particular, chitin metabolism); in metabolism of nitrogen, sulphur and
36
selenium, in phosphate metabolism, in amino acid metabolism (in particular in biosynthesis of
aspartate, methionine, lysine, valine, and degradation of isoleucine, valine and leucine); and in
metabolism of vitamins/cofactors and prosthetic groups. Interestingly, all these metabolic
functions have been reported as relevant for adaptation of C. glabrata to the human host [138, 142,
143]. Other functional classes that were found to comprise a significant number of genes mutated
in FFUL887 are the “transport” class, including several multidrug resistance transporters
previously implicated in resistance to antifungals (as discussed below), “signal transduction” and
“stress response”, these including several proteins belonging to well-described stress-responsive
signalling pathways such as the Hog1-kinase pathway or the PKC-pathway.
Figure 22 - Functional clustering of the proteins found to have different amino acid sequences in
FFUL887 and in CBS138 strains, according with MIPS functional catalogue. Enriched functional
classes (p-value below 0.01) are indicated with *.
It is hard, if not impossible, to predict the consequences for protein activity of the
mutations that were identified throughout the FFUL887 proteome and to assess how these
modifications impact the overall physiology of CBS138 and FFUL887 cells. However, in the case
37
of mutations leading to premature STOP codons such analysis is possible since this alteration
may lead to protein inactivation, depending on the region where the protein is prematurely
truncated. 67 proteins were found to harbour frameshift mutations in FFUL887 leading to
premature protein truncation and 6 proteins showed to increase in size as the result of frameshift
mutations. The full list of these proteins is available in Annex E and Annex F. In Table 11 it is
shown a list of 15 proteins whose proteins in the FFUL887 background are only translated at 20%
of the size of the CBS138 orthologue (Table 11). Most of the genes (31 out of 73) that seem to
be prematurely interrupted in the FFUL887 isolate do not have a known biological function (Annex
E and Annex F). Twenty-five of the genes that are predicted to be truncated in the FFUL887
isolate do not have annotated homologues in other Candida spp. nor in S. cerevisiae which
suggest that these DNA sequences may not actually corresponding to true coding sequence.
Among those that are associated to a given biological function (Figure 23). It is possible to
observe an enrichment of proteins involved in metabolism (in particular, in phosphate
metabolism); in cell cycle and DNA processing, transcription (in particular, mRNA synthesis and
processing); in protein fate, transport routes; and in biogenesis of cellular components (in
particular, cell wall, cytoskeleton/structural proteins and nucleus) (Figure 23). Consistently,
premature truncation of the CgSSK2 kinase, a component of the HOG1-signalling pathway, had
already been described to occur in other isolates [144].
Figure 23. Functional clustering of proteins found to have truncated sequences in FFUL887 compared
to CBS138 strains, according with MIPS functional catalogue [145]
38
Table 12 - Proteins affected by premature STOP codons at 20% of its sequence either originated by a nonsense mutation or frameshift mutations. The nomenclature
used for variation report is taken from www.hgvs.org/mutnomen/.
ORF name Description Nucleotide Mutation
Amino Acid modification
Protein size in
CBS138
STOP codon in FFUL887
% of truncated
protein
CAGL0A02552g Component of the septin ring that is required for cytokinesis 27_28insAC Val10fs 396 11 97.2
CAGL0A04213g Protein involved in G2/M phase progression and response to DNA damage
97_98insAA Pro33fs 639 59 90.8
CAGL0C01683g ATPase subunit of imitation-switch (SWI) class chromatin remodels
L58_59delTG Cys20fs 1115 22 98.0
CAGL0C02343g ATPase activity, role in cellular response to drug and ribosomal small subunit export from nucleus
275T>A Leu92* 720 92 87.2
CAGL0E00847g
ARF guanyl-nucleotide exchange factor activity and role in ER to Golgi vesicle-mediated transport, autophagic vacuole assembly, cellular response to drug, intra-Golgi vesicle-mediated transport
150_151insAACAA
Asp51fs 1821 77 95.8
CAGL0E05324g Role in mitochondrial DNA replication and mitochondrion localization
63_64insT Leu22fs 1284 41 96.8
CAGL0F01837g Protein with TBP-class protein binding, transcription cofactor activity
83_84insT Glu28fs 600 34 94.3
CAGL0H03751g Role in positive regulation of exit from mitosis and nucleus localization
291_292delGC Gln97fs 1021 101 90.1
CAGL0H04697g RNA polymerase I transcription factor binding 167delTinsGC Leu56fs 1010 101 90.0
CAGL0J02128g Protein of unknown function 8G>A Trp3* 94 3 96.8
CAGL0J09702g Protein with role in fungal-type cell wall organization, positive regulation of signal transduction and mitochondrion localization
2045delA; 1879_1880insC; 130_131insGTGCG
His682fs; Gly627fs; Lys44fs
695 56 91.9
CAGL0K11484g Protein of unknown function, without a known orthologue 26T>A; 171T>G Leu9*; Tyr57* 107 9 91.6
CAGL0M10153g Protein with MAP kinase kinase kinase kinase activity 210_211insTC Asn71fs 867 103 88.1
CAGL0M10829g (CgSSK2)
Protein with MAP kinase kinase kinase activity 227_228delCT Ala76fs 1667 79 95.3
39
3.5 Mutations occurring in genes associated with antifungal resistance in
C. glabrata
As said above it is not easy to predict how the genomic differences found in the CBS138
and in the FFUL887 isolate might contribute for the different resistance to antifungals of these two
strains. In order to get more insights into this, the set of genes having mutations in the FFUL887
genomic sequence was compared with a comprehensive list of 165 genes that had been
previously implicated in C. glabrata resistance to fluconazole, voriconazole and caspofungin,
based on numerous studies published in the literature exploring gene-by-gene or genome-wide
analyses. The deletion of any of the genes that were found to be truncated in the FFUL887 has
not been found to increase C. glabrata resistance to azoles or echinocandins indicating that the
premature interruptions of the proteins is not contributing for tolerance to antifungals in the
FFUL887 strain. Despite this, four genes found to be interrupted in the FFUL887 strain,
CAGL0A03872g (truncated at 92%), CAGL0H08217g (truncated at 61%), CAGL0I09746g
(truncated at 28%), CAGL0K11396g (truncated at 59%) are also found to encode proteins whose
deletion in other species led to azole resistance (Annex E). Up to now their involvement in C.
glabrata resistance to azoles was not demonstrated, however, it is possible that their deletion
could contribute to increase tolerance to C. glabrata to antifungals as demonstrated for their
orthologues.
106 of the “antifungal-resistance” genes were found to harbor non-synonymous
mutations in the FFUL887 isolate, 45 of these genes being associated with resistance to
fluconazole and/or voriconazole, 55 involved in resistance to caspofungin and 6 affecting C.
glabrata resistance to both classes of antifungals (Annex G, H and I). Among the genes that were
found to have mutations in FFUL887 were the FKS genes, the biological targets of echinocandins
(Figure 24). Although the mutations found in the FFUL887 isolate are located outside of the hot-
spot regions that are commonly mutated in echinocandin-resistant isolates (Figure 24), it is not
possible to know if these mutations are behind the higher tolerance to caspofungin of the
FFUL887 isolate (Annex 3). The Gly14Ser mutation occurring in the coding sequence of FFUL887
CgFKS1 occurs in the C-terminal region of the protein and creates a potential phosphorylation
site, according to the NetPhosYeast algorithm [146]. Phosphorylation of FKS1 has been described
to serve as an activating mechanism for this enzyme [147], although this has only been
demonstrated in S. cerevisiae and in regions located more close to the enzyme catalytic domain
[148]. In the case of CgFKS2 one of the mutations identified in the FFUL887, Thr926Pro, is located
in the catalytic domain of the protein (Figure 24), but in a region that is not conserved in all
Candida spp. (Figure 25). No mutations had been identified in the coding sequence of ERG11 of
FFUL887.
In the following sections it will be further detailed the mutations identified in the sets of
genes involved in resistance to echinocandins and/or azoles.
40
Figure 24 – Modification in the amino acid sequence of the glucan synthase genes CgFks1 and CgFks2 encoded by FFUL887, when compared to their counter-partners
encoded by the CBS138 strain. Domains from both proteins were predicted by Pfam Domain [3]. Grey boxes represent transmembrane domains predicted by TMHMM
SERVER [4].
Figure 25 - Partial alignment of C. glabrata CgFks2 protein sequence of CBS138 and FFUL887 to the paralogue CgFks1, and orthologue sequences of S. cerevisiae
S288c, C. albicans SC5314, C. parapsilosis CDC317 and C. tropicalis MYA-3404 using ClustalW2 [5]. Mutation found in CgFks2 of FFUL887 are highlighted.
41
3.5.1. Genes involved in azole and in echinocandin resistance
16 genes described to confer resistance to echinocandins and azoles in C. glabrata were
found to harbour mutations in the genome of the FFUL887 isolate, this set including two proteins
involved in chromatin remodelling and several proteins involved in the high-affinity calcium uptake
system (HACS) as detailed in Table 13 [7, 8, 149] and the full list is available in Annex G. None of
the mutations found in the coding sequence of CgCCH1 and in CgMID1 genes were found to be
located in the domains of these transporters that are known to be involved in Ca2+ uptake, as
shown in Figure 26. FFUL887 CgCCH1 has a mutation, Met1Ile, which alters the START codon.
Since no function has yet been attributed to the C-terminal region of CgCch1 and the most
proximal ATG codon starts at 7-9nt and keeps the coding frame, this mutation may not have
consequences to the protein function.
Table 13. Genes described to mediate resistance to azoles and echinocandins in C. glabrata that were
found to harbour mutant variations in the FFUL887 isolate. The nomenclature used for variation report
is taken from www.hgvs.org/mutnomen/.
Gene/ORF name
Function
Effect in antifungal resistance
Amino acid modification
found in FFUL887
CgSIN3
Component of both the Rpd3S and Rpd3L histone deacetylase complexes
- Deletion leads to fluconazole and caspofungin resistance[7, 8] - Deletion leads to decreased cell fitness [7]
Asn50Lys; Lys288Thr
CgSWI4
DNA binding component of the SBF complex (Swi4p-Swi6p)
- Deletion increases resistance to fluconazole, clotrimazole and ketoconazole [150] - Deletion increases susceptibility to caspofungin and micafungin [151]
Asn300Ile; Asn669Ser; Val324Ala; Arg715Lys
CgCCH1
Putative subunit of a plasma membrane gated channel involved in Ca2+ uptake (HACS)
- Deletion leads to fluconazole and micafungin susceptibility [8,
149] - Deletion increases susceptibility to fluconazole, voriconazole and caspofungin [7]
Met1Ile; Arg51Pro;
Glu1912Lys
CgMID1
Putative regulatory subunit of a plasma membrane gated channel involved in Ca2+ uptake (HACS)
Gly77Asp
42
Figure 26 - Modifications in the amino acid sequence of the protein domains of intervenient of the HACS, CgCch1 and CgMid1 encoded by FFUL887 when compared
to their counter-partners encoded by the CBS138 strain. The domains from CgMid1 were predicted based on the homology with S. cerevisiae Mid1 retrieved from Iida.
H., et al., 1994 [152] The S4 and P domains of CgCch1 were predicted based on the homology with S. cerevisiae Cch1 retrieved from Paidhungat, M. and Garret, S.,
1997 [153] and frizzled cysteine-rich domain was predicted by Phyre2 [154]. Grey boxes represent transmembrane domains predicted by TMHMM SERVER [4]. Peptide
signals were predicted by SignalP [155]
43
3.5.2 Genes involved in azole resistance: emphasis on the transcription factor
CgPdr1
Around 70% of the genes associated with resistance to azoles in C. glabrata were found
to harbor non-synonymous mutations in FFUL887. A selected set of these genes is shown in
Table 14 and the full list is available in Annex H. Functional clustering of these proteins reveals
an enrichment of proteins involved in chromatin remodeling and in multidrug resistance, including
several drug efflux pumps and various transcriptional regulators (Table 14). Two of the genes
found to harbor mutations in the FFUL887 isolate are the drug-efflux pumps CgPdh1 and CgQdr2,
which had been identified as determinants of C. glabrata tolerance to azoles [76, 77, 85], presumably
by helping reducing the internal accumulation of these drugs (Figure 27). Different studies
performed in C. albicans and in S. cerevisiae have been showing that mutations in the coding
sequences of MDR transporters affect a tolerance of these yeast species to drugs, including to
azoles [156]. In that sense, a further look was taken into the mutations that were found to occur in
the coding sequences of the CgPDH1 and CgQDR2 encoded by FFUL887 (Figure 27). The
mutations occurring in the coding sequence of CgPDH1 occur in cytoplasmic stretches of the
protein but outside of the ATP binding motifs (the Walker motifs) [157, 158]. The mutations observed
to occur in the coding sequence of CgQDR2 are also essentially located in the cytoplasmic
stretches, although one mutation, Ser212Ala, has been found to occur within the 5 and 6th
transmembrane domains of the protein (Figure 27). Notably, the Ile255Phe mutation in CgQDR2
occurs near a region that was found to influence the activity of the C. albicans orthologue
CaMDR1 against multiple drugs, including fluconazole [159]. Both Hst1 and Upc2A from FFUL887
show amino acid changes on known domains. Met70Lys from CgHst1 affects the regional
transcriptional silencer and Arg92Lys and Glu822Val modifications in Upc2A affect the DNA
binding domain and the activator domain.
44
Table 14. Genes described to mediate azole resistance and/or associated to multidrug resistance in C. glabrata that were found to harbour mutant variations in the
FFUL887 isolate. The nomenclature used for variation report is taken from www.hgvs.org/mutnomen/
Gene/ORF
name Function Effect in antifungal resistance
Amino acid modification found in
FFUL887
CgPDH1
Multidrug transporter of the ATP-binding
cassette Superfamily
- Deletion increases susceptibility to
fluconazole, voriconazole, ketoconazole, and
itraconazole [7, 160].
Lys438Gln; Glu839Asp
CgQDR2
Drug:H+ antiporter of the Major Facilitator
Superfamily
- Deletion increases susceptibility to
miconazole, tioconazole, clotrimazole and
ketoconazole [83]
Asn38Ile; Ala69Thr; Ser212Ala; Ile255Phe;
Arg304His;Leu307Ile Leu309Ile;
Asn417Asp
CAGL0L04400g
(YRR1)
Zinc finger transcription factor involved in
transcriptional regulation of MDR genes.
Orthologue of S. cerevisiae ScYRR1
- Deletion increases susceptibility to
fluconazole and ketoconazole [6]
Cys24Gly; Ala58Val;
Ile137Leu; Asp229Glu; Ile346Val;
Glu574Lys; Ile593Leu; Glu710Asp;
Ala933Val
CgUPC2A
Zinc finger transcription factor required for
transcriptional regulation of genes involved in
uptake and biosynthesis of ergosterol - Deletion increases susceptibility to
fluconazole [74] Arg92Lys; Asn304Ser; Glu822Val
CgPDR1
Zinc finger transcription factor, activator of drug
resistance genes
- Deletion increases susceptibility to
fluconazole, voriconazole, itraconazole and
ketoconazole [6-8, 87]
A; B; C; X
CgHST1 Histone deacetylase that regulates gene
expression in niacin-limiting conditions
- Deletion increases tolerance to
fluconazole[161] Met70Lys
CgSUM1
CAGL0J10956g
Transcriptional repressor involved in chromatin
silencing
- Deletion increases tolerance to fluconazole
[161]
Asn284Asp; Ser307Gly; Val478Ile;
Ser531Thr
CgBEM2
Protein with GTPase activator activity with role
in actin cytoskeleton organization and negative
regulation of Rho protein signal transduction
- Deletion increases susceptibility to
fluconazole, clotriconazole and
ketoconazole[68]
Ala139Thr; Leu169Met; Thr171Ala;
Ser240Asn; Thr405Ser; Lys461Arg;
Leu879His; Phe1347Ser
45
Figure 27 - Modifications in the amino acid sequence of the proteins involved in azole resistance
encoded by FFUL887, when compared to their counter-partners encoded by the CBS138 strain.
Neutral mutations are shown in grey. The domains shown for CgPdh1 were retrieved from Miyazaki,
H., et al., 1998 [85], CgUpc2A and CgHst1 domains were predicted based on the homologue domains
from S. cerevisiae described by Davies, B., 2005 [162] and Kadosh, D. and Struhl, K., 1998 [163] using
ClustalW2 [5]. Grey boxes represent transmembrane domains, as predicted by the TMHMM algorithm
[4].
A transcription factor found to harbor mutations in the FFUL887 strain was CgPDR1.
Three of the mutations (A, B and C) found in the CgPdr1 transcription factor encoded by FFUL887
(Figure 28) had been previously described in isolates resistant and susceptible to azoles [13],
indicating that these mutations are not likely to underlie the azole-resistance phenotype of the
strain. Notably, the X mutation has never been described, although this mutation is mapped in a
region where other GOF mutation had been identified [13] (Figure 28). Analysis of the secondary
structure of the CgPdr1 protein encoded by FFUL887 and CBS138, performed using the algorithm
Phyre2 [154], shows that the mutation A in the FFUL887 strain may induce a change in the protein
structure. This mutation is located in a region of the CgPdr1 protein that is only found in the C.
glabrata protein being absent from ScPdr1.
46
Figure 28 - Modifications in the amino acid sequence of the CgPdr1 transcription factor encoded by
FFUL887, when compared to its counter-partners encoded by the CBS138 strain. Neutral mutations
are represented in grey. CgPdr1 domains were retrieved from Tsai, H., 2010 [14].
The identification of this new mutation in CgPDR1 sequence led us to hypothesize that
the FFUL887 CgPdr1 could be a new gain-of-function mutant of this protein. In order to confirm
this hypothesis the resistance to azoles of the FFUL887 strain was compared to the resistance
exhibited by another C. glabrata clinical isolate described to encode a GOF CgPdr1 mutant. In
specific, the MIC values of fluconazole and voriconazole were compared in 4 strains: FFUL887;
DSY562, a strain devoid of CgPDR1; SFY114, a strain which has the same genetic background
of DSY562 but in which a wt CgPDR1 has been introduced; and SFY115, a strain that has the
same genetic background of DSY562 but in which it was introduced a L328F mutation in CgPDR1
sequence, this being a GOF mutation found in azole-resistant isolates [13, 81] (Figure 30). The
results obtained showed that the DSY562 (pdr1) and SFY114 (wt CgPdr1) strains are both
susceptible to fluconazole and voriconazole, indicating that CgPdr1 function was not required for
tolerance to these azoles in these genetic backgrounds (Figure 29). Strain SFY115 (GOF
CgPdr1) was found to be resistant to fluconazole and was more tolerant to voriconazole than the
parental strain SFY114 (wt CgPdr1), indicating that the GOF mutation had a more drastic impact
in augmenting tolerance to fluconazole. Differently, the FFUL887 strain was found to be resistant
to both fluconazole and voriconazole.
47
Figure 29 - MIC values for fluconazole and voriconazole evaluated by the EUCAST dilution method
for the DSY562, SFY114, SFY115 and FFUL887 strains.
To fully elucidate if the FFUL887 CgPdr1 indeed corresponded to a GOF mutant the
transcriptomes of the FFUL887 and CBS138 strains were compared using DNA microarrays (in
collaboration with the group of Professor Geraldine Butler, from University College of Dublin). For
this, cells of the two strains were cultivated in RPMI growth medium until mid-exponential phase,
after which total RNA was obtained. Around 544 genes were found to be differently expressed
(above or below a 1.5-fold threshold level) between the CBS138 and FFUL887 strains.
Significantly, 21 genes found to be more expressed in the FFUL887 isolate correspond to
documented targets of CgPdr1, these being shown in Table 15. Among these set of well-
described CgPdr1-regulated genes are the CgPDR1 gene itself and the drug-efflux pumps
CgPDH1 and CgCDR1.
To confirm the results of the microarrays the higher expression of some CgPDR1
regulated genes in the FFUL887 strain that are known to be involved in azole resistance was
confirmed by real time RT-PCR. The results obtained confirmed the up-regulation of CgCdr1,
CgPdh1, CgQdr2 and CgPdr1 genes in the FFUL887 strain (Figure 30).
Figure 30 - Comparison of the transcript levels of CgPDR1, CgCDR1, CgPDH1 and CgQDR2 genes
in FFUL887 and in the CBS138 strains, as revealed by real time RT-PCR.
48
Table 15. Expression of CgPDR1 documented target genes in C. glabrata FFUL887 and in CBS138 strains, as revealed by microarray analysis. The set of documented
CgPdr1 targets is accoting to the results of Claude, K., et al., 2011[16], Tsai, F., et al., 2010 [14] and Vermitsky, J. et al., 2006 [17].
Functional group S. cerevisiae orthologue
Gene name/ORF name Function Fold Change,
FUL887 vs CBS138
Transport
OAC1 CAGL0K11616g Putative mitochondrial inner membrane transporter. 2.32 ± 0.08
PDR5 CAGL0M01760g (CgCDR1) Multidrug transporter of ABC-superfamily. Involved in fluconazole and voriconazole resistance.
4.61 ± 0.35
PDR15 CAGL0F02717g (CgPDH1) Multidrug transporter of ABC-superfamily. Involved in fluconazole resistance
3.21 ± 0.31
QDR2 CAGL0G08624g Drug:H+ antiporter of the MFS-superfamily 3.95 ± 0.63
YOR1 CAGL0G00242g Putative ABC transporter involved in multidrug efflux 2.33 ± 0.38
Lipid metabolism PLB3 CAGL0J11770g Phospholipase B 1.88 ± 0.16
Cell stress and homeostasis
HSP12 CAGL0J04202g Heat shock protein 1.89 ± 0.001
TSL1 CAGL0H02387g Putative trehalose-6-phosphate synthase/phosphatase subunit 1.57 ± 0.15
YML131W CAGL0K12958g Putative stress-induced alcohol dehydrogenase 1.60 ± 0.09
Transcription
PDR1 CAGL0A00451g Zinc finger transcription factor, activator of drug resistance genes via pleiotropic drug response elements. Involved in fluconazole and voriconazole resistance
1.96 ± 0.21
SIP3 CAGL0I01980g Putative role in retrograde transport of sterols from the plasma membrane to the ER
1.62 ± 0.19
RNA processing RTC3 CAGL0H02101g Protein with role in RNA metabolic process 2.85 ± 0.05
Signal transduction CDC25 CAGL0D06512g Putative membrane bound guanine nucleotide exchange factor 2.33 ± 0.53
Mitochondrial POR1 CAGL0J09900g Mitochondrial voltage-dependent anion channel 1.56 ± 0.01
UTH1 CAGL0L05434g Protein with role in fungal-type cell wall biogenesis 1.65 ± 0.04
Cell wall SED1 CAGL0K10164g Putative adhesin-like protein 3.09 ± 0.09
- CAGL0E01771g (CgYPS5) Putative aspartic protease 2.75 ± 0.06
Amino acid metabolism
MET8 CAGL0K06677g Putative bifunctional dehydrogenase and ferrochelatase 2.46 ± 0.07
MET17 CAGL0D06402g O-acetyl homoserine sulfhydrylase 5.14 ± 0.13
Other Metabolism INP1 CAGL0H05137g Aldehyde dehydrogenase [NAD(P)+] activity 1.89 ± 0.001
DNA damage response
DDR48 CAGL0H08844g Putative adhesin-like protein 2.21 ± 0.23
YIM1 CAGL0M09713g Putative protein involved in DNA damage response 1.53 ± 0.13
Unknown Function
ALD5 CAGL0F07777g Putative aldehyde dehydrogenase 2.69 ± 0.08
GAS5 CAGL0G02717g Protein with glucan 1,4-alpha-glucosidase activity 1.70 ± 0.30
- CAGL0E04554g Putative protein 1.74 ± 0.20
49
3.5.3. Mutations in genes involved in resistance to caspofungin
55 genes found to contribute for resistance of C. glabrata to echinocandins were found
to harbor non-synonymous mutations in FFUL887, a selected set of these being shown in Table
16 and the full list being available in Annex I. A substantial amount of these echinocandin-
resistance genes that have mutations in FFUL887 are involved in cell wall biosynthesis and
assembly including CAGL0G00440g (CgLCB1), CAGL0J11506g (CgCHS2), CAGL0M04169g
(CgKRE1) and PKC pathway signalling pathway genes. CgPKC1, encoding the kinase that
responds to perturbations in cell wall structure [164] and was also demonstrated to be involved in
S. cerevisiae response to caspofungin [164], also shows modifications in FFUL887 (Figure 31).
Other genes of the PKC pathway were also found to harbour SNP mutations in the FFUL887
although it is not possible to determine if these mutations affect protein activity and thereby
contribute for the phenotype of tolerance to caspofungin of this strain. Both CgRlm1 and CgSwi4
show amino acid changes on known domains. Pro233Leu modification affects the regulatory
domain of CgRlm1 and Asn300Ile modification is in ankyrin repeat domain of CgSwi4.
Figure 31 - Modifications in the amino acid sequence of the PKC signalling pathway proteins encoded
by FFUL887, when compared to its counter-partners encoded by the CBS138 strain. Pkc1 domains
were predicted by Pfam Domain [3]. The regulatory and activation domains from Rlm1, the
inhibitory/Swi6 and Slt2 interaction domain from Swi4 were predicted based on the homologue
domains from S. cerevisiae described by Watanabe, Y., et al. 1997 [165] and Siegmud, R. and Nasmyth,
K., 1996 [166] using ClustalW2 [54].
50
Table 16. Genes described to mediate resistance to echinocandins in C. glabrata found to harbour mutant variations in the FFUL887 isolate. The nomenclature used
for variation report is taken from www.hgvs.org/mutnomen/.
Gene/ORF name
Function Effect in antifungal resistance Amino acid modification found in
FFUL887
CAGL0B01441g (RPD3)
Histone deacetylase, component of both the Rpd3S and Rpd3L complexes
- Deletion leads to caspofungin susceptibility [7] - Deletion leads to decreased cell fitness [7]
Thr414Ala
PKC signalling pathway
CAGL0L03520g (BCK1)
MAP kinase kinase kinase of cell integrity pathway - Deletion increases susceptibility to caspofungin [7]
Ile22Met; Ala550Val
CgRLM1
Putative transcription factor with a predicted role in cell wall integrity
- Deletion increases susceptibility to caspofungin and micafungin [7, 167]
Ala103Thr; Pro233Leu; Gln309Arg
CgSWI6
Transcription cofactor component of the SBF complex (Swi4p-Swi6p)
- Deletion increases susceptibility to caspofungin and micafungin [151]
Glu100Asp
HOG signalling pathway
CgSTE20
Putative signal transducing kinase of the PAK (p21-activated kinase) family, involved in maintaining cell wall integrity and osmotic stress response
- Deletion creates caspofungin susceptible phenotype [7]
Val279Ile; Arg352Lys
CAGL0B00858g (STE50)
Adaptor protein for various signalling pathways involved in mating response, invasive/filamentous growth and osmotolerance
Glu40Asp
CAGL0L05632g (PBS2)
MAP kinase kinase of the HOG signaling pathway, activated under severe osmotic stress
Leu178Pro
CAGL0G03597g (SHO1)
Transmembrane osmosensor for filamentous growth and HOG pathways
Thr224Ala
51
4. Final Discussion
The goal of this work was to analyse the increasing problem of acquired resistance to
antifungals in the human pathogen Candida glabrata using a comparative genomics approach.
For that the genome sequence of a clinical isolate FFUL887 found to be resistance to fluconazole
and voriconazole and showing increased tolerance to caspofungin, was compared with the
genome of the susceptible reference strain CBS138. A first analysis of the data revealed a
remarkably high number of proteins involved in adhesion that were confirmed to have different
sequences in FFUL887 and in CBS138 strains. In some cases the number of variations identified
reached 50, which is a quite high number considering that most genes harboured 5 variations or
less. Proteins involved in adhesion play a key role in shaping C. glabrata recognition, adhesion
and further adaptation in the host [139]. A very high mutational rate of this class of proteins had
been reported before to be an important mechanism of pathogenicity evolution in C. glabrata,
mostly attributed to chromosomal rearrangements of increasing number of tandem repeat
sequences and subtelomeric location which enhance recombination frequency [138]. This rapid
evolution helps to further diversify the function of adhesion genes, which are considered important
pathogenicity genes. Differences in the ability of FFUL887 and CBS138 strains to adhere in vitro
to different biotic surfaces were registered. It was particularly prominent the higher capacity of the
FFUL887 strain to adhere to vitronectin. FFUL887 was recovered from an urine sample and
therefore it is possible that is increased adherence to vitronectin could result from an adaptive
response considering that vitronectin is relevant in the human urinary tract were it is involved in
anchoring basal urothelial cells to the underlining basement membrane against constant bladder
cyclin [140]. Also, the distribution of soluble form of vitronectin in circulation or as an ECM-bound
protein was shown to overlap C. albicans dissemination [168]. FFUL887 cells were also found to
have a lower adherence to fibronectin than the CBS138 strain. Interestingly, studies have shown
that decreased adherence to fibronectin could help Candida spp. to evade the action of the host
immune system [169, 170]. It can’t be determined if and how the differences found in adhesive
properties of the two strains correlate with the differences registered in the sequence of adhesins,
however, further studies should be performed in order to understand how these differences affect
the protein activity this contributing for a better understanding of the mechanisms underlying
adhesion of C. glabrata to the human host.
Several genes were found to harbour frameshift mutations in the FFUL887 that result in
protein inactivation. Most of these genes do not have an attributed biological function and
therefore it is not possible to assess what could be the advantage of such truncation for the
FFUL887 isolate. None of the genes found to be prematurely truncated in the FFUL887 isolate
has been reported to increase C. glabrata resistance to antifungals. Nevertheless, the deletion of
4 genes interrupted in FFUL887 strain, CAGL0A03872g, CAGL0H08217g, CAGL0I09746g and
CAGL0K11396g led to increased resistance to azoles in S. cerevisiae. It will thus be interesting
to examine if the deletion of these genes also augments tolerance to antifungals in C. glabrata.
A new gain-of-function mutation had been identified in the coding sequence of the
transcriptional regulator CgPdr1 encoded by the FFUL887 strain. One mutation identified in the
52
coding sequence of FFUL887 CgPdr1 has not been described to occur in susceptible C. glabrata
clinical isolates. This mutation is located in a domain of CgPdr1 in which other GOF mutations
had been described. Transcriptomic analysis of CBS138 and FFUL887 revealed the over-
expression in the later isolate of a significant number of documented targets of CgPdr1 thereby
sustaining the idea that the X mutation identified is indeed a GOF mutation. It is of notice the fact
that the transcriptomic analysis had been conducted in the absence of any antifungal drug which
means that the FFUL887 CgPdr1 transcription factor is, somehow, in a constitutively active form.
Since this mutation occurs in the transactivation domain of the protein it can be hypothesized that
the mutation might lead to an enhanced ability of the transcription factor to recruit transcriptional
machinery, an idea that needs to be further demonstrated. The set of documented targets of
CgPdr1 that was found to be up-regulated in FFUL887 coincides, in a large extent, to those that
had been described in other GOF CgPdr1 mutants, which is another evidence sustaining the
hypothesis that this mutation is indeed a GOF mutation. Among the CgPdr1-targets that were
found to be up-regulated in FFUL887 are the CgPDR1 gene itself and also the drug-efflux pumps
CgPDH1 and CgCDR1 previously found to contribute for resistance to fluconazole and/or
voriconazole [6-8, 83, 87, 160]. Up-regulation of CgCDR1 in other azole-resistant isolates has also
been reported [60, 160]. Other genes not regulated by CgPdr1 had also been found to be up-
regulated in the FFUL887 strain including CgTIR3, CgAUS1, and CAGL0E01353g,
CAGL0E06248g (Annex J). It is possible that the higher expression of these genes in the
FFUL887 isolate could also contribute for the higher tolerance of this strain to azoles.
Besides azoles, the FFUL887 strain was also found to be more tolerant than CBS138 to
caspofungin. Several mutations were identified in genes encoding proteins that are known to
contribute for tolerance to caspofungin in C. glabrata. It is not possible to assess if these mutations
are behind the tolerance phenotype of the strain. In particular, several mutations in components
of the PKC and of the HOG1 signalling pathway had been identified which suggest that the activity
of these two signalling systems may differ in the FFUL887 and in the CBS138 strain. Indeed,
inspection of the transcriptomes of FFUL887 and CBS138 revealed that several genes that are
documented to be regulated by Rlm1 and SFB complex (Swi4/Swi6) of C. albicans and S.
cerevisiae, two transcription factors that are the final effectors of the PKC pathway, were found
to be up-regulated in the FFUL887 strain (data not shown) which might indicate a higher activity
of this pathway in this strain. Nevertheless, the killing effect exerted by caspofungin was
significantly higher in CBS138 than in FFUL887, which is an important contributing factor to
improve tolerance in this later strain. The molecular mechanisms by which C. glabrata cells lose
viability upon caspofungin exposure are not known although, in C. albicans this has been
demonstrated to involve both apoptotic and necrotic-induced death [171]. The genes mediating
these mechanisms of programmed cell death induced by caspofungin have not been identified
and therefore it is not possible to know if the CBS138 and the FFUL887 might have differences
that could justify the observed resistance of the later strain to caspofungin-induced killing,
although this an hypothesis that deserves further investigation.
53
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Annex A Table 17 - List of isolates tested and the local where the sample was collected. To some cases a MIC
of fluconazole was tested previously on the hospital. The washed sample corresponds to
bronchoalveolar lavage fluids. Isolates signalized with an asterisk were isolated from patients with
AIDS that followed fluconazole treatment.
Isolate Sample MIC Fluconazole Isolate Sample MIC Fluconazole
18 Mouth 497 Urine
24 Anal 495 Urine
46 Anal 496 Urine
48 Uretral 497 Urine
75 Anal 498 Urine
76 Anal 499 Urine
91 Urina 500 Urine
92 Anal 612 Urine
93 Anal 613 Feaces
96 Urine 614 Pharynx
97 Anal 644* Washed 16
98 Anal 696* Washed 16
246 Anal 648* Washed 4
247 Anal 665* Washed >32
249 Urine 668* Washed >32
267 Anal 670 Washed 32
268 Anal 674* Washed >32
277 Toungue 677* Washed >32
279 Toungue 679* Washed 0,5
281 Anal 696 Washed 16
282 Anal 737* Washed 8
314 Urine 830 Urine
412* Washed >64 834 Urine 16
443* Washed >64 862 Urine 4
464 Urine 866 Urine 64
465 Urine 878 Urine 64
466 Urine 887 Urine 64
467 Urine 4012
468 Urine 8093
473 Urine
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Annex B Figure 32 - Measured MIC values for the antifungals fluconazole and voriconazole of the cohort clinical isolates used in this study and the reference strain CBS138
(white). Resistant isolates are market in black, while isolates with sensitive/intermediate resistance are marked in grey.
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Annex C Figure 33 - Measured MIC values for the antifungals anidulafungin and caspofungin of the cohort clinical isolates used in this study and the reference strain CBS138
(white). Resistant isolates are market in black, while isolates with sensitive/intermediate resistance are marked in grey.
63
Annex D Table 18 - Putative and known adhesion-like proteins with modifications found in FFUL887.
Gene/ORF name Function
CAGL0C00110g (CgEPA6) Adhesin with a role in cell adhesion
CAGL0C00209g (CgAWP7) Putative adhesin-like cell wall protein
CAGL0C00253g Putative cell wall adhesin
CAGL0C00847g (CgEPA8) Putative adhesin-like protein
CAGL0C01133g Putative cell wall adhesin
CAGL0C03575g Putative adhesin-like protein
CAGL0E01661g Adhesin-like protein
CAGL0E06600g Putative adhesin-like protein
CAGL0E06644g (CgEPA1) Adhesin with a role in cell adhesion
CAGL0E06666g (CgEPA2) Epithelial adhesion protein
CAGL0E06688g (CgEPA3) Epithelial adhesion protein
CAGL0F08833g Putative adhesin-like protein
CAGL0F09273g Putative adhesin-like protein
CAGL0G00968g Protein with actin binding activity
CAGL0G10175g (CgAWP6) Putative GPI-linked cell wall protein
CAGL0G10219g Adhesin-like protein
CAGL0H10626g Predicted cell wall adhesin
CAGL0I00220g (CgEPA23) Predicted adhesin-like protein
CAGL0I07293g Adhesin-like cell wall protein
CAGL0I10098g Adhesin-like protein
CAGL0I10340g(CgPWP5) Cell wall adhesin
CAGL0I10362g (CgPWP4) Cell wall adhesin
CAGL0J00253g Putative adhesin-like protein
CAGL0J01727g Putative adhesion protein
CAGL0J02508g (CgAWP1) Adhesin-like protein
CAGL0J02530g Putative adhesion protein
CAGL0J02552g Adhesin-like protein
CAGL0J11968g (CgEPA15) Putative adhesin-like cell wall protein
CAGL0K00110g (CgAWP2) Putative adhesin
CAGL0K13024g (CgAED1) Adhesin-like protein required for adherence to endothelial cells
64
CAGL0L00157g Putative adhesin-like cell wall protein
CAGL0L10092g Putative adhesin-like cell wall protein
CAGL0L13332g (CgEPA12) Lectin-like adhesin with a role in cell adhesion
CAGL0M00132g (CgEPA12) Putative adhesin-like cell wall protein
CAGL0M03773g Predicted GPI-linked adhesin-like protein
CAGL0M14069g (CgPWP6) Adhesin-like protein
65
Appendix E Table 19 - Genes affected by premature STOP codon as result of frameshift and nonsense mutations found in FFUL887. The nomenclature used for variation report is
taken from www.hgvs.org/mutnomen/. Premature truncation of the protein and if or not GPI-anchor signal is eliminated or introduced are also summarized.
Gene/ORF name
Function Nucleotide
modification found in FFUL887
Amino acid modification
found in FFUL887
Protein size
Premature STOP codon
position
% of Truncated
Protein
CaglfMp05 (Cgai1)
Putative endonuclease encoded by the first exon and part of the first intron of the mitochondrial COX1 gene
980_981delAT His327fs 451 328 27.3
CaglfMp07 (Cgai3)
Putative endonuclease encoded by the first three exons and part of the third intron (a group I intron) of the mitochondrial COX1 gene
717_718insAT Asn240fs 603 245 59.4
CAGL0A02552g Component of the septin ring that is required for cytokinesis
27_28insAC Val10fs 396 11 97.2
CAGL0A03872g Role in actin cortical patch assembly 1405_1406insAG Pro469fs 510 508 0.4
CAGL0A04213g Protein involved in G2/M phase progression and response to DNA damage
97_98insAA Pro33fs 639 59 90.8
CAGL0B00572g Protein of unknown function, without a known orthologue
580G>T Glu194* 202 194 4.0
CAGL0B03509g Role in protein phosphorylation, regulation of meiosis, regulation of mitosis, stress-activated protein kinase signalling cascade
1325_1326delCTinsG Pro442fs 597 488 18.3
CAGL0C00209g (CgAWP7)
Putative adhesin-like cell wall protein - - 437 - -
CAGL0C00253g Putative cell wall adhesin - - 1608 - -
CAGL0C01265g (CgGLM4)
FG-nucleoporin component of central core of the nuclear pore complex
3498C>A Cys1166* 1501 1166 22.3
CAGL0C01683g ATPase subunit of imitation-switch (SWI) class chromatin remodelers
L58_59delTG Cys20fs 1115 22 98.0
CAGL0C02343g ATPase activity, role in cellular response to drug and ribosomal small subunit export from nucleus
275T>A Leu92* 720 92 87.2
66
CAGL0C05401g Protein of unknown function, without a known orthologue
123_124insTT; 137_138insT;
145delC; 155T>G
Met42fs; Gly46fs; Gln49fs;
Leu52* 87 52 40.2
CAGL0D00374g Protein of unknown function, without a known orthologue
189T>G; 268C>T Tyr63*; Gln90* 103 63 38.8
CAGL0D01254g Protein of unknown function, without a known orthologue
172_173insCA; Ala58fs 227 71 68.7
CAGL0E00847g
ARF guanyl-nucleotide exchange factor activity and role in ER to Golgi vesicle-mediated transport, autophagic vacuole assembly, cellular response to drug, intra-Golgi vesicle-mediated transport
150_151insAACAA Asp51fs 1821 77 95.8
CAGL0E05324g Role in mitochondrial DNA replication and mitochondrion localization
63_64insT Leu22fs 1284 41 96.8
CAGL0F04543g Protein of unknown function, without a known orthologue
41_42insT Pro14fs 91 22 75.8
CAGL0F07095g Role in mRNA polyadenylation. 386_387insGC Gln129fs 531 198 62.7
CAGL0F01837g Protein with TBP-class protein binding, transcription cofactor activity
83_84insT Glu28fs 600 34 94.3
CAGL0G00924g (CgGLM5)
Required for biogenesis of the small ribosomal subunit, has a possible role in the osmoregulatory glycerol response
1143_1147delTGATG Asp381fs 1196 482 59.7
CAGL0G03949g Protein of unknown function, without a known orthologue
252T>G Tyr84* 167 84 49.7
CAGL0G03993g Protein of unknown function, without a known orthologue
255delT His85fs 117 88 24.8
CAGL0G06446g Protein of unknown function, without a known orthologue
172_173insTG Tyr57fs 108 67 38.0
CAGL0G07040g Dolichyl-diphosphooligosaccharide-protein glycotransferase activity, role in protein N-linked glycosylation via asparagine
905_906insC Phe302fs 331 323 2.4
CAGL0G07183g.2
Putative retrotransposon TYA Gag and TYB Pol genes
4159A>T; T2729G>A Lys1387*; Trp910*
1504 910 39.5
CAGL0G07293g Protein complex scaffold activity, role in ER-associated protein catabolic process, mRNA splicing, via spliceosome, positive regulation of protein
2723_2724insGC; 2809C>T
Gln908fs; Gln937*
942 924 1.9
67
oligomerization and Hrd1p ubiquitin ligase ERAD-L complex localization
CAGL0G09108g Protein of unknown function 643_644delAC Thr215fs 346 236 31.8
CAGL0H02783g Putative core subunit of the RENT complex, involved in nucleolar silencing and telophase exit.
2954delC Ala985fs 1785 1022 42.7
CAGL0H03751g Role in positive regulation of exit from mitosis and nucleus localization
291_292delGC Gln97fs 1021 101 90.1
CAGL0H04697g RNA polymerase I transcription factor binding 167delTinsGC Leu56fs 1010 101 90.0
CAGL0H06435g Protein of unknown function with mitochondrion localization
292_293insCTCGGGAA; 298G>T
Thr98fs; Gly100* 103 100 2.9
CAGL0H08756g Protein of unknown function, without a known orthologue
102delT Ser34fs 82 72 12.2
CAGL0I00396g Protein of unknown function, without a known orthologue
54_55delCT Ser19fs 115 28 75.7
CAGL0I02794g Protein of unknown function 330_331delCCinsAT Tyr110_Leu111
delins*Leu 252 110 56.3
CAGL0I02860g Role in tRNA export from nucleus and cytoplasm 1793_1794insTACAC
CAG Ser598fs 676 662 2.1
CAGL0I03432g Component of the heterotetrameric MHF histone-fold complex
96G>A; 115A>T Trp32*; Lys39* 149 32 78.5
CAGL0I04378g Protein with mannosylphosphate transferase activity, role in cell wall mannoprotein biosynthetic process. ScKTR2
415delA; 1065delA Ile139fs; Leu355fs
424 154 63.7
CAGL0I07051g Protein with cyclin-dependent protein serine/threonine kinase regulator activity, role in positive regulation of macroautophagy
797_798insAC Leu266fs 448 312 30.4
CAGL0H08217g
Protein with ubiquitin binding activity, role in mitotic DNA integrity checkpoint and condensed nuclear chromosome kinetochore. Orthologue of ScBUB3 that when deleted leads to an increase in azole resistance.
382A>T Lys128STOP 325 128 60.6
CAGL0I08591g Protein of unknown function 422_423insCA Gln141fs 517 144 72.1
68
CAGL0I09746g Involved in ER to Golgi vesicle-mediated transport Orthologue of ScSLY41 that when deleted leads to an increase in azole resistance.
964_965insTG Ala322fs 463 335 27.6
CAGL0J00253g Putative adhesin-like protein - - 497 - -
CAGL0J00825g Protein with GTPase activator activity involved in vesicle mediated protein transport
216_217insGA Glu73fs 437 122 72.1
CAGL0J01507g Protein with a role in attachment of spindle microtubules to kinetochore
2624_2625insTA His875fs 1267 891 29.7
CAGL0J02046g Protein of unknown function, without a known orthologue
51delCinsAT Phe17fs 79 26 67.1
CAGL0J02128g Protein of unknown function 8G>A Trp3* 94 3 96.8
CAGL0J02552g Adhesin-like protein; predicted GPI anchor - - 895 - -
CAGL0J03982g Protein of unknown function, without a known orthologue
260delAinsCC Tyr87fs 115 111 3.5
CAGL0J04114g Protein with dicarboxylic acid transmembrane transporter activity and role in mitochondrial transport.
674delG Ser225fs 303 227 25.1
CAGL0J09702g Protein with role in fungal-type cell wall organization, positive regulation of signal transduction and mitochondrion localization
2045delA; 1879_1880insC;
130_131insGTGCG
His682fs; Gly627fs; Lys44fs
695 56 91.9
CAGL0K03641g
Subunit of the nuclear inner membrane Asi ubiquitin ligase complex. Asi complex targets both misfolded proteins and regulators of sterol biosynthesis for ubiquitin-mediated degradation
1414_1415insAT Asp472fs 597 489 18.1
CAGL0K04675g Protein of unknown function, without a known orthologue
244delC Arg82fs 144 86 40.3
CAGL0K07502g Protein of unknown function, without a known orthologue
294delA Thr98fs 213 107 49.8
CAGL0K08470g Protein of unknown function, without a known orthologue
139delC Leu47fs 59 48 18.6
SIR4 CAGL0K11396g
Protein involved in subtelomeric silencing and regulation of biofilm formation. Orthologue ScSIR4 when delected creates a fluconazole resistance phenotype
2563C>T Arg855* 1447 855 40.9
69
CAGL0K11484g Protein of unknown function, without a known orthologue
26T>A; 171T>G Leu9*; Tyr57* 107 9 91.6
CAGL0K12166g Protein with a role in Golgi to plasma membrane transport.
1506delA Gly502fs 764 524 31.4
CAGL0L03388g Protein of unknown function, without a known orthologue
476G>A; Tyr164fs Trp159*; Tyr164fs
204 159 22.1
CAGL0L04836g Protein of unknown function, without a known orthologue
17_18insG Ser6fs 64 54 15.6
CAGL0L05852g Structural constituent of nuclear pore activity. NUP49 369_370insGCAGGA
AATAACAC Ser124fs 504 240 52.4
CAGL0L12518g Protein of unknown function and without a known orthologue
- - 116 - -
CAGL0M00132g (CgEPA12)
EPA12 Putative adhesin-like cell wall protein -
- 922 - -
CAGL0M00792g Protein of unknown function, without a known orthologue
247delAinsGG Thr83fs 100 90 10.0
CAGL0M02629g Protein involved in assembly of iron-sulfur clusters 198_199insGGTGCC
AT Gly67fs 213 72 66.2
CAGL0M04543g Protein of unknown function, without a known orthologue
578_579delAT Asn193fs 280 195 30.4
CAGL0M05115g Protein of unknown function 1213C>T; 1291delA Arg405*; Arg431fs
433 405 6.5
CAGL0M05599g Covalently-bound cell wall protein of unknown function
493_494insCTTCCCAAGCT
Ser165fs 446 235 47.3
CAGL0M07744g Protein of unknown function, without a known orthologue
280delA; 247delG Met94fs; Glu83fs 234 90 61.5
CAGL0M10153g Protein with MAP kinase kinase kinase kinase activity 210_211insTC Asn71fs 867 103 88.1
CAGL0M10829g (CgSSK2)
Protein with MAP kinase kinase kinase activity 227_228delCT Ala76fs 1667 79 95.3
CAGL0M12452g Protein of unknown function, without a known orthologue
100G>T Gly34* 204 34 83.3
70
Annex F Table 20 - Proteins with size increased has result of frameshift mutations found in FFUL887. The nomenclature used for variation report is taken from
www.hgvs.org/mutnomen/.
Gene/ORF name
S. cerevisiae orthologue
Function
Nucleotide modification
found in FFUL887
Amino acid modification
found in FFUL887
Protein size
Premature STOP codon
position
CAGL0C01309g HOS4 Role in histone deacetylation, negative regulation of meiosis and Set3 complex localization
3216delA Thr1072fs 1110 1116
CAGL0F09273g Putative adhesin-like protein - - 154 -
CAGL0G01969g VHS3 Phosphopantothenoylcysteine decarboxylase activity, role in cellular monovalent inorganic cation homeostasis
1589_1590insGA Asp530fs 538 553
CAGL0G02871g Protein of unknown function, without a known orthologue 319_320insAT Ser107fs 107 118 CAGL0G07645g Protein of unknown function, without a known orthologue 259_260insGC Gln87fs 93 109 CAGL0J00869g RPC25 Protein with role in tRNA transcription. 546_547delTC His182fs 216 224
71
Annex G Table 21 - Genes described to mediate fluconazole and/or voriconazole and caspofungin resistance in C. glabrata that were found to harbour mutant variations in the
FFUL887 isolate. The nomenclature used for variation report is taken from www.hgvs.org/mutnomen/.
Gene/ORF name
S. cerevisiae orthologue
Function Amino acid modification
found in FFUL887
CAGL0A04565g (CgSWI4) SWI4 DNA binding component of the SBF complex (Swi4p-Swi6p) Asn300Ile; Asn669Ser; Val324Ala;
Arg715Lys
CAGL0B02211g (CgCCH1) CCH1 Putative subunit of a plasma membrane gated channel involved in Ca2+ uptake (HACS)
Met1Ile; Arg51Pro Glu1912Lys
CAGL0C04048g MNT3 Protein with predicted role in protein glycosylation Ser36Thr
CAGL0E02475g (CgSIN3) SIN3 Component of both the Rpd3S and Rpd3L histone deacetylase complexes
Asn50Lys;Lys288Thr
CAGL0H01287g SSD1 Protein with mRNA 5'-UTR binding and translation repressor activity Pro90Ala
CAGL0M03597g (CgMID1) MID1 Putative regulatory subunit of a plasma membrane gated channel involved in Ca2+ uptake (HACS)
Gly77Asp
72
Annex H Table 22 - Genes described to mediate fluconazole and/or voriconazole resistance in C. glabrata that were found to harbour mutant variations in the FFUL887 isolate.
The nomenclature used for variation report is taken from www.hgvs.org/mutnomen/.
Gene/ORF name
S. cerevisiae orthologue
Function Amino acid modification found in FFUL887
CAGL0A00451g (CgPDR1)
PDR1 Zinc finger transcription factor, activator of drug resistance genes A; B; C; X
CAGL0A01452g CWH41 Predicted glucosidase I Thr176Ser; Asn812Ser; Met30Ile
CAGL0A04785g (CgRTG2)
RTG2 Non-DNA binding transcription factor Met537Ile
CAGL0B01353g (CgSPT20)
ST20 Subunit of the SAGA transcriptional regulatory complex Ser35Tyr
CAGL0B03421g Predicted zinc finger transcription factor Met139Val; Ala274Thr; Ala306Thr; Asn321Asp
CAGL0B04631g (CgINP53)
INP53 Protein with polyphosphatidylinositol phosphatase involved in trans Golgi network-to-early endosome pathway
Asn943Asp; Asn1063Thr
CAGL0B04741g (Cg PGS1)
PGS1 Phosphatidylglycerolphosphate synthase Glu458Asp; Asn484Ser
CAGL0C01199g (CgUPC2A)
UPC2 Zinc finger transcription factor required for transcriptional regulation of genes involved in uptake and biosynthesis of ergosterol
Arg92Lys; Asn304Ser; Glu822Val
CAGL0C03509g FPK1 Protein with serine/threonine kinase activity and role in positive
regulation of phospholipid translocation, protein autophosphorylation
Val117Ala; Val298Gly; Asn356Thr
CAGL0C05357g (CgHST1)
HST1 Histone deacetylase Met70Lys
CAGL0E00627g (CgSRB8)
SRB8 Subunit of the RNA polymerase II mediator complex Tyr399Cys; His1051Asn; Gln1322His
CAGL0E05060g (CgNUT1)
NUT1 Component of the RNA polymerase II mediator complex Asp247Glu; Ile537Val; Met540Val
CAGL0F02717g (CgPDH1)
PDR15 Multidrug transporter of ABC-superfamily Lys438Gln; Glu839
CAGL0F02519g YJL206C Predicted zinc finger transcription factor Ser191Asn; Gly298Asp; Gly719Asp; Met731Ile;
Ala769Thr
73
CAGL0F03025g ARO80 Predicted zinc finger transcription factor orthologue of S.
cerevisiae ARO80 involved ranscription of aromatic amino acid catabolic genes
Pro812Leu; Thr818Ser
CAGL0F06347g (CgSTV7)
STV7 Vacuolar proton-transporting V-type ATPase Ser391Pro
CAGL0F06743g DAL81 Zinc finger transcription factor, ortholog of S. cerevisiae DAL81
involved in positive regulation of genes in multiple nitrogen degradation pathways
Ile499Val
CAGL0F08129g (CgSDA1)
SDA1 Protein required for actin organization and passage through Start Ala244Val; Arg512Lys
CAGL0I02552g (SgSTB5)
STB5 Zinc finger transcription factor involved in regulating multidrug resistance and oxidative stress response
Val390Ile
CAGL0I06512g (CgBEM2)
BEM2 Protein with GTPase activator activity with role in actin cytoskeleton organization and negative regulation of Rho protein signal transduction
Ala139Thr; Leu169Met; Thr171Ala; Ser240Asn; Thr405Ser; Lys461Arg; Leu879His; Phe1347Ser
CAGL0F09229g TOG1 zinc finger transcription factor, orthologue of S. cerevisiae TOG1
activator of oleate genes Val517Leu
CAGL0G00286g (CgGAS1)
GAS1 Predicted glycoside hydrolase of the Gas/Phr family Ile10Thr
CAGL0G08624g (CgQDR2)
QDR1 Drug:H+ antiporter of the Major Facilitator Superfamily Asn38Ile; Ala69Thr; Ser212Ala; Ile255Phe; Arg304His;Leu307Ile Leu309Ile; Asn417Asp
CAGL0G09757g YLR278C Predicted zinc finger transcription factor Met1310Ile; Thr949Pro; Pro544His
CAGL0H03377g (CgDBP3)
DBP3 Protein with ATP-dependent RNA helicase activity Asp41Glu; Asp44Glu; Asn58Lys; Asp59Glu; Lys61Asn; Asn143Lys; Asp229Glu
CAGL0J07150g PIP2 zinc finger transcription factor, orthologue of S. cerevisiae PIP2
oleate-activated transcription factor Ile223Met
CAGL0J10956g (CgSUM1)
SUM1 Transcriptional repressor involved in chromatin silencing Asn284Asp; Ser307Gly; Val478Ile; Ser531Thr
CAGL0K03817g (CgPOM152)
POM152 Protein with anchor activity and role in nuclear pore organization Phe197Leu; Asp1079Asn
CAGL0K05379g (CgVMA13)
VMA13 Part of the electrogenic proton pump found throughout the endomembrane system
Thr174Ala; His244Gln
CAGL0K05841g (CgHAP1)
HAP1 Zinc finger transcription factor, orthologue of S. cerevisiae HAP1 involved in the complex regulation of gene expression in response to levels of heme and oxygen
Ala364Val; Asn378Thr; Ala676Gly; Ser743Ala; Ser894Ala; Gly1219Val
74
CAGL0K11902g LYS14 Zinc finger transcription factor, orthologue of S. cerevisiae LYS14
involved in regulating lysine biosynthesis Cys256Phe; Pro274Gln; Leu642Pro
CAGL0L03377g
Predicted zinc finger transcription factor Gly134Asp; Gly172Ser; Lys252Arg; Ile347Met; Ala695Gly; Lys813Arg; Asp819Glu; Thr867Met
CAGL0L09383g SUT1 Predicted zinc finger transcription factor Ser116Asn; Ile185Val; Met202Val
CAGL0L12386g (CgSUV3)
SUV3 ATP-dependent RNA helicase with a role in maturation of cytochrome c oxidase subunit transcripts
Ser94Cys; Ala248Thr; Asn273Asp; Ile597Leu
CAGL0L13112g (CgRGR1)
RGR1 Subunit of the RNA polymerase II mediator complex Met401Lys
CAGL0M00748g (CgECM7)
ECM7 Predicted integral membrane protein required for high-affinity Ca2+ influx (HACS)
Lys394Arg; Asn356Thr; Val298Gly
CAGL0M01518g (CgSHE9)
SHE9 Mitochondrial inner membrane protein of unknown function Ile355Thr
CAGL0M02651g RDS2 Zinc finger transcription factor, orthologue of S. cerevisiae RDS2
involved in regulating gluconeogenesis Met63Ile; Met159Leu; Asp205Glu
CAGL0L04400g YRR1
Zinc finger transcription factor involved in transcriptional regulation of MDR genes
Cys24Gly; Ala58Val; Ile137Leu; Asp229Glu; Ile346Val; Glu574Lys;
Ile593Leu; Glu710Asp; Ala933Val
CAGL0M07381g KTR6 Protein with mannosylphosphate transferase activity, role in
fungal-type cell wall organization Leu60Phe; Val417Ile
CAGL0H06215g (CgGAl11A)
GAL11 Component of the transcriptional Mediator complex that provides interfaces between RNA polymerase II and upstream activator proteins
Ser134Asn; Ser965Gly; Ser1084Asn
CAGL0B03421g Predicted zinc finger transcription factor Met139Val; Ala274Thr; Ala306Thr; Asn321Asp
75
Annex I
Table 23 - Genes described to mediate caspofungin resistance in C. glabrata that were found to harbour mutant variations in the FFUL887 isolate. The nomenclature
used for variation report is taken from www.hgvs.org/mutnomen/.
Gene/ORF
name
S. cerevisiae orthologue
Function Amino acid modification found in
FFUL887
CAGL0A01892g Protein of unknown function His11Asn; Gly14Arg
CAGL0B00858g STE50 Adaptor protein for various signalling pathways Glu40Asp
CAGL0B01166g SWI6 Transcription cofactor component of the SBF complex (Swi4p-Swi6p) Glu100Asp
CAGL0B01441g (CgRPD3)
RPD3 Histone deacetylase, component of both the Rpd3S and Rpd3L complexes
Thr414Ala
CAGL0B01947g (CgINO2)
INO2 Transcriptional regulator involved in de novo inositol biosynthesis Tyr131Asn; Tyr132His; Ser186Tyr
CAGL0B01991g SWF1 Protein with palmitoyltransferase activity and role in ascospore wall
assembly, cortical actin cytoskeleton organization and establishment of cell polarity
Met93Thr
CAGL0C05335g RTG1 Transcription factor (bHLH) involved in interorganelle communication Thr100Ser
CAGL0D01364g CYC8 General transcriptional co-repressor Asn761Lys; Lys769Glu; Glu828Gln;
Thr863Ala; Ala955Val
CAGL0E02783g SLA1 Cytoskeletal protein binding protein, required for assembly of the
cortical actin cytoskeleton Asn718Asp
CAGL0E03894g (CgFPS2)
FPS2 Glycerol transporter Asn18Asp; His128Asn; Ala409Thr; Lys475Arg; Asp517Tyr
CAGL0E06028g ALG5 UDP-glucose:dolichyl-phosphate glucosyltransferase Gly53Ser; Ala20Val
CAGL0F01837g SPT8 Subunit of the SAGA transcriptional regulatory complex Glu28fs; Glu46Asp; Leu415Val
CAGL0F08283g GCN5 H3 histone acetyltransferase activity Gly60Glu; Thr93Ser; Ala122del; Thr129Ala; Met496Leu
CAGL0F08371g (CgTNA1)
TNA1 High-affinity nicotinic acid transporter Lys78Arg
CAGL0G01034g (CgFKS1)
FKS1 Putative 1,3-beta-glucan synthase component; functionally redundant with Fks2p
Gly14Ser
CAGL0G05896g DSE2 Putative adhesin-like protein -
76
CAGL0H05621g RLM1 Putative transcription factor with a predicted role in cell wall integrity Ala103Thr; Pro233Leu; Gln309Arg
CAGL0H06347g DEP1 Role in histone deacetylation involved in negative regulation of chromatin silencing at rDNA
Val111Ile; Gly147Asp; Arg162Gln; Glu193Val; Thr248Ser; Lys266Glu; Ser284Asn; Ser346Leu; Ser355Phe
CAGL0H06875g ARG81 Zinc finger transcription factor, orthologue of S. cerevisiae ARG81
involved in arginine-responsive genes Leu284Gln
CAGL0H07513g (CgIFA38)
IFA38 Predicted ketoreductase involved in production of very long chain fatty acid for sphingolipid biosynthesis
Ser284Phe
CAGL0H07711g DEP1 Subunit of RNAPII-associated chromatin remodeling Paf1 complex Arg122Lys
CAGL0I03498g Signal transducing MAP kinase kinase Ala28Thr
CAGL0I05236g (CgBCY1)
BCY1 cAMP dependent protein kinase Ala48Val; Asp94Glu; Glu287Asp
CAGL0I07513g STE7 Protein with tyrosine kinase activity Ala121Val; Val159Leu; Ser640Thr; Ile936Thr
CAGL0I10769g RTF1 Transcription factor involved in cell-type-specific transcription and
pheromone response Gln196Pro
CAGL0I09130g PTR3 Sensor of external amino acids Val237Leu; Ser385Leu
CAGL0J06072g CKB1 Protein with serine/threonine kinase activity Ser153Pro
CAGL0J07942g
Protein with predicted phospholipid binding activity Ile190Leu; Ala415Ser; Glu705Gly; Ile892Val; Ile1007Met; Asn1015Ser; Val1024Ala;
Arg1026Lys; Leu1046Phe; Phe1114Tyr; Thr28Ser; Glu395Asp; Asn417Tyr; Lys469Thr; Phe471Val; Ala710Thr;
Thr716Ser
CAGL0J08734g PMT3 Protein with dolichyl-phosphate-mannose-protein mannosyltransferase
activity Ala41Val; Met266Val; Val378Ile; Ile703Val
CAGL0J11506g CHS1 Protein with chitin synthase activity Asn115His; Asp588Tyr
CAGL0K00605g CDC6 ATP-binding protein required for DNA replication Arg117Lys; Val163Ala; Lys268Arg;
Arg380Lys
CAGL0K02673g (CgSTE20)
STE20 Putative signal transducing kinase of the PAK (p21-activated kinase) family
Val279Ile; Arg352Lys
CAGL0K04037g (CgFKS2)
FKS2 Putative 1,3-beta-glucan synthase component; functionally redundant with Fks1p
Thr926Pro
CAGL0K08690g YIR016W Putative protein of unknown function Arg183Lys
CAGL0K11231g MNN10 Protein have alpha-1,6-mannosyltransferase activity and role in ascospore formation
Asp17Glu
77
CAGL0G00440g LCB1 Protein with serine C-palmitoyltransferase activity, role in sphingolipid biosynthetic
Ser37Phe
CAGL0G00858g Protein of unknown function Ala30Val
CAGL0L02123g SNF5 Subunit of the SWI/SNF chromatin remodeling complex Ile115Asn; Pro930Ser; Lys975Asn; Tyr1004Ser; Thr1009Ala
CAGL0L03520g BCK1 Protein MAP kinase kinase kinase activity Ile22Met; Ala550Val
CAGL0G03597g SHO1 Transmembrane osmosensor for filamentous growth and HOG pathways
Thr224Ala
CAGL0L05632g PBS2 Protein with MAP kinase kinase activity Leu178Pro
CAGL0L06226g HEK2 Protein with role in intracellular mRNA localization Asn56Lys; Ala73Thr; Glu167Gln; Asn289Ser
CAGL0L07326g PBS2 Protein with kinase activity with role in DNA damage checkpoint Ile80Val; Ile163Val; His506Gln
CAGL0L11880g RPH1 JmjC domain-containing histone demethylase Asp56Glu; Glu60Asp; Asn304Asp;
Pro386Ala; Lys432Arg; Asp440Asn; Glu443Gly; Thr469Ser
CAGL0L12562g MET31 Protein with predicted nucleic acid binding activity Val17Ile; Val122Thr
CAGL0M00220g PMT4 Protein with dolichyl-phosphate-mannose-protein mannosyltransferase
activity Pro8Ser; Met526Val
CAGL0M00528g MNS1 Protein with mannosyl-oligosaccharide 1,2-alpha-mannosidase activity Ser412Asn
CAGL0M02585g SPP1 histone methyltransferase activity (H3-K4 specific) Ser141Pro
CAGL0M04169g KRE1 Putative cell wall protein with predicted role in cell wall biogenesis and
organization Gln116Pro
CAGL0M05643g YFH1 Protein with iron chaperone activity Ser29Pro
CAGL0M05841g KRE2 Protein with predicted mannosyltransferase activity, role in protein
glycosylation and membrane localization Thr402Lys
CAGL0M09361g PKC1 Protein serine/threonine kinase Thr70Pro; Val652Asp
CAGL0M09669g RIM3 Protein with calcium-dependent cysteine-type endopeptidase activity Ala63Ser; Leu244Phe
CAGL0M13739g ATM1 Mitochondrial inner membrane ATP-binding cassette (ABC) transporter Val507Ile; Ala118Ser
78
Annex J Table 24 - Expression of genes described to mediate fluconazole and/or voriconazole or caspofungin resistance in C. glabrata in FFUL887 and in CBS138 strains, as
revealed by microarray analysis.
Gene/ORF name S. cerevisiae orthologue
Function Fold Change, FUL887
vs CBS138
Fluconazole and/or Voriconazole Resistance
CAGL0A00451g (CgPDR1) PDR5 Zinc finger transcription factor, activator of drug resistance genes via pleiotropic drug response elements
1.96 ± 0.21
CAGL0C03872g (CgTIR3) TIR3 Putative GPI-linked cell wall protein involved in sterol uptake 1.80 ± 0.001
CAGL0E01353g Predicted zinc finger transcription factor 2.52 ± 0.67
CAGL0E06248g RCN1 Positive regulator of calcineurin 1.57 ± 0.13
CAGL0F02717g (CgPDH1) PDR15 Multidrug transporter of ABC-superfamily 3.21 ± 0.31
CAGL0F01419g (CgAUS1) AUS1 ATP-binding cassette transporter involved in sterol uptake 1.64 ± 0.07
CAGL0M01760g (CgCDR1) CDR1 Multidrug transporter of ABC-superfamily 4.61 ± 0.35
Caspofungin Resistance genes
CAGL0B01947g (CgINO2) INO2 Transcriptional regulator involved in de novo inositol biosynthesis 1.55 ± 0.18
CAGL0E02629g ALG6 Alpha 1,3 glucosyltransferase 1.66 ± 0.21
CAGL0M13739g ATM1 Mitochondrial inner membrane ATP-binding cassette (ABC) transporter 1.55 ± 0.13