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Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates
International Journal of Medical Microbiology 302 (2012) 155–161
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Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates Bettina Schulz ∗ , Mathias Knobloch, Kai Weber, Markus Ruhnke Charité – Universitätsmedizin Berlin, Charité Campus Mitte, Department of Oncology and Hematology, Charitéplatz 1, 10117 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 19 November 2011 Received in revised form 26 April 2012 Accepted 29 April 2012 Keywords: Candida glabrata Petite mutants Doxorubicin Resistance ABC transporter Fluconazole susceptibility
a b s t r a c t Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 g/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 g/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy. © 2012 Elsevier GmbH. All rights reserved.
Introduction Fungal infections in humans are increasing due to numerous factors, particularly the rising number of immunocompromised patients as a result of mucosal barrier disruption, defects in neutrophil numbers and function or in cell-mediated immunity, metabolic dysfunction, and extremes of age (Pfaller and Diekema, 2007; Segal et al., 2006). Broad-spectrum antibiotics, cytotoxic chemotherapies and transplantations further increase the risk of both common and uncommon opportunistic fungal infections (Nucci and Marr, 2005; Pfaller and Diekema, 2004). More than 17 different Candida species have been reported to cause invasive fungal infections in humans. C. glabrata has emerged as the second most common causative species in the last decade and is even the predominant pathogen in some groups of patients such as elderly or diabetic patients (Goswami et al., 2006; Pfaller et al., 2010a,b; Safdar et al., 2001). C. glabrata is less intrinsically susceptible to azoles, and many clinical isolates have a 16–64 times higher fluconazole (FLU) minimum inhibitory concentration (MIC) than C.
∗ Corresponding author. Tel.: +49 30 450 513 383; fax: +49 30 450 513 976. E-mail address: bettina [email protected] (B. Schulz). 1438-4221/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2012.04.002
albicans (Safdar et al., 2001). C. glabrata ATP-binding cassette (ABC) transporters regulated by the transcription factor gene CgPDR1 are among the primary causes of both intrinsic and acquired resistance of C. glabrata to azole drugs (Sanglard et al., 1999, 2001; Torelli et al., 2008). The most prominent ABC transporter genes of the CgPDR1-regulated network are CgCDR1, CgCDR2 and CgSNQ2. Furthermore, C. glabrata is capable of developing a respiratorydeficient phenotype when exposed to certain substances such as acriflavine, ethidium bromide, FLU or statins (Bouchara et al., 2000; Brun et al., 2004; Bulder, 1964; Westermeyer and Macreadie, 2007). The so-called petite mutants are highly resistant to azoles and may be associated with antifungal treatment failure (Bouchara et al., 2000; Brun et al., 2003). Azole-resistance in C. albicans is also often mediated by ABC transporter proteins, the encoding genes of which are regulated by the transcription factor CaTAC1 – an equivalent of CgPDR1. We recently demonstrated a CaTAC1-dependent induction of ABC transporter expression by DOX in C. albicans. DOX upregulated CaCDR1 and CaCDR2 in FLU-susceptible C. albicans isolates but did not increase efflux pump expression in a tac1/ mutant. Moreover, FLU MIC values were elevated in C. albicans isolates exposed to DOX (Kofla et al., 2011). The same phenomenon was observed in C. dubliniensis (Schulz et al., 2012). The present study
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Table 1 Overview of C. glabrata isolates used for this study. Designation
Abbreviation
MICfluconazole [g/ml]
DSM 11950
–
4
RKI 06-0367
Cg1
8
RKI 05-0559-02
Cg2
4
RKI 05-0445-01
Cg3
64
Origin DSMZ Braunschweig Robert Koch Institute, Berlin Robert Koch Institute, Berlin Robert Koch Institute, Berlin
investigated the influence of short- and long-term DOX exposure on ABC transporter expression and FLU susceptibility in C. glabrata. Materials and methods
and harvested for RNA isolation. All isolates were concomitantly cultured without the drug and used as controls. This experiment was performed on two different occasions. In a second experiment, the isolates were incubated with 20 g/ml of DOX for 10 days, starting on day 0. Medium supplemented with fresh DOX was changed on days 1, 2, 3, 6 and 8. All isolates were concomitantly cultured without the drug and used as controls. Samples were taken for RNA extraction and MIC determination on the indicated days as well as on day 10. They were washed twice with 1× PBS. The cell pellet needed for RNA extraction was frozen at −80 ◦ C until further use. For MIC determination, the yeast cells were resuspended in 10% glycerol and stored at −20 ◦ C. This experiment was performed on two different occasions. To assess respiratory deficiency, the long-term experiment was performed again as described above and repeated twice. Gene expression analysis
Isolates and culture conditions Three clinical isolates (Cg1, Cg2, Cg3; Robert Koch Institute, Berlin, Germany) and one reference strain of C. glabrata (DSM 11950; DSMZ, Braunschweig, Germany) were used in this study (Table 1). MIC values of isolates DSM 11950, Cg1 and Cg2 indicated dose-dependent susceptibility to FLU. Isolate Cg3 was FLU-resistant. Isolates were kept as glycerol stocks at −80 ◦ C. They were subcultured overnight at 30 ◦ C on Sabouraud dextrose agar plates (BD, Heidelberg, Germany) and then kept at room temperature.
RNA isolation, cDNA synthesis and gene expression analyses were performed as previously described (Kofla et al., 2011). Expression levels of the following genes were analyzed: CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Primers and probes (TIB Molbiol, Berlin, Germany) are listed in Table 2. All cDNA samples were analyzed in duplicate, and no-template controls were included in all runs. Crossing points (CP ) were calculated using the LightCycler® 480 software (Roche), and relative quantification was done by the CP method (Schulz et al., 2012). MIC determination
Chequerboard experiment The chequerboard assay was performed as previously described (Schulz et al., 2012). FLU was used in concentrations of 128–0.25 g/ml, and DOX was sequentially diluted from 80 to 2.5 g/ml. The fractional inhibitory concentration (FIC) index was determined as described elsewhere (Dougherty et al., 1977; Odds, 2003). The chequerboard experiment was performed twice.
Samples drawn from the 10-day DOX exposure assay were processed for MIC determination. MIC values of all selected petite mutants and controls were additionally determined. FLU concentrations ranged from 0.25 to 128 g/ml, and MIC values were determined according to the Clinical and Laboratory Standards Institute (CLSI) M27 A2 guideline, including all necessary controls (Clinical and Laboratory Standards Institute, 1997).
DOX treatment assays
Detection of petite mutants
For gene expression analyses, C. glabrata isolates were cultured overnight at 30 ◦ C in yeast peptone dextrose (YPD) broth (Qbiogene, Heidelberg, Germany) with shaking. The overnight culture was used to inoculate fresh YPD broth, and cells were allowed to grow until mid-log phase. After adding DOX (20 g/ml final concentration), samples destined for treatment were incubated for 90 min
Diluted C. glabrata isolates drawn from the 10-day DOX assay were plated on solid YPD agar (Qbiogene, Heidelberg, Germany) allowing growth of both respiratory-competent and respiratorydeficient clones. Since petite mutants grow slowly, plates were incubated overnight at 37 ◦ C and then kept at room temperature for 24 h. In order to differentiate and quantify petite mutants and
Table 2 Sequences of C. glabrata primers and probes used for gene expression analysis. Sequence 5 –3
Gene
Accession number
Assay efficiency
CDR1
AF109723
97.9%
Primer sense Primer antisense Probe
AgC AAC TCA gAC CCg gAT TAC TAC ggT ATT CgA TAT CAg CAg ATT CAC C TTg CgA CCA AAT CCT TCC AgT AAC AgC C
CDR2
AF251023
97.7%
Primer sense Primer antisense Probe
CCC TgA CTT CTT gAC TTC gAT C TTC TCT TAg TTg CTT ATA TTC CTC g CAT ATC CAA Tgg CgT CTg Tgg CA
SNQ2
AF251022
91.4%
Primer sense Primer antisense Probe
CAA TCT gTC TTT TgA gCA CAg ACC TA CAg TTC TgT TCA gAC CAg ACA gg CTg AAg CAT Tgg gAA gTA CTA TCg CCA A
PDR1
AY700584
93.2%
Primer sense Primer antisense Probe
CAg AgT gCC AAA gTA TgC AgC CAA gAA TTT TTT TgA ATg ACA ACg TA AgC ATC ggA ggT AgA TCT gCA Agg TCA
ACT1
AF069746
96.7%
Primer sense Primer antisense Probe
gAg CCg TCT TCC CTT CCA TC TCg TCA CCg ACg TAA gAg TCC T CAT ACC gAC CAT gAT ACC TTg gTg TCT Tg
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Fig. 1. Overview of results of gene expression analyses obtained during long-term exposure of isolates to 20 g/ml of DOX. Fresh YPD medium supplemented with DOX was changed on days 1, 2, 3, 6, and 8. Samples for further investigations were also taken on those days as well as on day 10. Gene expression was normalized to CgACT1 expression and then related to that of untreated control isolates. The y-axes are presented on a log 2 scale. Results are means of two biological replicates (total of four technical replicates) ± standard deviation.
respiratory-competent clones, colonies were transferred onto yeast peptone glycerol (YPG) agar containing 1% yeast extract, 2% peptone, 2% agar, and 3% glycerol as the sole carbon source. Since petite mutants are not capable of fermenting glycerol, they do not grow on YPG agar. The transfer was achieved by lightly tapping a velvetcovered stamp onto the YPD plate. Colonies adhered to the velvet of the stamp, which was then placed on a fresh YPG plate. YPG plates were incubated overnight at 37 ◦ C. YPD and the corresponding YPG plates were compared, and respiratory-competent clones and petite mutants were counted and calculated as percentages of the total colony count. Gene expression analyses of petite mutants Twenty-four respiratory-deficient mutants selected from C. glabrata isolates DSM 11950, Cg1 and Cg2 during all three 10-day experiments were used for further investigations. They were subcultured on YPD agar plates and checked for respiratory deficiency on YPG agar. Controls were 23 DOX-treated respiratory-competent clones (grown on YPD and YPG agar) as well as 6 untreated clones selected from all three DOX-treatment experiments, respectively. For gene expression analyses, petite mutants and controls were cultured in YPD broth overnight at 30 ◦ C. The overnight culture was used to inoculate fresh YPD broth, and cells were allowed to grow until mid-log phase. RNA extraction, cDNA synthesis and gene expression analyses were conducted as previously described (Kofla et al., 2011). All cDNA samples were analyzed in duplicate, and no-template controls were included in all runs. CP values were
calculated, and relative quantification was done by the CP method (normalization to CgACT1 expression) (Kofla and Ruhnke, 2007). Results Combination of FLU and DOX had no effect on FLU MIC values The presence of DOX alone affected cell growth of all isolates tested. A DOX concentration of 40 g/ml was lethal for FLUsusceptible isolates and a concentration of 80 g/ml was lethal for the FLU-resistant isolate. The combination of FLU and DOX revealed that DOX did not alter FLU MIC values in any of the isolates tested, regardless of the exposure concentration (data not shown). Indifference was confirmed by a FIC index of 2.0 for all isolates. DOX induced the expression of efflux pumps and CgPDR1 during long-term exposure Short-term exposure to 20 g/ml of DOX for 90 min did not induce the expression of ABC transporters or CgPDR1 in any of the C. glabrata isolates (data not shown). However, long-term DOX exposure (up to 10 days) increased the expression of ABC transporters and CgPDR1 in isolates Cg1, Cg2 and DSM 11950 (Fig. 1). CgCDR1 expression was up to 6.2 times higher in DSM 11950, 8.5 times higher in Cg1 and 19.3 times higher in Cg2 than in untreated control isolates. Genes CgCDR2, CgSNQ2 and CgPDR1 were also overexpressed when exposed to DOX, but to a lesser extent. An
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Table 3 MIC values for FLU in g/ml determined during long-term exposure of all tested isolates to 20 g/ml of DOX. Untreated control isolates are given in parentheses. Day
DSM 11950
Cg1
Cg2
Cg3
0 1 2 3 6 8 10
4 4 (4) 4 (4) 2 (4) 4 (4) 4 (4) 4 (4)
8 8 (4) 8 (8) 8 (8) 32 (8) 32 (8) 64 (8)
4 8 (4) 8 (8) 8 (4) 16 (4) 16 (4) 32 (4)
32 32 (32) 32 (32) 32 (32) 32 (32) 32 (32) 32 (32)
approximately 2-fold increase of gene expression for the three above-mentioned genes was observed on most test days. On several days, gene expression remained uninfluenced by DOX in all three isolates. The reason for this phenomenon was the increased gene expression of investigated genes in respective untreated control isolates especially considering CgPDR1 (data not shown). Since gene expressions of treated and untreated isolates were related to each other an increased expression in the control isolates relativised the expression of DOX-treated isolates. Gene expression data were generated from samples of the growing culture between day 0 and day 10. Since DOX is capable to select for respiratory deficiency a mixed population of respiratorycompetent and respiratory-deficient C. glabrata cells might have been present in the later days of the experiment. The changed composition of cultures might be an explanation why generally, isolates DSM 11950, Cg1 and Cg2 showed a heterogeneous pattern of gene expression between day 1 and day 10 with a clear DOX-induced overexpression of only CgCDR1. DOX did not affect gene expression in the FLU-resistant isolate Cg3. Except on day 2 slight increases of CgCDR1, CgCDR2 and CgSNQ2 expressions were observed (Fig. 1). FLU MIC values increased in Cg1 and Cg2 during long-term DOX exposure Prolonged DOX exposure increased FLU MIC values in isolates Cg1 and Cg2 (Table 3). The rise in MIC values was first observed
on day six, and susceptibility decreased until day 10. MIC values ranged from 8 (days 0–3) to 32 g/ml (day 10) for Cg1 and from 4 (day 0) to 64 g/ml (day 10) for Cg2. MIC values did not change during the experiment for isolates Cg3 or DSM 11950 or for untreated control isolates. DOX induced the petite mutant phenotype in FLU-susceptible isolates Petite mutants occurred in the three FLU-susceptible isolates Cg1, Cg2 and DSM 11950 after six days of DOX treatment (Fig. 2). Isolate Cg3 as well as untreated control isolates did not develop clones with respiratory deficiency. Occurrence of respiratory-deficient clones differed greatly between isolates (Fig. 3). Three independent experiments revealed that the percentage of petite mutants after ten days of DOX exposure was 2.2–2.3% for isolate DSM 11950, 3.8–4.6% for Cg1, and 21.3–65.0% for Cg2. Petite mutants overexpressed ABC transporter genes and CgPDR1 and were highly resistant to FLU Single clones of all three FLU-susceptible C. glabrata isolates derived from three independent experiments were selected for respiratory deficiency and processed for gene expression analysis. They showed marked upregulation of drug efflux pumps CgCDR1 and CgCDR2 compared to respiratory-competent DOXtreated clones or untreated control clones (Fig. 4). In the selected clones, CgCDR1 was overexpressed up to 87-fold compared to untreated control clones. Compared to respiratory competent DOXtreated clones CgCDR1 expression was even increased up to the 129-fold. CgCDR2 expression levels were also much higher in petite mutants (up to 83 times higher than in DOX-treated nonpetites and up to 66 times higher than in untreated control cells). The ABC transporter gene CgSNQ2 and its transcriptional regulator CgPDR1 were upregulated as well, but to a lesser extent. The outcome of gene expression analyses correlated with FLU MIC values determined for petite mutants versus
Fig. 2. Assessment of the petite phenotype during long-term incubation with 20 g/ml of DOX. Samples were taken on days indicated and plated on solid YPD medium. Grown colonies were transferred onto solid YPG medium with glycerol as the sole carbon source using a velvet-covered stamp. Petite mutants do not grow on glycerol agar and can be counted when comparing colony growth on YPG and the corresponding YPD plate. The petite mutant colony count was related to the total number of colonies. The figure shows the results of three independent experiments.
B. Schulz et al. / International Journal of Medical Microbiology 302 (2012) 155–161
Fig. 3. Comparison of colony growth and assessment of the petite phenotype of isolates DSM 11950, Cg1 and Cg2. After 10 days of incubation with 20 g/ml DOX a sample of each cell suspension was washed with PBS, diluted and spread on solid YPD medium. Grown colonies were transferred onto solid YPG medium with glycerol as the sole carbon source using a velvet-covered stamp. Petite mutants (black circles) do not grow on glycerol agar and can be distinguished from normal respiratory colonies.
respiratory-competent cells and untreated control cells. All petite mutants derived from the three FLU-susceptible C. glabrata isolates showed MIC values of ≥64 g/ml, whereas respiratorycompetent and untreated control cells remained dose-dependently susceptible (data not shown). Discussion Doxorubicin (DOX) is an anthracycline antibiotic agent effective against a variety of malignancies (Carter, 1975). However, it can also induce the petite phenotype in yeasts. Several reports deal with its effect on the respiratory competence of the haploid yeast Saccharomyces cerevisiae, a close relative of Candida glabrata (Buschini et al., 2003; Hixon et al., 1980). S. cerevisiae is widely used as a model for investigating the cytotoxic side effects of DOX on eukaryotic cells (Buschini et al., 2003; Hixon et al., 1980). In this study, C. glabrata was examined as a cause of severe invasive fungal infections in immunocompromised individuals. Fungal cells may be exposed to DOX in the course of anticancer chemotherapy. Therefore, a therapeutic DOX serum concentration of 20 g/ml was used for incubation. Higher concentrations were
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not applicable, since a 40 g/ml DOX concentration was lethal for all FLU-susceptible C. glabrata isolates tested in this study (data not shown). The manipulative effect of DOX on efflux pump expression was previously demonstrated in C. albicans and C. dubliniensis (Kofla et al., 2011; Schulz et al., 2012). Both species showed DOX-induced ABC transporter gene expression and increased MIC values when cultured together with fluconazole (FLU), indicating direct DOX involvement in efflux pump gene expression. In C. albicans, this induction was dependent on the transcription factor CaTAC1, since DOX-induced efflux pump expression was abolished in a tac1/ mutant (Kofla et al., 2011). In the case of C. dubliniensis, little is known about the regulation of ABC transporter pumps. However, the C. dubliniensis ortholog, CdTAC1, shares 88% amino acid identity with CaTAC1 and very likely performs the same functions (Coleman et al., 2010). In C. glabrata, similar mechanisms were identified for structurally different drugs and xenobiotics such as ketoconazole, oligomycin and cycloheximide (Thakur et al., 2008; Tsai et al., 2006). The C. glabrata transcription factor Pdr1p might bind directly to the agents with subsequent induction of efflux pump expression (Thakur et al., 2008). However, not all substances had this effect. Dexamethasone, for example, did not induce the Pdr1p/Pdr3p network of S. cerevisiae (Thakur et al., 2008). Considering DOX, there are two principle mechanisms by which DOX could affect C. glabrata efflux pump expression and thus FLU susceptibility. Firstly, induction would lead to increased FLU transport out of the cells on exposure to DOX as was observed for C. albicans and C. dubliniensis. Secondly, if DOX has an inhibitory effect on C. glabrata cells and is itself transported by the efflux pumps, prolonged growth in its presence could select for efflux pump overexpressing mutants that are resistant. We tested the FLU susceptibility of isolates exposed to DOX. The drug combination study revealed that DOX exposure did not alter FLU MIC values. Hence, DOX might not directly modulate efflux pump gene expression in C. glabrata. Some observations suggest that DOX has an inhibitory effect on the cells and activates the detoxifying machinery in response to cellular stress. DOX cytotoxicity previously shown in S. cerevisiae leads to the assumption that DOX might also have a cytotoxic effect on C. glabrata (Hixon et al., 1980; Kule et al., 1994). This hypothesis is supported by the effect of short-term DOX exposure on cells. The fact that DOX did not induce expression of either ABC transporter gene investigated substantiated the hypothesis that DOX might not directly affect the expression of C. glabrata ABC transporters. If DOX directly activated efflux pump expression, an induction would be visible after 90 min (Kofla et al., 2011; Thakur et al., 2008). Moreover, MIC values from samples collected during the 10-day treatment began to gradually increase on day 6, which coincided with the first occurrence of petite mutants. Taken together, these observations rather lead to the hypothesis that prolonged growth in the presence of DOX selects for efflux pump overexpressing mutants that are resistant – as previously shown for S. cerevisiae (Hixon et al., 1980) – and that there is no direct involvement of DOX considering the induction of ABC transporter expression as was observed for C. albicans and C. dubliniensis. Although DOX might not be a direct regulator of ABC transporter expression, other inducing pathways must exist, since the genes investigated were upregulated in the three FLU-susceptible isolates during the 10-day exposure – even during the first few days when cells were still respiratory-competent. An explanation may lie in DOX cytotoxicity and the subsequent production of reactive oxygen species (ROS) (Kule et al., 1994). The resulting oxidative stress activates a variety of defence mechanisms to secure cell survival. One of these mechanisms has been demonstrated for S. cerevisiae: the induction of pleiotropic drug resistance genes encoding ABC transporter proteins (Ro et al., 2008). Hence, DOX may activate the expression of C. glabrata ABC transporters indirectly, probably by
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Fig. 4. Expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1 genes was analyzed in selected petite mutants of all three FLU-susceptible C. glabrata isolates derived from three independent experiments. Gene expression was normalized to CgACT1 expression. The corresponding gene expression values of DOX-treated respiratory-competent clones and untreated C. glabrata isolates were included for control purposes.
producing ROS, but further experiments are necessary to confirm this hypothesis. Since mitochondria are involved in ROS production, DOX cytotoxicity is dependent on cells possessing functional mitochondria. Cells without mitochondria or cells harboring mitochondria that lose their function become resistant to DOX (Buschini et al., 2003; Hixon et al., 1980; Kule et al., 1994). As mentioned above, respiratory-deficient cells arising after DOX treatment were not induced but selected from the pre-existing population of spontaneously derived petite mutants (Hixon et al., 1980). The fact that petite mutants do not appear until day 6 in the present study could possibly be due to the incubation of cells in rich medium. Hixon et al. showed that growth inhibition rather than cell death did not exert sufficient selective pressure in S. cerevisiae to reveal the resistance of petite mutants to drug toxicity, in contrast to the effect after incubation in water or buffer (Hixon et al., 1980). The FLU-resistant C. glabrata isolate showed no respiratorydeficient cells at all throughout the long-term experiment. The activated resistance mechanisms of this isolate seem to be sufficient to overcome DOX cytotoxicity. The switch between states of mitochondrial competence and incompetence of C. glabrata cells reported by Kaur et al. (2004) cannot explain this phenomenon, since the FLU-resistant isolate Cg3 was respiratory-competent throughout the experiments. The isolated petite mutants of all three FLU-susceptible C. glabrata isolates were highly resistant to FLU and overexpressed both CgPDR1 and ABC transporter genes. A study previously published by Tsai et al. (2006) showed that mitochondrial dysfunction increases CgPDR1 expression, which may be one explanation for the highly resistant phenotype of respiratory-deficient cells. Petite mutation in C. glabrata has been demonstrated not only in vitro but also in vivo (Bouchara et al., 2000; Ferrari et al., 2011). A recently published study by Ferrari et al. (2011) described a highly resistant respiratory-deficient C. glabrata mutant from a patient undergoing azole therapy. Since this isolate was selected in vivo and showed greater fitness in mice than its parental precursor,
mitochondrial dysfunction may confer an advantage on yeast cells in host tissues and may be the reason for antifungal treatment failure.
Conflicts of interest The authors declare that they have no conflicts of interest in relation to this work.
References Bouchara, J.P., Zouhair, R., Le, B.S., Renier, G., Filmon, R., Chabasse, D., Hallet, J.N., Defontaine, A., 2000. In vivo selection of an azole-resistant petite mutant of Candida glabrata. J. Med. Microbiol. 49, 977–984. Brun, S., Aubry, C., Lima, O., Filmon, R., Berges, T., Chabasse, D., Bouchara, J.P., 2003. Relationships between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 47, 847–853. Brun, S., Berges, T., Poupard, P., Vauzelle-Moreau, C., Renier, G., Chabasse, D., Bouchara, J.P., 2004. Mechanisms of azole resistance in petite mutants of Candida glabrata. Antimicrob. Agents Chemother. 48, 1788–1796. Bulder, C.J., 1964. Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts. Anton. Van Leeuw. 30, 1–9. Buschini, A., Poli, P., Rossi, C., 2003. Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones. Mutagenesis 18, 25–36. Carter, S.K., 1975. Adriamycin—a review. J. Natl. Cancer Inst. 55, 1265–1274. Clinical Laboratory Standards Institute, 1997. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. National Committee for Clinical Laboratory Standards, Wayne, PA. Coleman, D.C., Moran, G.P., McManus, B.A., Sullivan, D.J., 2010. Mechanisms of antifungal drug resistance in Candida dubliniensis. Future Microbiol. 5, 935–949. Dougherty, P.F., Yotter, D.W., Matthews, T.R., 1977. Microdilution transfer plate technique for determining in vitro synergy of antimicrobial agents. Antimicrob. Agents Chemother. 11, 225–228. Ferrari, S., Sanguinetti, M., De, B.F., Torelli, R., Posteraro, B., Vandeputte, P., Sanglard, D., 2011. Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice. Antimicrob. Agents Chemother. 55, 1852–1860. Goswami, D., Goswami, R., Banerjee, U., Dadhwal, V., Miglani, S., Lattif, A.A., Kochupillai, N., 2006. Pattern of Candida species isolated from patients with diabetes mellitus and vulvovaginal candidiasis and their response to single dose oral fluconazole therapy. J. Infect. 52, 111–117.
B. Schulz et al. / International Journal of Medical Microbiology 302 (2012) 155–161 Hixon, S.C., Ocak, A., Thomas, J.E., Daugherty, J.P., 1980. Resistance to adriamycin cytotoxicity among respiratory-deficient mutants in yeast. Antimicrob. Agents Chemother. 17, 443–449. Kaur, R., Castano, I., Cormack, B.P., 2004. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob. Agents Chemother. 48, 1600–1613. Kofla, G., Ruhnke, M., 2007. Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression. J. Microbiol. Methods 68, 178–183. Kofla, G., Turner, V., Schulz, B., Storch, U., Froelich, D., Rognon, B., Coste, A.T., Sanglard, D., Ruhnke, M., 2011. Doxorubicin induces drug efflux pumps in Candida albicans. Med. Mycol. 49, 132–142. Kule, C., Ondrejickova, O., Verner, K., 1994. Doxorubicin, daunorubicin, and mitoxantrone cytotoxicity in yeast. Mol. Pharmacol. 46, 1234–1240. Nucci, M., Marr, K.A., 2005. Emerging fungal diseases. Clin. Infect. Dis. 41, 521–526. Odds, F.C., 2003. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 52, 1. Pfaller, M.A., Castanheira, M., Messer, S.A., Moet, G.J., Jones, R.N., 2010a. Variation in Candida spp. distribution and antifungal resistance rates among bloodstream infection isolates by patient age: report from the SENTRY Antimicrobial Surveillance Program (2008–2009). Diagn. Microbiol. Infect. Dis. 68, 278–283. Pfaller, M.A., Diekema, D.J., 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42, 4419–4431. Pfaller, M.A., Diekema, D.J., 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin. Microbiol. Rev. 20, 133–163. Pfaller, M.A., Diekema, D.J., Gibbs, D.L., Newell, V.A., Ellis, D., Tullio, V., Rodloff, A., Fu, W., Ling, T.A., 2010b. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J. Clin. Microbiol. 48, 1366–1377. Ro, D.K., Ouellet, M., Paradise, E.M., Burd, H., Eng, D., Paddon, C.J., Newman, J.D., Keasling, J.D., 2008. Induction of multiple pleiotropic drug resistance genes in
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yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. BMC Biotechnol. 8, 83. Safdar, A., Chaturvedi, V., Cross, E.W., Park, S., Bernard, E.M., Armstrong, D., Perlin, D.S., 2001. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob. Agents Chemother. 45, 2129–2133. Sanglard, D., Ischer, F., Bille, J., 2001. Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 45, 1174–1183. Sanglard, D., Ischer, F., Calabrese, D., Majcherczyk, P.A., Bille, J., 1999. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob. Agents Chemother. 43, 2753–2765. Schulz, B., Knobloch, M., Moran, G.P., Weber, K., Ruhnke, M., 2012. Influence of doxorubicin on fluconazole susceptibility and efflux pump gene expression of Candida dubliniensis. Med. Mycol. 50, 421–426. Segal, B.H., Kwon-Chung, J., Walsh, T.J., Klein, B.S., Battiwalla, M., Almyroudis, N.G., Holland, S.M., Romani, L., 2006. Immunotherapy for fungal infections. Clin. Infect. Dis. 42, 507–515. Thakur, J.K., Arthanari, H., Yang, F., Pan, S.J., Fan, X., Breger, J., Frueh, D.P., Gulshan, K., Li, D.K., Mylonakis, E., Struhl, K., Moye-Rowley, W.S., Cormack, B.P., Wagner, G., Naar, A.M., 2008. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452, 604–609. Torelli, R., Posteraro, B., Ferrari, S., La, S.M., Fadda, G., Sanglard, D., Sanguinetti, M., 2008. The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata. Mol. Microbiol. 68, 186–201. Tsai, H.F., Krol, A.A., Sarti, K.E., Bennett, J.E., 2006. Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants. Antimicrob. Agents Chemother. 50, 1384–1392. Westermeyer, C., Macreadie, I.G., 2007. Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA in Candida glabrata. FEMS Yeast Res. 7, 436–441.
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International Journal of Medical Microbiology journal homepage: www.elsevier.de/ijmm
Doxorubicin selects for fluconazole-resistant petite mutants in Candida glabrata isolates Bettina Schulz ∗ , Mathias Knobloch, Kai Weber, Markus Ruhnke Charité – Universitätsmedizin Berlin, Charité Campus Mitte, Department of Oncology and Hematology, Charitéplatz 1, 10117 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 19 November 2011 Received in revised form 26 April 2012 Accepted 29 April 2012 Keywords: Candida glabrata Petite mutants Doxorubicin Resistance ABC transporter Fluconazole susceptibility
a b s t r a c t Candida infections are a permanent threat to immunocompromised individuals such as cancer patients, and Candida glabrata has emerged as a major problem in recent years. Resistance may develop during lengthy antifungal therapies and is often mediated by upregulation of fungal drug efflux pumps. During chemotherapy the yeast cell is also exposed to cytotoxic agents that may affect its drug susceptibility. Four C. glabrata isolates, three susceptible and one resistant to fluconazole (FLU), were incubated with 20 g/ml of doxorubicin (DOX) for 90 min. In a second experiment, the isolates were cultured with DOX for ten days. Samples were taken on subsequent days to determine the minimal inhibitory concentration (MIC) of FLU and to analyze expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Samples were also used to assess the petite phenotype. Short-term DOX exposure did not induce efflux pump gene expression, but genes were consistently overexpressed in FLU-susceptible isolates during long-term exposure. An increase in MIC values on day 6 in two of the isolates coincided with the first occurrence of petite mutants in all susceptible isolates. The respiratory deficiency of selected petite mutants was confirmed by culturing mutants on agar containing glycerol as the sole carbon source. FLU MIC values for respiratory-deficient clones were ≥64 g/ml, and efflux pump gene expression was greatly increased. The resistant isolate did not develop mitochondrial dysfunction. In summary, the cytotoxic agent DOX selects for FLU-resistant respiratory-deficient C. glabrata mutants, which may affect antifungal therapy. © 2012 Elsevier GmbH. All rights reserved.
Introduction Fungal infections in humans are increasing due to numerous factors, particularly the rising number of immunocompromised patients as a result of mucosal barrier disruption, defects in neutrophil numbers and function or in cell-mediated immunity, metabolic dysfunction, and extremes of age (Pfaller and Diekema, 2007; Segal et al., 2006). Broad-spectrum antibiotics, cytotoxic chemotherapies and transplantations further increase the risk of both common and uncommon opportunistic fungal infections (Nucci and Marr, 2005; Pfaller and Diekema, 2004). More than 17 different Candida species have been reported to cause invasive fungal infections in humans. C. glabrata has emerged as the second most common causative species in the last decade and is even the predominant pathogen in some groups of patients such as elderly or diabetic patients (Goswami et al., 2006; Pfaller et al., 2010a,b; Safdar et al., 2001). C. glabrata is less intrinsically susceptible to azoles, and many clinical isolates have a 16–64 times higher fluconazole (FLU) minimum inhibitory concentration (MIC) than C.
∗ Corresponding author. Tel.: +49 30 450 513 383; fax: +49 30 450 513 976. E-mail address: bettina [email protected] (B. Schulz). 1438-4221/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijmm.2012.04.002
albicans (Safdar et al., 2001). C. glabrata ATP-binding cassette (ABC) transporters regulated by the transcription factor gene CgPDR1 are among the primary causes of both intrinsic and acquired resistance of C. glabrata to azole drugs (Sanglard et al., 1999, 2001; Torelli et al., 2008). The most prominent ABC transporter genes of the CgPDR1-regulated network are CgCDR1, CgCDR2 and CgSNQ2. Furthermore, C. glabrata is capable of developing a respiratorydeficient phenotype when exposed to certain substances such as acriflavine, ethidium bromide, FLU or statins (Bouchara et al., 2000; Brun et al., 2004; Bulder, 1964; Westermeyer and Macreadie, 2007). The so-called petite mutants are highly resistant to azoles and may be associated with antifungal treatment failure (Bouchara et al., 2000; Brun et al., 2003). Azole-resistance in C. albicans is also often mediated by ABC transporter proteins, the encoding genes of which are regulated by the transcription factor CaTAC1 – an equivalent of CgPDR1. We recently demonstrated a CaTAC1-dependent induction of ABC transporter expression by DOX in C. albicans. DOX upregulated CaCDR1 and CaCDR2 in FLU-susceptible C. albicans isolates but did not increase efflux pump expression in a tac1/ mutant. Moreover, FLU MIC values were elevated in C. albicans isolates exposed to DOX (Kofla et al., 2011). The same phenomenon was observed in C. dubliniensis (Schulz et al., 2012). The present study
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Table 1 Overview of C. glabrata isolates used for this study. Designation
Abbreviation
MICfluconazole [g/ml]
DSM 11950
–
4
RKI 06-0367
Cg1
8
RKI 05-0559-02
Cg2
4
RKI 05-0445-01
Cg3
64
Origin DSMZ Braunschweig Robert Koch Institute, Berlin Robert Koch Institute, Berlin Robert Koch Institute, Berlin
investigated the influence of short- and long-term DOX exposure on ABC transporter expression and FLU susceptibility in C. glabrata. Materials and methods
and harvested for RNA isolation. All isolates were concomitantly cultured without the drug and used as controls. This experiment was performed on two different occasions. In a second experiment, the isolates were incubated with 20 g/ml of DOX for 10 days, starting on day 0. Medium supplemented with fresh DOX was changed on days 1, 2, 3, 6 and 8. All isolates were concomitantly cultured without the drug and used as controls. Samples were taken for RNA extraction and MIC determination on the indicated days as well as on day 10. They were washed twice with 1× PBS. The cell pellet needed for RNA extraction was frozen at −80 ◦ C until further use. For MIC determination, the yeast cells were resuspended in 10% glycerol and stored at −20 ◦ C. This experiment was performed on two different occasions. To assess respiratory deficiency, the long-term experiment was performed again as described above and repeated twice. Gene expression analysis
Isolates and culture conditions Three clinical isolates (Cg1, Cg2, Cg3; Robert Koch Institute, Berlin, Germany) and one reference strain of C. glabrata (DSM 11950; DSMZ, Braunschweig, Germany) were used in this study (Table 1). MIC values of isolates DSM 11950, Cg1 and Cg2 indicated dose-dependent susceptibility to FLU. Isolate Cg3 was FLU-resistant. Isolates were kept as glycerol stocks at −80 ◦ C. They were subcultured overnight at 30 ◦ C on Sabouraud dextrose agar plates (BD, Heidelberg, Germany) and then kept at room temperature.
RNA isolation, cDNA synthesis and gene expression analyses were performed as previously described (Kofla et al., 2011). Expression levels of the following genes were analyzed: CgCDR1, CgCDR2, CgSNQ2 and CgPDR1. Primers and probes (TIB Molbiol, Berlin, Germany) are listed in Table 2. All cDNA samples were analyzed in duplicate, and no-template controls were included in all runs. Crossing points (CP ) were calculated using the LightCycler® 480 software (Roche), and relative quantification was done by the CP method (Schulz et al., 2012). MIC determination
Chequerboard experiment The chequerboard assay was performed as previously described (Schulz et al., 2012). FLU was used in concentrations of 128–0.25 g/ml, and DOX was sequentially diluted from 80 to 2.5 g/ml. The fractional inhibitory concentration (FIC) index was determined as described elsewhere (Dougherty et al., 1977; Odds, 2003). The chequerboard experiment was performed twice.
Samples drawn from the 10-day DOX exposure assay were processed for MIC determination. MIC values of all selected petite mutants and controls were additionally determined. FLU concentrations ranged from 0.25 to 128 g/ml, and MIC values were determined according to the Clinical and Laboratory Standards Institute (CLSI) M27 A2 guideline, including all necessary controls (Clinical and Laboratory Standards Institute, 1997).
DOX treatment assays
Detection of petite mutants
For gene expression analyses, C. glabrata isolates were cultured overnight at 30 ◦ C in yeast peptone dextrose (YPD) broth (Qbiogene, Heidelberg, Germany) with shaking. The overnight culture was used to inoculate fresh YPD broth, and cells were allowed to grow until mid-log phase. After adding DOX (20 g/ml final concentration), samples destined for treatment were incubated for 90 min
Diluted C. glabrata isolates drawn from the 10-day DOX assay were plated on solid YPD agar (Qbiogene, Heidelberg, Germany) allowing growth of both respiratory-competent and respiratorydeficient clones. Since petite mutants grow slowly, plates were incubated overnight at 37 ◦ C and then kept at room temperature for 24 h. In order to differentiate and quantify petite mutants and
Table 2 Sequences of C. glabrata primers and probes used for gene expression analysis. Sequence 5 –3
Gene
Accession number
Assay efficiency
CDR1
AF109723
97.9%
Primer sense Primer antisense Probe
AgC AAC TCA gAC CCg gAT TAC TAC ggT ATT CgA TAT CAg CAg ATT CAC C TTg CgA CCA AAT CCT TCC AgT AAC AgC C
CDR2
AF251023
97.7%
Primer sense Primer antisense Probe
CCC TgA CTT CTT gAC TTC gAT C TTC TCT TAg TTg CTT ATA TTC CTC g CAT ATC CAA Tgg CgT CTg Tgg CA
SNQ2
AF251022
91.4%
Primer sense Primer antisense Probe
CAA TCT gTC TTT TgA gCA CAg ACC TA CAg TTC TgT TCA gAC CAg ACA gg CTg AAg CAT Tgg gAA gTA CTA TCg CCA A
PDR1
AY700584
93.2%
Primer sense Primer antisense Probe
CAg AgT gCC AAA gTA TgC AgC CAA gAA TTT TTT TgA ATg ACA ACg TA AgC ATC ggA ggT AgA TCT gCA Agg TCA
ACT1
AF069746
96.7%
Primer sense Primer antisense Probe
gAg CCg TCT TCC CTT CCA TC TCg TCA CCg ACg TAA gAg TCC T CAT ACC gAC CAT gAT ACC TTg gTg TCT Tg
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Fig. 1. Overview of results of gene expression analyses obtained during long-term exposure of isolates to 20 g/ml of DOX. Fresh YPD medium supplemented with DOX was changed on days 1, 2, 3, 6, and 8. Samples for further investigations were also taken on those days as well as on day 10. Gene expression was normalized to CgACT1 expression and then related to that of untreated control isolates. The y-axes are presented on a log 2 scale. Results are means of two biological replicates (total of four technical replicates) ± standard deviation.
respiratory-competent clones, colonies were transferred onto yeast peptone glycerol (YPG) agar containing 1% yeast extract, 2% peptone, 2% agar, and 3% glycerol as the sole carbon source. Since petite mutants are not capable of fermenting glycerol, they do not grow on YPG agar. The transfer was achieved by lightly tapping a velvetcovered stamp onto the YPD plate. Colonies adhered to the velvet of the stamp, which was then placed on a fresh YPG plate. YPG plates were incubated overnight at 37 ◦ C. YPD and the corresponding YPG plates were compared, and respiratory-competent clones and petite mutants were counted and calculated as percentages of the total colony count. Gene expression analyses of petite mutants Twenty-four respiratory-deficient mutants selected from C. glabrata isolates DSM 11950, Cg1 and Cg2 during all three 10-day experiments were used for further investigations. They were subcultured on YPD agar plates and checked for respiratory deficiency on YPG agar. Controls were 23 DOX-treated respiratory-competent clones (grown on YPD and YPG agar) as well as 6 untreated clones selected from all three DOX-treatment experiments, respectively. For gene expression analyses, petite mutants and controls were cultured in YPD broth overnight at 30 ◦ C. The overnight culture was used to inoculate fresh YPD broth, and cells were allowed to grow until mid-log phase. RNA extraction, cDNA synthesis and gene expression analyses were conducted as previously described (Kofla et al., 2011). All cDNA samples were analyzed in duplicate, and no-template controls were included in all runs. CP values were
calculated, and relative quantification was done by the CP method (normalization to CgACT1 expression) (Kofla and Ruhnke, 2007). Results Combination of FLU and DOX had no effect on FLU MIC values The presence of DOX alone affected cell growth of all isolates tested. A DOX concentration of 40 g/ml was lethal for FLUsusceptible isolates and a concentration of 80 g/ml was lethal for the FLU-resistant isolate. The combination of FLU and DOX revealed that DOX did not alter FLU MIC values in any of the isolates tested, regardless of the exposure concentration (data not shown). Indifference was confirmed by a FIC index of 2.0 for all isolates. DOX induced the expression of efflux pumps and CgPDR1 during long-term exposure Short-term exposure to 20 g/ml of DOX for 90 min did not induce the expression of ABC transporters or CgPDR1 in any of the C. glabrata isolates (data not shown). However, long-term DOX exposure (up to 10 days) increased the expression of ABC transporters and CgPDR1 in isolates Cg1, Cg2 and DSM 11950 (Fig. 1). CgCDR1 expression was up to 6.2 times higher in DSM 11950, 8.5 times higher in Cg1 and 19.3 times higher in Cg2 than in untreated control isolates. Genes CgCDR2, CgSNQ2 and CgPDR1 were also overexpressed when exposed to DOX, but to a lesser extent. An
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Table 3 MIC values for FLU in g/ml determined during long-term exposure of all tested isolates to 20 g/ml of DOX. Untreated control isolates are given in parentheses. Day
DSM 11950
Cg1
Cg2
Cg3
0 1 2 3 6 8 10
4 4 (4) 4 (4) 2 (4) 4 (4) 4 (4) 4 (4)
8 8 (4) 8 (8) 8 (8) 32 (8) 32 (8) 64 (8)
4 8 (4) 8 (8) 8 (4) 16 (4) 16 (4) 32 (4)
32 32 (32) 32 (32) 32 (32) 32 (32) 32 (32) 32 (32)
approximately 2-fold increase of gene expression for the three above-mentioned genes was observed on most test days. On several days, gene expression remained uninfluenced by DOX in all three isolates. The reason for this phenomenon was the increased gene expression of investigated genes in respective untreated control isolates especially considering CgPDR1 (data not shown). Since gene expressions of treated and untreated isolates were related to each other an increased expression in the control isolates relativised the expression of DOX-treated isolates. Gene expression data were generated from samples of the growing culture between day 0 and day 10. Since DOX is capable to select for respiratory deficiency a mixed population of respiratorycompetent and respiratory-deficient C. glabrata cells might have been present in the later days of the experiment. The changed composition of cultures might be an explanation why generally, isolates DSM 11950, Cg1 and Cg2 showed a heterogeneous pattern of gene expression between day 1 and day 10 with a clear DOX-induced overexpression of only CgCDR1. DOX did not affect gene expression in the FLU-resistant isolate Cg3. Except on day 2 slight increases of CgCDR1, CgCDR2 and CgSNQ2 expressions were observed (Fig. 1). FLU MIC values increased in Cg1 and Cg2 during long-term DOX exposure Prolonged DOX exposure increased FLU MIC values in isolates Cg1 and Cg2 (Table 3). The rise in MIC values was first observed
on day six, and susceptibility decreased until day 10. MIC values ranged from 8 (days 0–3) to 32 g/ml (day 10) for Cg1 and from 4 (day 0) to 64 g/ml (day 10) for Cg2. MIC values did not change during the experiment for isolates Cg3 or DSM 11950 or for untreated control isolates. DOX induced the petite mutant phenotype in FLU-susceptible isolates Petite mutants occurred in the three FLU-susceptible isolates Cg1, Cg2 and DSM 11950 after six days of DOX treatment (Fig. 2). Isolate Cg3 as well as untreated control isolates did not develop clones with respiratory deficiency. Occurrence of respiratory-deficient clones differed greatly between isolates (Fig. 3). Three independent experiments revealed that the percentage of petite mutants after ten days of DOX exposure was 2.2–2.3% for isolate DSM 11950, 3.8–4.6% for Cg1, and 21.3–65.0% for Cg2. Petite mutants overexpressed ABC transporter genes and CgPDR1 and were highly resistant to FLU Single clones of all three FLU-susceptible C. glabrata isolates derived from three independent experiments were selected for respiratory deficiency and processed for gene expression analysis. They showed marked upregulation of drug efflux pumps CgCDR1 and CgCDR2 compared to respiratory-competent DOXtreated clones or untreated control clones (Fig. 4). In the selected clones, CgCDR1 was overexpressed up to 87-fold compared to untreated control clones. Compared to respiratory competent DOXtreated clones CgCDR1 expression was even increased up to the 129-fold. CgCDR2 expression levels were also much higher in petite mutants (up to 83 times higher than in DOX-treated nonpetites and up to 66 times higher than in untreated control cells). The ABC transporter gene CgSNQ2 and its transcriptional regulator CgPDR1 were upregulated as well, but to a lesser extent. The outcome of gene expression analyses correlated with FLU MIC values determined for petite mutants versus
Fig. 2. Assessment of the petite phenotype during long-term incubation with 20 g/ml of DOX. Samples were taken on days indicated and plated on solid YPD medium. Grown colonies were transferred onto solid YPG medium with glycerol as the sole carbon source using a velvet-covered stamp. Petite mutants do not grow on glycerol agar and can be counted when comparing colony growth on YPG and the corresponding YPD plate. The petite mutant colony count was related to the total number of colonies. The figure shows the results of three independent experiments.
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Fig. 3. Comparison of colony growth and assessment of the petite phenotype of isolates DSM 11950, Cg1 and Cg2. After 10 days of incubation with 20 g/ml DOX a sample of each cell suspension was washed with PBS, diluted and spread on solid YPD medium. Grown colonies were transferred onto solid YPG medium with glycerol as the sole carbon source using a velvet-covered stamp. Petite mutants (black circles) do not grow on glycerol agar and can be distinguished from normal respiratory colonies.
respiratory-competent cells and untreated control cells. All petite mutants derived from the three FLU-susceptible C. glabrata isolates showed MIC values of ≥64 g/ml, whereas respiratorycompetent and untreated control cells remained dose-dependently susceptible (data not shown). Discussion Doxorubicin (DOX) is an anthracycline antibiotic agent effective against a variety of malignancies (Carter, 1975). However, it can also induce the petite phenotype in yeasts. Several reports deal with its effect on the respiratory competence of the haploid yeast Saccharomyces cerevisiae, a close relative of Candida glabrata (Buschini et al., 2003; Hixon et al., 1980). S. cerevisiae is widely used as a model for investigating the cytotoxic side effects of DOX on eukaryotic cells (Buschini et al., 2003; Hixon et al., 1980). In this study, C. glabrata was examined as a cause of severe invasive fungal infections in immunocompromised individuals. Fungal cells may be exposed to DOX in the course of anticancer chemotherapy. Therefore, a therapeutic DOX serum concentration of 20 g/ml was used for incubation. Higher concentrations were
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not applicable, since a 40 g/ml DOX concentration was lethal for all FLU-susceptible C. glabrata isolates tested in this study (data not shown). The manipulative effect of DOX on efflux pump expression was previously demonstrated in C. albicans and C. dubliniensis (Kofla et al., 2011; Schulz et al., 2012). Both species showed DOX-induced ABC transporter gene expression and increased MIC values when cultured together with fluconazole (FLU), indicating direct DOX involvement in efflux pump gene expression. In C. albicans, this induction was dependent on the transcription factor CaTAC1, since DOX-induced efflux pump expression was abolished in a tac1/ mutant (Kofla et al., 2011). In the case of C. dubliniensis, little is known about the regulation of ABC transporter pumps. However, the C. dubliniensis ortholog, CdTAC1, shares 88% amino acid identity with CaTAC1 and very likely performs the same functions (Coleman et al., 2010). In C. glabrata, similar mechanisms were identified for structurally different drugs and xenobiotics such as ketoconazole, oligomycin and cycloheximide (Thakur et al., 2008; Tsai et al., 2006). The C. glabrata transcription factor Pdr1p might bind directly to the agents with subsequent induction of efflux pump expression (Thakur et al., 2008). However, not all substances had this effect. Dexamethasone, for example, did not induce the Pdr1p/Pdr3p network of S. cerevisiae (Thakur et al., 2008). Considering DOX, there are two principle mechanisms by which DOX could affect C. glabrata efflux pump expression and thus FLU susceptibility. Firstly, induction would lead to increased FLU transport out of the cells on exposure to DOX as was observed for C. albicans and C. dubliniensis. Secondly, if DOX has an inhibitory effect on C. glabrata cells and is itself transported by the efflux pumps, prolonged growth in its presence could select for efflux pump overexpressing mutants that are resistant. We tested the FLU susceptibility of isolates exposed to DOX. The drug combination study revealed that DOX exposure did not alter FLU MIC values. Hence, DOX might not directly modulate efflux pump gene expression in C. glabrata. Some observations suggest that DOX has an inhibitory effect on the cells and activates the detoxifying machinery in response to cellular stress. DOX cytotoxicity previously shown in S. cerevisiae leads to the assumption that DOX might also have a cytotoxic effect on C. glabrata (Hixon et al., 1980; Kule et al., 1994). This hypothesis is supported by the effect of short-term DOX exposure on cells. The fact that DOX did not induce expression of either ABC transporter gene investigated substantiated the hypothesis that DOX might not directly affect the expression of C. glabrata ABC transporters. If DOX directly activated efflux pump expression, an induction would be visible after 90 min (Kofla et al., 2011; Thakur et al., 2008). Moreover, MIC values from samples collected during the 10-day treatment began to gradually increase on day 6, which coincided with the first occurrence of petite mutants. Taken together, these observations rather lead to the hypothesis that prolonged growth in the presence of DOX selects for efflux pump overexpressing mutants that are resistant – as previously shown for S. cerevisiae (Hixon et al., 1980) – and that there is no direct involvement of DOX considering the induction of ABC transporter expression as was observed for C. albicans and C. dubliniensis. Although DOX might not be a direct regulator of ABC transporter expression, other inducing pathways must exist, since the genes investigated were upregulated in the three FLU-susceptible isolates during the 10-day exposure – even during the first few days when cells were still respiratory-competent. An explanation may lie in DOX cytotoxicity and the subsequent production of reactive oxygen species (ROS) (Kule et al., 1994). The resulting oxidative stress activates a variety of defence mechanisms to secure cell survival. One of these mechanisms has been demonstrated for S. cerevisiae: the induction of pleiotropic drug resistance genes encoding ABC transporter proteins (Ro et al., 2008). Hence, DOX may activate the expression of C. glabrata ABC transporters indirectly, probably by
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Fig. 4. Expression of CgCDR1, CgCDR2, CgSNQ2 and CgPDR1 genes was analyzed in selected petite mutants of all three FLU-susceptible C. glabrata isolates derived from three independent experiments. Gene expression was normalized to CgACT1 expression. The corresponding gene expression values of DOX-treated respiratory-competent clones and untreated C. glabrata isolates were included for control purposes.
producing ROS, but further experiments are necessary to confirm this hypothesis. Since mitochondria are involved in ROS production, DOX cytotoxicity is dependent on cells possessing functional mitochondria. Cells without mitochondria or cells harboring mitochondria that lose their function become resistant to DOX (Buschini et al., 2003; Hixon et al., 1980; Kule et al., 1994). As mentioned above, respiratory-deficient cells arising after DOX treatment were not induced but selected from the pre-existing population of spontaneously derived petite mutants (Hixon et al., 1980). The fact that petite mutants do not appear until day 6 in the present study could possibly be due to the incubation of cells in rich medium. Hixon et al. showed that growth inhibition rather than cell death did not exert sufficient selective pressure in S. cerevisiae to reveal the resistance of petite mutants to drug toxicity, in contrast to the effect after incubation in water or buffer (Hixon et al., 1980). The FLU-resistant C. glabrata isolate showed no respiratorydeficient cells at all throughout the long-term experiment. The activated resistance mechanisms of this isolate seem to be sufficient to overcome DOX cytotoxicity. The switch between states of mitochondrial competence and incompetence of C. glabrata cells reported by Kaur et al. (2004) cannot explain this phenomenon, since the FLU-resistant isolate Cg3 was respiratory-competent throughout the experiments. The isolated petite mutants of all three FLU-susceptible C. glabrata isolates were highly resistant to FLU and overexpressed both CgPDR1 and ABC transporter genes. A study previously published by Tsai et al. (2006) showed that mitochondrial dysfunction increases CgPDR1 expression, which may be one explanation for the highly resistant phenotype of respiratory-deficient cells. Petite mutation in C. glabrata has been demonstrated not only in vitro but also in vivo (Bouchara et al., 2000; Ferrari et al., 2011). A recently published study by Ferrari et al. (2011) described a highly resistant respiratory-deficient C. glabrata mutant from a patient undergoing azole therapy. Since this isolate was selected in vivo and showed greater fitness in mice than its parental precursor,
mitochondrial dysfunction may confer an advantage on yeast cells in host tissues and may be the reason for antifungal treatment failure.
Conflicts of interest The authors declare that they have no conflicts of interest in relation to this work.
References Bouchara, J.P., Zouhair, R., Le, B.S., Renier, G., Filmon, R., Chabasse, D., Hallet, J.N., Defontaine, A., 2000. In vivo selection of an azole-resistant petite mutant of Candida glabrata. J. Med. Microbiol. 49, 977–984. Brun, S., Aubry, C., Lima, O., Filmon, R., Berges, T., Chabasse, D., Bouchara, J.P., 2003. Relationships between respiration and susceptibility to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 47, 847–853. Brun, S., Berges, T., Poupard, P., Vauzelle-Moreau, C., Renier, G., Chabasse, D., Bouchara, J.P., 2004. Mechanisms of azole resistance in petite mutants of Candida glabrata. Antimicrob. Agents Chemother. 48, 1788–1796. Bulder, C.J., 1964. Induction of petite mutation and inhibition of synthesis of respiratory enzymes in various yeasts. Anton. Van Leeuw. 30, 1–9. Buschini, A., Poli, P., Rossi, C., 2003. Saccharomyces cerevisiae as an eukaryotic cell model to assess cytotoxicity and genotoxicity of three anticancer anthraquinones. Mutagenesis 18, 25–36. Carter, S.K., 1975. Adriamycin—a review. J. Natl. Cancer Inst. 55, 1265–1274. Clinical Laboratory Standards Institute, 1997. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Approved Standard M27-A. National Committee for Clinical Laboratory Standards, Wayne, PA. Coleman, D.C., Moran, G.P., McManus, B.A., Sullivan, D.J., 2010. Mechanisms of antifungal drug resistance in Candida dubliniensis. Future Microbiol. 5, 935–949. Dougherty, P.F., Yotter, D.W., Matthews, T.R., 1977. Microdilution transfer plate technique for determining in vitro synergy of antimicrobial agents. Antimicrob. Agents Chemother. 11, 225–228. Ferrari, S., Sanguinetti, M., De, B.F., Torelli, R., Posteraro, B., Vandeputte, P., Sanglard, D., 2011. Loss of mitochondrial functions associated with azole resistance in Candida glabrata results in enhanced virulence in mice. Antimicrob. Agents Chemother. 55, 1852–1860. Goswami, D., Goswami, R., Banerjee, U., Dadhwal, V., Miglani, S., Lattif, A.A., Kochupillai, N., 2006. Pattern of Candida species isolated from patients with diabetes mellitus and vulvovaginal candidiasis and their response to single dose oral fluconazole therapy. J. Infect. 52, 111–117.
B. Schulz et al. / International Journal of Medical Microbiology 302 (2012) 155–161 Hixon, S.C., Ocak, A., Thomas, J.E., Daugherty, J.P., 1980. Resistance to adriamycin cytotoxicity among respiratory-deficient mutants in yeast. Antimicrob. Agents Chemother. 17, 443–449. Kaur, R., Castano, I., Cormack, B.P., 2004. Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob. Agents Chemother. 48, 1600–1613. Kofla, G., Ruhnke, M., 2007. Development of a new real-time TaqMan PCR assay for quantitative analyses of Candida albicans resistance genes expression. J. Microbiol. Methods 68, 178–183. Kofla, G., Turner, V., Schulz, B., Storch, U., Froelich, D., Rognon, B., Coste, A.T., Sanglard, D., Ruhnke, M., 2011. Doxorubicin induces drug efflux pumps in Candida albicans. Med. Mycol. 49, 132–142. Kule, C., Ondrejickova, O., Verner, K., 1994. Doxorubicin, daunorubicin, and mitoxantrone cytotoxicity in yeast. Mol. Pharmacol. 46, 1234–1240. Nucci, M., Marr, K.A., 2005. Emerging fungal diseases. Clin. Infect. Dis. 41, 521–526. Odds, F.C., 2003. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 52, 1. Pfaller, M.A., Castanheira, M., Messer, S.A., Moet, G.J., Jones, R.N., 2010a. Variation in Candida spp. distribution and antifungal resistance rates among bloodstream infection isolates by patient age: report from the SENTRY Antimicrobial Surveillance Program (2008–2009). Diagn. Microbiol. Infect. Dis. 68, 278–283. Pfaller, M.A., Diekema, D.J., 2004. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 42, 4419–4431. Pfaller, M.A., Diekema, D.J., 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin. Microbiol. Rev. 20, 133–163. Pfaller, M.A., Diekema, D.J., Gibbs, D.L., Newell, V.A., Ellis, D., Tullio, V., Rodloff, A., Fu, W., Ling, T.A., 2010b. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion. J. Clin. Microbiol. 48, 1366–1377. Ro, D.K., Ouellet, M., Paradise, E.M., Burd, H., Eng, D., Paddon, C.J., Newman, J.D., Keasling, J.D., 2008. Induction of multiple pleiotropic drug resistance genes in
161
yeast engineered to produce an increased level of anti-malarial drug precursor, artemisinic acid. BMC Biotechnol. 8, 83. Safdar, A., Chaturvedi, V., Cross, E.W., Park, S., Bernard, E.M., Armstrong, D., Perlin, D.S., 2001. Prospective study of Candida species in patients at a comprehensive cancer center. Antimicrob. Agents Chemother. 45, 2129–2133. Sanglard, D., Ischer, F., Bille, J., 2001. Role of ATP-binding-cassette transporter genes in high-frequency acquisition of resistance to azole antifungals in Candida glabrata. Antimicrob. Agents Chemother. 45, 1174–1183. Sanglard, D., Ischer, F., Calabrese, D., Majcherczyk, P.A., Bille, J., 1999. The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents. Antimicrob. Agents Chemother. 43, 2753–2765. Schulz, B., Knobloch, M., Moran, G.P., Weber, K., Ruhnke, M., 2012. Influence of doxorubicin on fluconazole susceptibility and efflux pump gene expression of Candida dubliniensis. Med. Mycol. 50, 421–426. Segal, B.H., Kwon-Chung, J., Walsh, T.J., Klein, B.S., Battiwalla, M., Almyroudis, N.G., Holland, S.M., Romani, L., 2006. Immunotherapy for fungal infections. Clin. Infect. Dis. 42, 507–515. Thakur, J.K., Arthanari, H., Yang, F., Pan, S.J., Fan, X., Breger, J., Frueh, D.P., Gulshan, K., Li, D.K., Mylonakis, E., Struhl, K., Moye-Rowley, W.S., Cormack, B.P., Wagner, G., Naar, A.M., 2008. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452, 604–609. Torelli, R., Posteraro, B., Ferrari, S., La, S.M., Fadda, G., Sanglard, D., Sanguinetti, M., 2008. The ATP-binding cassette transporter-encoding gene CgSNQ2 is contributing to the CgPDR1-dependent azole resistance of Candida glabrata. Mol. Microbiol. 68, 186–201. Tsai, H.F., Krol, A.A., Sarti, K.E., Bennett, J.E., 2006. Candida glabrata PDR1, a transcriptional regulator of a pleiotropic drug resistance network, mediates azole resistance in clinical isolates and petite mutants. Antimicrob. Agents Chemother. 50, 1384–1392. Westermeyer, C., Macreadie, I.G., 2007. Simvastatin reduces ergosterol levels, inhibits growth and causes loss of mtDNA in Candida glabrata. FEMS Yeast Res. 7, 436–441.