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Unveiling the mechanisms of evolution towards fluconazole resistance of a Candida glabrata clinical

isolate: a transcriptomics approach

Mafalda Banazol de Santa Rita Cavalheiro

Instituto Superior Técnico, Lisboa, Portugal

October 2015

Abstract

Candida glabrata clinical isolates are often found to exhibit azole antifungal drug resistance, hampering the success of therapeutic procedures. Further insights into the molecular mechanisms underlying the development of resistance towards individual azole drugs, as thus required.

In this work, a transcriptomics analysis of the evolution of a C. glabrata clinical isolate from azole susceptibility to posaconazole resistance (21st day), clotrimazole resistance (31st day) and fluconazole and voriconazole resistance (45th day), induced by longstanding incubation with fluconazole, was carried out. The acquisition of posaconazole resistance involved increased levels of expression of genes associated to transcription, translation and cell cycle, while the posazonazole/clotrimazole resistance development was triggered by an up regulation of CgERG11 gene and of adhesin encoding genes. Finally, the posaconazole/clotrimazole/fluconazole/voriconazole resistant strain, found to exhibit a GOF mutation in the CgPDR1 gene, displayed an up regulation of genes encoding multidrug resistance transporters and the eisosome component CgPil1. Along this evolution, C. glabrata cells displayed the ability to accumulate progressively lower levels of azole drugs, while their ergosterol concentration was found to change only to lower levels in the final strain. Based on the obtained global results, a deeper analysis of the role of adhesins and eisosomes in azole drug resistance was undertaken, leading to the characterization of the adhesin CgEpa3 and the eisosome component CgPil1 as new determinants of azole drug resistance.

Altogether, this study highlights the multifactorial nature of azole drug resistance acquisition and that new players have to be considered in this context.

Keywords: Candida glabrata, azole resistance, CgEpa3, CgPil1.

1. Introduction

C. glabrata is nowadays the second most common cause of candidiasis in immunocompromised patients. The most frequent type of the disease is the mucosae infection. When entering the bloodstream, C. glabrata can also lead to systemic infection, which has high mortality rates [1]–[3]. The treatment frequently fails due to the condition of the patient’s immune system, the antifungal agent characteristics and the development of resistance towards it [4]. The acquisition of

azole resistance has been reported to be linked to specific molecular mechanisms developed within C. glabrata clinical isolates. Among these mechanisms are the alterations in the sequence and expression of ERG11 gene, which codifies the target of azole drugs [5]–[7], and the upregulation of transporters from the ATP-binding cassette (ABC) superfamily [8], [9] and the major facilitator superfamily (MFS) [10], [11]. According to some studies, the upregulation of certain transporters seems to be present for the development of resistant to one specific azole and not others, like the case of the CgQdr2 transporter important for the

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development of clotrimazole resistance but not for fluconazole, in C. glabrata [10].

Another molecular mechanism known corresponds to a specific phenotype called petite, which characteristics are absence of growth on non-fermentable carbon sources, deficient growth in media supplemented with glucose, reduced oxygen consumption and partial or total mtDNA deletion [3], [12]. This phenotype promotes azole resistance, since the biosynthesis of P-450-dependent 14α-sterol demethylase, codified by ERG11 gene, is stimulated by anaerobic conditions [13]. Finally, some isolates of C. glabrata have shown the capacity of sterol uptake, another mechanism which enables the overcome of the blockage of ergosterol biosynthesis caused by azole action [5], [14], [15].

Azole resistance is achieved in most cases by a few different molecular mechanisms, alone or in combination. However, some of the described mechanisms work specifically against certain azoles and not others. The challenge is to distinguish mechanisms that are specific to a given azole from those that are general for most azoles. Thereby, specific therapeutics could be developed for the eradication of Candida infections exhibiting specific patterns of azole resistance.

Based on the results described in this study, two new players in azole drug resistance were unravelled: adhesins and eisosomes.

Adhesins are cell wall proteins which bind to amino acid or sugar residues on the surface of other cells. Cell adhesion is their main function although they are also related to cell shape maintenance, limiting permeability, hydrophobicity, cell wall biosynthesis and remodelling, biofilm formation and antigenicity [16], [17].

The major group of adhesin-like proteins belongs to the EPA family. The CgEpa1,Cg Epa6 and CgEpa7 adhesins have been shown to be involved in kidney colonization of C. glabrata, while CgEpa3, has been reported as important in biofilm formation [18]–[20]. This gives evidence of the role of this family of adhesins in C. glabrata virulence.

On the other hand, eisosomes are assemblies of different protein components localized in specific membrane domains rich in ergosterol, called membrane compartment of Can1 (MCC) [21]. The major components of eisosomes are Pil1, Lsp1 and Seg1. Pil1 is necessary for the eisosome’s biogenesis [22], and Seg1 is needed for the assembly of the components, determining the length of the eisosome [23]. Pil1 and Lsp1 components have Bin/amphiphysin/Rvs (BAR) domains, which allow the formation of filaments and

binding of membranes [21]. Although eisosome’s function is not yet clear, there several clues for the importance of this assemblies. First of all, they are connected with membrane domains rich in ergosterol promoting membrane integrity. Secondly, eisosomes have been linked to endocytosis [24] and seem to be involved in osmotic shock and dehydration [25]. More specifically, Pil1 is involved in biofilm formation in Candida albicans [26], while Sur7, another component of eisosomes, has a role in cell wall synthesis, decreased virulence in a mouse model of systemic candidiasis and defective in endocytosis, also in the same organism [27], [28].

2. Background

C. glabrata azole resistance is developed based on different molecular mechanisms that may be specific to a given azole but that can also work together to achieve resistance. The difficulty resides in discovering which specific mechanisms are responsible for a given azole resistance, in order to improve therapeutic approaches for candidiasis.

The evolution of a particular C. glabrata clinical isolate (044) from azole susceptibility to posaconazole resistance at the 21st day (044Fluco21 strain), clotrimazole resistance at the 31st day (044Fluco31 strain) and fluconazole and voriconazole resistance at the 45th day (044Fluco45 strain), induced by longstanding incubation with constant concentration of fluconazole, was performed at Centro Hospitalar S. João, in Porto, by the team led by Prof. Acácio Rodrigues. The acquisition of fluconazole and voriconazole resistance was accompanied by a gain-of-function (GOF) mutation in CgPDR1 gene.

This particular case study was undertaken herein, in order to unravel the mechanisms responsible for the acquisition of each azole resistance. Therefore, a transcriptomics analysis was performed, comparing the three evolved strains obtained from each acquisition of azole resistance, with the initial 044 clinical isolate. Based on the results, several paths were pursuit in order to specify the molecular mechanisms involved in azole resistance.

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3. Experimental Procedures

3.1. Cell Cultures 3.1.1. Candida glabrata strains

The C. glabrata strains used in this master

this were the 044 clinical isolate and the

evolved strains 044Fluco21, 044Fluco31 and

044Fluco45, kindly provided by the team of

Prof. Acácio Rodrigues, Centro Hospitalar of S.

João, Porto. And also the strain KUE100 wild-

type, and deletion mutants ΔCAGL0G05093g,

Δcgpil1, Δcglsp1, Δcgseg1, Δcgepa1,

Δcgepa3, Δcgepa10, kindly provided by Prof.

Hiroji Chibana, Medical Mycology Research

Center, Chiba University, Chipa, Japan.

3.1.2. Growth media

Cells were batch-cultured at 30ºC with

orbital agitation (250 rpm) in different growth

media according to the following protocols. The

yeast extract peptone dextrose (YEPD) growth

media was used as a rich medium, with the

following composition (per liter): 20 g glucose

(Merck), 20 g yeast extract (Difco) and 10 g

bacterial-peptone (LioChem). The minimal

growth medium (MMG) used contained per

liter: 20 g glucose (Merck); 2.7 g (NH4)2SO4

(Merck); 1.7 g yeast nitrogen base without

amino acids or (NH4)2SO4 (Difco). The Roswell

Park Memorial Institute (RPMI) 1640 medium

at pH of 4 was used with 18 g glucose (Merck);

10.4 g RPMI-1640 (Sigma); 34.53 g MOPS

(Sigma) per liter. Also used was Sabouraud’s

dextrose broth (SDB) containing 40 g glucose

(Merck) and 10 g peptone (LioChem) per liter.

1.1.1. Antifungal drugs

The stock solutions of antifungal drugs used

in the studies present herein, were obtained

from Sigma and dissolved in Dimethyl

Sulfoxide (DMSO) also from Sigma. The stock

solutions were prepared in order to have the

concentrations present in Table 2.

1.2. Transcriptomic analysis

1.2.1. RNA extraction

The pre-inoculum for the 044 clinical isolate

was grown overnight, following the preparation

of the inoculum, which was incubated for 5 h in

order to obtain the final optical density (OD) of

0.8. The cells were recovered using the J2-MC

centrifuge, Beckman, following their

resuspension in 1.5 mL of medium. Another

centrifugation was performed in eppendorfs, in

the Scanspeed mini centrifuge, Labogene. The

supernatant was removed and the cells were

frozen at -80ºC.

RNA extraction was performed using the

RiboPure™ RNA Isolation Kit (Ambion, Life

Technologies, California, USA). The recovered

cell pellet was resuspended in 480 μL of lysis

buffer. After this 48 μl of SDS and 480 μl of a

mixture of Phenol:Chloroform:IAA were added

to the mixture and the suspension was

transferred to one of tubes containing 750 μL

cold Zirconia Beads prepared previously. The

sample tubes were horizontally positioned on

the vortex. The vortex was set at maximum

speed, for 10 min. The lysate obtained from

this disruption process was centrifuged for

5 min at 16,100 g at room temperature, in order

to separate the aqueous phase, containing the

RNA, from the organic phase. The aqueous

phase was collected and added to 1.90 ml of

Binding Buffer and 1.25 mL of cold 100%

Ethanol. The total volume was centrifuged

through a filter cartridge and washed with

700 μL of Wash Solution 1. After new

centrifugation, the filter was washed two times

with 500 μL of Wash Solution 2/3 followed by

an extra min to ensure the complete removal of

wash solution. Total RNA obtained was eluted

in two times 50 μL of Elution Solution,

previously heated at 95°C. The isolated RNA

was treated with DNase I to remove traces of

chromosomal DNA. Therefore, 100 μl RNA

sample (50 μl + 50 μl) was added to 8 U of

DNase I and 10 μl of 10X DNase I Buffer. The

mixture was incubated at 37°C during 30 min.

After this incubation, 10 μl of DNase

Inactivation Reagent were added to the

mixture, which was then vortexed and left for 5

min at room temperature. The purified RNA (in

the supernatant fraction) was collected into a

fresh tube by centrifugation (>10000 rpm,

3 min).

1.2.2. Microarray data analysis

At each fluconazole induction time for which

the C. glabrata clinical isolate acquired

resistance to each azole drug, the RNA

extraction was performed and a

transcriptomics analysis was assessed in

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collaboration with Geraldine Butler, from

University College Dublin. Therefore, a sample

of RNA was obtained at the 21st day of

fluconazole induction corresponding to the

gained of posaconazole resistance, following a

sample at the 31st day of fluconazole induction,

concerning clotrimazole resistance, and finally,

a RNA sample at the 45th day was performed,

for which the isolate had already acquired

fluconazole and voriconazole resistance. The

microarray analysis was conducted for these 3

RNA samples and each sample was analysed

in comparison with a RNA sample from the

initial susceptible isolate (control). In Table 1

are represented by letters the several samples

and respective controls considered in the

microarray analysis performed for each

acquired resistance.

Table 1. RNA samples and respective controls of C.

glabrata 044 clinical isolate considered in the microarrays

analysis relative to each resistance acquisition of the

clinical isolate. Respective fluconazole induction time

(days).

Clinical

Isolate Azole

Resistance

Induction

Time

(days)

Samples Controls

Posaconazole 21 D, E, F A1, B1,

C1

Clotrimazole 31 G, H A2, B2

Fluconazole

and

Voriconazole

45 J, K, L A3, B3,

C3

The microarray data was analysed for each

resistance case, selecting the up regulated and

down regulated genes according to two criteria.

The first criterion was based on the p-value of

each hybridization, which was established as

being ≤ 0.05. The second criterion established

that the logarithm to base 2 of the fold change

of the two probes for each gene could not differ

more than 30%.

With the selection performed, the several

up regulated and down regulated genes of

each time of induction were submitted to

several analysis using different databases and

bioinformatic tools.

1.2.2.1. Expression profiles analysis

From all the up regulated and down

regulated genes selected previously, the

genes were grouped according to the cellular

functions in which they belong. From this

organization, the genes concerning multiple

drug resistance (MDR), cell wall and lipid

metabolism were selected in order to

development an evaluation of their expression

profiles over the time of induction, considering

the fold change of each gene, at the each

specific acquisition of azole resistance.

1.3. 3H-clotrimazole accumulation assays

In order to assess whether azole drug resistance was related to an efficient export of azole drugs, several strains in study were evaluated regarding the intracellular accumulation of 3H-clotrimazole. The accumulation assays were started by growing the cells in MMG medium at 30ºC, with an orbital agitation of 250 rpm. Achieving an O.D.600nm of 1±0.08, cells were harvested by filtration and resuspended in MMG medium with vortex, obtaining an O.D.600nm of 0.7. Afterwards, 30 mg/L of cold clotrimazole (Sigma) and 0.1 µM 3H-clotrimazole (Moravek Biochemicals) were added to the cellular suspension, which was incubated at 30ºC for a period of 30 min with orbital agitation of 180 rpm. In this period, measurements of the intracellular and extracellular concentrations of the 3H-clotrimazole drug were performed at certain time-points.

For the evaluation of intracellular accumulation of 3H-clotrimazole, 200 µL of the cellular suspension were collected by filtration using pre-wetted glass microfibers (Whatman, GF/C) at each time-point. Each filter was washed with 10 mL of ice cold TM buffer at pH 4.5 and immersed in 7 mL of scintillation liquid (Beckman).

In the case of extracellular accumulation of 3H-clotrimazole, 70 µL of the cellular suspension were collected at each time-point and centrifuged at 13200 rpm for 1 min. The supernatant was harvested and immersed in 7 mL of scintillation liquid (Beckman).

The radioactivity for each sample of intracellular and extracellular accumulation, at each time-point, was measured in the Beckman LS 5000TD scintillation counter.

1.4. Quantification of Biofilm Formation

The crystal-violet method [29] was used to study the capacity of biofilm formation in the C. glabrata strains. Cells were grown in SDB medium and collected by centrifugation at mid-exponential phase. Cells were then inoculated in 96-well polystyrene titter plates (Greiner), which were previously filled with the appropriated medium, SDB or RPMI at pH 4, in

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order to have an initial OD600nm = 0.05±0.005. Afterwards, cells were cultivated at mild orbital shaking (70 rpm), for 15 h, at 30ºC.

Subsequently, each well was washed three times with 200 µL of deionized water to remove the cells that were not attached to the formed biofilm. After a washing step, 200 µL of 1% crystal-violet (Merk) alcoholic solution was added in each well in order to stain the formed biofilm (15 min of incubation). Then, each well was washed with 250 µL of deionized water. Finally, in each well 200 µL of 96% (v/v) ethanol was added, to elute the stained biofilm, following absorbance reading in a microplate reader at the wavelength of 590 nm (SPECTROstar Nano, BMG Labtech).

1.5. Quantification of total cellular ergosterol

Total ergosterol content was extracted from C. glabrata cells using the method of physical disruption [30] with some adjustments. Cells were cultivated in 100 mL of YEPD and with an orbital agitation of 250 rpm until stationary phase was reached. Cells were harvested by centrifugation and ressuspended in 5 mL of methanol. Colesterol, used as an internal standard to allow quantification of the yield of ergosterol extraction, was added in order to have a final concentration of 1.25 mg/mL in each sample. Afterwards, glass beads were added approximately in the same weight as the cell pellet. Then, each sample was homogenized in 30 sec, following an orbital agitation of 320 rpm for 1 h. The samples were centrifuged at 8000 rpm for 7 min at 4ºC. 1.7 mL of supernatant was extracted to an eppendorf, following another centrifugation at 11000 rpm for 10 min at 4ºC. 1 mL of the supernatant was then collected and stored until analysis.

The extracts obtained were analysed by High Pressure Liquid Chromatography with a 250 mm x 4 mm C18 column (LiChroCART Purospher STAR RP-18 end- capped 5 mm) at 30ºC. The samples were eluted in 100% methanol at a flow rate of 1 mL methanol per min. Colesterol was detected at 210 nm corresponding to a retention time of 13.77±0.67 min. Ergosterol was detected at 282 nm with a retention time of 11.33±0.18 min.

The corresponding results are presented as the ratio between the average concentration of ergosterol of the KUE100 strain or the clinical isolate, according to each case, and the concentration of the other samples tested.

1.6. Susceptibility Assays The susceptibility of the 044 clinical isolate,

the evolved strains, the wild-type KUE100 and derived mutants: Δpil1, Δlsp1, Δseg1, Δepa1, Δepa3, Δepa10 and ΔCAGL0G05093g was assessed to all antifungal drugs prepared by spot assays. The cell suspensions of the clinical strains were batch-cultured at 30ºC with orbital agitation (250 rpm) in MMG liquid medium until the standardized culture OD600nm

of 0.6±0.05 was reached in the absence of antifungal drugs. Afterwards, the cell suspensions were diluted to a standardized OD600nm of 0.05±0.005 in 1 mL of deionised water, following sequential dilutions of 1:5 and 1:25. The cellular suspensions were applied as spots (4 μL) onto the surface of the agarized MMG medium plates, supplemented with adequate concentration of the antifungal drugs tested. Each plate was prepared adding a precise volume of the stock solution to 25 mL of MMG medium, in order to have the desired concentration. The range of drug concentrations tested, as well as the concentration of the stock solutions used are present in table 2.

Table 2. Range of drug concentrations used in the

spot assays.

Antifungal Drug

Concentration of the Stock

Solution

Range of the drug

concentration (µg/mL)

Amphotericin B 10mg/10mL 1-1.5

5-Flucytosine 10mg/10mL 0.01

Clotrimazole 10mg/250µL 25-45

Miconazole 10mg/10mL 0.65-0.75

Tioconazole 10mg/10mL 0.5-2

Fluconazole 10mg/mL 200-220

Ketoconazole 10mg/mL 60-100

Following the inoculation, the plates were

incubated at 30ºC for 2-5 days.

4. Results and Discussion

4.1. Stepwise evolution of the 044 clinical isolate to fluconazole resistance

Following the initial study described in the Background section, susceptibility assays revealed that the 044 clinical isolate was more susceptible to fluconazole, clotrimazole, ketoconazole, miconazole and tioconazole, when compared to the evolved strains. Also,

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the 044Fluco45 strain, was found to exhibit slightly higher resistance to amphotericin B, than the remaining strains, suggesting that in this case fluconazole exposure led to increased resistance to all the other tested azole drugs and also to cross-resistance against amphotericin B. Moreover, the 044 clinical isolate showed being more resistant to 5-flucytosine.

Along the directed evolution of this isolate, it appears to have progressively led to a stepwise increased resistance to each of these azoles. This is consistent with the MIC values obtained for clotrimazole and fluconazole.

4.2. Identification of transcriptome-

wide changes occurring in a clinical isolate of Candida glabrata, subject to fluconazole stress: general features

In order to understand the phenotypic changes undergone by the 044 C. glabrata clinical isolate leading to differential acquisition of resistance to four azoles, a transcriptomics analysis was performed in collaboration with Prof. Geraldine Butler, from University College Dublin. The transcriptomes of cells collected at the 21st, 31st and 45th day of fluconazole induction, corresponding to cells exhibiting posaconazole, clotrimazole/posaconazole and fluconazole/voriconazole/clotrimazole/posaconazole resistance, respectively, were analysed by microarray hybridization. Each sample was analysed in comparison with the azole susceptible C. glabrata 044 clinical isolate (control), in triplicates. The obtained microarray data was analysed for each comparison (posaconazole resistant vs control, posaconazole/clotrimazole resistant vs control or posaconazole/clotrimazole/fluconazole/voriconazole resistant vs control), enabling the identification of the up and down regulated genes, and taking into consideration that for each gene, two expression level measurements were obtained, as the microarray design included two probes for each gene in the C. glabrata genome [31]. The total number of genes whose transcript levels was two-fold up or down regulated in the resistant strains when compared to the azole susceptible strain, with an associated p-value<0.05, are present in Table 3.

Table 3. Total number of up or down regulated genes in the resistant C. glabrata evolved strains, when compared to the azole susceptible one.

Clinical Isolate Azole

Resistance

Down

regulated

Genes

Up

regulated

genes

Posaconazole 355 299

Clotrimazole/ Posaconazole 73 199

Fluconazole/Voriconazole/

Clotrimazole/ Posaconazole 6 27

4.3. Decreased accumulation of azole drugs and ergosterol content during the evolution of the Candida glabrata 044 clinical isolate towards fluconazole resistance

A manual classification of the up or down regulated genes in each considered strain was performed. For this analysis, the up regulated and down regulated genes expressed in each acquisition of resistance were grouped according to the main cellular functions to which they belong. Afterwards, the analysis of the expression profiles of genes, with relevance in this study, was developed.

Figure 1. Multiple drug resistance genes expression profile of C. glabrata 044 clinical isolate with the respective fold change for each fluconazole induction time correspondent to the acquisition of posaconazole, clotrimazole and fluconazole and voriconazole resistance.

Focusing first on the genes involved in multidrug resistance (MDR) (Figure 1), the expression profiles show that the GAGL0M01760g, GAGL0M07293g, CAGL0F02717g and CAGL0E03674g genes, homologues to PDR5, PDR12, PDR15 and TPO1, respectively, from S. cerevisiae, involved in MDR, have a highest fold-change of expression at 45th day of induction. Interestingly, in a transcriptome analysis of C. glabrata oropharyngeal isolates exhibiting a GOF mutation in CgPdr1 transcription factor, which gained azole resistance when exposed to fluconazole, the homologues of PDR5 and

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PDR15 genes also exhibited an increase of expression consistent with the results present herein [32].

In general, it is possible to conclude that

the expression of transporters is mainly

observed for the 044Fluco45 strain, probably

helping the acquisition of azole resistance in

the 044 clinical isolate. Therefore, since most

of these transporters are involved in the export

of azole drugs, 3H-clotrimazole accumulation

assays were performed for the 044 clinical

isolate and the resistant strains 044Fluco21,

044Fluco31 and 044Fluco45, in order to

compare the capacity of each strain to

decrease the accumulation of azole drugs

(Figure 2).

Based on the time-course accumulation of 3H-clotrimazole, it is possible to conclude that C. glabrata 044 clinical isolate accumulates 5- to 10-fold more azole drugs than the evolved strains (Figure 2). After 21 days of fluconazole exposure, when only resistance to posaconazole had been reached, 3H-clotrimazole accumulation was already 5-fold lower in the evolved 044Fluco21 strain than that registered in the parental strain. Strains 044Fluco31 (resistant to posaconazole and clotrimazole) and 044Fluco45 (resistant to posaconazole, clotrimazole, fluconazole and voriconazole) displayed an even lower level of 3H-clotrimazole accumulation. These results show that azole resistant strains have indeed lower accumulation of these drugs, as expected. Nevertheless, since 044Fluco45 strain is the only one exhibiting higher expression of multidrug transporter encoding genes, it would be interesting to understand which molecular mechanisms allow decreased accumulation of azole drug in the remaining azole resistant strains.

According to the expression profiles studied

regarding the genes involved in the ergosterol biosynthesis, it is possible to observe an increase of expression of the CgERG11 gene present in the 044Fluco31 strain (Figure 3). The fold change of CgERG11 gene drastically decreases when fluconazole and voriconazole resistance is further acquired. Also, all the other ERG genes whose expression did not change significantly, seem to exhibit slightly decreased expression in the 044Fluco45 strain. Although not expected, one particular study describes a C. glabrata clinical isolate which is resistant to fluconazole, voriconazole and amphotericin B, and deficient in ergosterol content. This isolate exhibited a single-amino-acid substitution in CgERG11 gene, causing the loss of function of CgErg11 protein. When supplemented with cholesterol, this isolate exhibited sterol uptake, which allowed a higher resistance to the previous antifungal agents [5]. According to this report, the sterol uptake and alterations in membrane composition might be the key mechanisms allowing azole resistance in ergosterol deficient conditions. These observations are not in accordance with other reports where CgERG11 gene is described as up regulated in clinical isolates of C. glabrata when induced with fluconazole [6], [33], [34].

Since ergosterol content in yeast has been

linked to different domains on the membrane

which might confer increased membrane

stability and impermeability [27], [28], the total

ergosterol content along the temporal evolution

of the 044 clinical isolate was quantified, in

order to assess its relevance in acquisition of

azole resistance.

Figure 2. Time-course accumulation of 3H-clotrimazole in C. glabrata 044 clinical isolate (●) and evolved strains 044Fluco21 (■), 044Fluco31 (▲) and 044Fluco45 (▼). The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviations. *p<0.05; **p<0.01

Figure 3. Ergosterol biosynthesis genes expression profile of C. glabrata 044 clinical isolate with the respective fold change for each fluconazole induction time correspondent to the acquisition of posaconazole, clotrimazole and fluconazole and voriconazole resistance.

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The results only show a significant difference between the 044 clinical isolate and the 044Fluco45 strain, which exhibits a decreased ergosterol content when compared with the first. This is consistent with the expression profile regarding ergosterol biosynthesis, showing that the strain resistant to all four azoles in study has, indeed, less ergosterol content.

4.4. Screening for the possible role of

eisosomes in the acquisition of azole resistance in the Candida glabrata 044 clinical isolate

Among the genes found to be up regulated in the 044Fluco45 strain, when compared to the parental strain, is CgPIL1, encoding an essential component of eisosomes. The expression levels of the remaining constituents of eisosomes, encoded by CgLSP1 and CgSEG1 genes, suffered no alterations during the same period of fluconazole induction.

In order to assess the importance of these genes in antifungal resistance in C. glabrata, the susceptibility to antifungal drugs of the corresponding deletion mutants Δpil1, Δlsp1 and Δseg1, kindly provided by Professor Hiroji Chibana, was compared to that of the parental KUE100 wild-type strain. The results showed that CgPil1 is important for C. glabrata

resistance to clotrimazole, ketoconazole and tioconazole, while the other two components had no significant effect in azole drug resistance.

Therefore, the role of CgPil1 in 3H-clotrimazole accumulation was analysed (Figure 5), as well as, the ergosterol content in the deletion mutant Δpil1 (Figure 6).

The Δpil1 deletion mutant was observed to exhibit a higher accumulation of 3H-clotrimazole than the wild-type KUE100 strain (Figure 5). According to these results, the role of CgPIL1 gene in clotrimazole resistance may

Figure 4. Temporal evolution of ergosterol content in the C. glabrata 044 clinical isolate during azole resistance in vitro induction assay. The cells of the 044 clinical isolate and the evolved strains were harvested after 15h of growth in YPED medium, following the extraction and quantification through HPLC of total ergosterol. Relative ergosterol content was calculated using ergosterol content of the initial 044 clinical isolate as a reference. In the scatter dot plot represented each dot corresponds to ergosterol content (µg) per mg of cells harvested in each sample. The average of ergosterol content per cells is indicated by a black line (-) corresponds to at least 3

independent experiments. *p<0.05.

Figure 5. Time-course accumulation of 3H-clotrimazole in C. glabrata KUE100 (●) and Δpil1 (■). The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviations. *p<0.05; **p<0.01.

Figure 6. Ergosterol content in the C. glabrata KUE100 and the deletion mutant Δpil1. The cells were harvested after 15h of growth in YPED medium, following the extraction and quantification through HPLC of total ergosterol. Relative ergosterol content was calculated using ergosterol content of KUE100 as a reference. In the scatter dot plot represented each dot corresponds to ergosterol content (µg) per mg of cells harvested in each sample. The average of ergosterol content per cells is indicated by a black line (-) corresponds to at least 3 independent experiments. *p<0.05.

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correlate with its direct or indirect involvement in controlling the intracellular concentration of clotrimazole in C. glabrata.

A significant increase in ergosterol content was observed for the Δpil1 deletion mutant, when compared to the wild-type strain (Figure 6). According to this observation, the absence of CgPIL1 gene leads cells to increased ergosterol accumulation, while its overexpression in the 044Fluco45 strain appears to correlate with decreased ergosterol levels.

4.5. Screening for the possible role of

adhesins in the acquisition of azole resistance in the Candida glabrata 044 clinical isolate

Among the observations in the transcriptomics analysis, 11 adhesin encoding genes where found to be overexpressed in the 044Fluco31 clotrimazole/posaconazole resistant strain, when compared to the 044 parental strain. Interestingly, the expression levels of these genes dropped back to nearly basal values in the 044Fluco45 fluconazole resistant strain (Figure 7).

Since adhesins are responsible for the first step of biofilm formation, adhesion, the quantification of biofilm formation was assessed in the 044 clinical isolate and evolved strains.

Although no differences were found between the evolved strains and the clinical isolate, it is important to consider that several adhesins, which are overexpressed in 044Fluco31, are encoded by EPA genes which are involved in adherence to mammalian cells and not plastic structures [35], [36].

Afterwards, the cell-to-cell aggregation variability was assessed in the 044 clinical isolate and the evolved strains, by microscopic

observation. The number of aggregates was quantified considering that an aggregate is a set of 10 or more cells.

Figure 8. Scatter dot plot of percentage of aggregates in the temporal evolution of the C. glabrata clinical isolate 044 and evolved strains obtained after fluconazole induction at day 0 (clinical isolate 044), day 21 (044Fluco21 - posaconazole resistant), day 31 (044Fluco31 – clotrimazole resistant), day 45 (044Fluco45 – fluconazole and voriconazole). For this evaluation it was considered that an aggregate was equal to the aggregation of 10 cells. Standard deviations are represented by ****p<0.0001.

According to Figure 8, the 044Fluco31 strain has a significant higher number of aggregates when compared to the 044 clinical isolate. These observations are consistent with the transcriptomics data which shows an up regulation of the expression of several adhesion encoding genes in this strain.

Furthermore, the specific role of three

selected adhesins, CgEpa1, CgEpa3 and

CgEpa10, was studied, regarding azole

resistance. Susceptibility assays of the

deletion mutants Δepa1, Δepa3 and Δepa10,

kindly provided by Professor Hiroji Chibana,

Chiba University, Japan were performed. The

results give evidence that CgEpa3 has a role in

azole resistance in C. glabrata since it is

necessary for miconazole, tioconazole,

ketoconazole, clotrimazole, fluconazole,

itraconazole and 5-flucytosine resistance.

Given their natural role, the effect of the

deletion of CgEPA1, CgEPA3 or CgEPA10

genes in biofilm formation was also analysed,

in a polystyrene surface (Figure 9).

Figure 7. Adhesins expression profile of C. glabrata clinical isolate with the respective fold change for each fluconazole induction time correspondent to the acquisition of posaconazole, clotrimazole and fluconazole and voriconazole resistance.

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The relative amount of biofilm formation in

the deletion mutant Δepa3 is significantly lower

than that registered for the wild-type strain,

suggesting that this adhesin play an important

role in biofilm formation on a polystyrene

surface. In fact, other studies have reported the

increased expression of CgEPA3 gene in

C. glabrata biofilms [20], [37].

Since the previous results highlight the

importance of the adhesin CgEpa3 in

antifungal resistance and biofilm formation, its

role in 3H-clotrimazole intracellular

accumulation was then analysed.

The time-course accumulation of 3H-

clotrimazole (Figure 10) exhibits a clear

difference between the C. glabrata KUE100

wild-type strain and Δepa3 mutant, the mutant

showing a higher accumulation of the drug. In

fact, there are statistically significant

differences between the wild and the mutant

that suggests once again the importance of this

adhesin in the resistance phenotype observed

in the 044Fluco31 strain, which acquired

resistance to clotrimazole.

In the future, it would be interesting to study

the possible role in azole resistance of more of

the adhesins whose expression was found to

increase in the clotrimazole resistant

044Fluco31 strain. It appears to be likely that

as CgEpa3, others adhesins, as well as the

ability to grow as cell aggregates, might be

related to the resistant phenotype observed

herein.

5. Conclusions

The analysis performed herein allowed some insights in the temporal evolution of the 044 clinical isolate towards azole resistance.

In the acquisition of posaconazole resistance, the strain obtained had maintained ergosterol content and cell-to-cell aggregation in comparison to the initial isolate. The intracellular accumulation of azole drugs was found to be 5-fold lower in these cells, than in the azole susceptible 044 clinical isolate, although there is no clear molecular mechanism responsible for the posaconazole resistance in this strain.

Regarding the acquisition of clotrimazole resistance, the up regulation of the MDR transporters CgCdr2 and CgTpo1, correlate with the observed ability of these strains to accumulate 10-fold less radiolabelled clotrimazole than the 044 clinical isolate. Despite the observed overexpression of the CgERG11 gene, the ergosterol concentration in these cells remained unchanged, when compared to the parental strain. The 044Fluco31 strain was found to display higher cell-to-cell aggregation, but not an increased ability to form biofilm in polystyrene microplates, when compared to the remaining strains.

Moreover, the Epa3 adhesin was found to confer resistance to several azoles, enhance the ability of C. glabrata cells to form biofilm and to promote cell-to-cell aggregation. Given that significant differences in azole

Figure 10. Time-course accumulation of 3H-clotrimazole in C. glabrata KUE100 (●) and Δepa3 (■). Error bars were calculated based on the standard deviation for a sample of n=3. The indicated values are averages of at least three independent experiments. Error bars represent the corresponding standard deviations. *p<0.05.

Figure 9. Biofilm formation followed by crystal violet staining and measurements of absorbance at 590 nm for the KUE100 and deletion mutants Δepa1, Δepa3, and Δepa10. Cells were grown for 15 h and the experiment was performed in SDB medium pH 5.6. In the scatter dot plot represented each dot corresponds to the level of biofilm formed in each sample. The average level of formed biofilm indicated by a black line (-) corresponds to at least 4 independent experiments. Standard deviations are represented by *p<0.05.

11

accumulation could be observed when comparing the wild-type strain and the Δepa3 deletion mutant, we hypothesize that the role of the increased expression of CgEPA3 and other adhesins in clotrimazole resistance, as found in the 044Fluco31 strain, may be to protect the cells from the extracellular concentration of clotrimazole by promoting cell aggregation.

Finally, the 044Fluco45 strain exhibited an overexpression of CgCDR1, CgCDR2, CgPDR12 and CgTPO1, which appears to be consistent with the observed emergence of a CgPDR1 mutation in this final strain. It would be important to assess the role of this mutation in the acquisition of azole resistance in clinical isolates of C. glabrata. Strongly correlated with the up regulation of the referred MDR transporters, is the clear ability to accumulate lower concentrations of clotrimazole, exhibited by these multiazole resistance cells.

Surprisingly, however, the 044Fluco45 strain was found to exhibit decreased ergosterol content. This result was not expected since azole resistant strains exhibit usually higher expression levels of CgERG11 or other ERG genes [6], [33], [34]. Nevertheless, in a particular study an azole resistant clinical isolate was described as being ergosterol-deficient, resulting from a single-amino-acid loss of function mutation in CgERG11 gene [5]. In this case study, since the primary target of azoles is no longer exhibiting function, azoles have low or no affinity for this enzyme. Additionally, the authors suggest that this isolate might be able to survive through growth on lanosterol-type sterols and/or perform sterol uptake, with changes in membrane composition. It would be interesting to explore the causes behind the decrease in ergosterol content, and whether this decrease may have a positive impact in azole resistance in C. glabrata.

Finally, the CgPil1 component of eisosomes was shown to be important for ketoconazole, clotrimazole and tioconazole resistance, and appears to be involved, direct or indirectly, in clotrimazole efflux. None of the eisosome-embeded transporters identified so far is directly linked to drug extrusion, but rather to the import of amino acids and nucleotides. Thus it would be interesting to gain further insights into how eisosomes affect the intracellular concentration of azole drugs. Still, the Δpil1 mutant was found to exhibit higher ergosterol content, which might be eventually due to a different membrane organization exhibited by these cells [22], an observation that does not help to explain the azole resistance phenotype exhibited by these cells.

Nonetheless, it is worth pointing out that while the deletion of CgPIL1 gene leads cells to increased ergosterol accumulation, the overexpression of this gene in the 044Fluco45 strain appears to correlate with decreased ergosterol levels.

Altogether, this study shows the global transcriptional alterations that were experienced by a cell population as it adapted to multiazole resistance.

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