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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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
ii
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.
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).
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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.
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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
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)
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
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].
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
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
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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.
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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
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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 - -
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
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
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
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
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
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
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