Journal de Mycologie Médicale (2014) 24, e169—e177
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride on in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata ´ s d’une hypoxie et d’une hypoxie au chlorure de Effets compare ´ rence, la formation de biofilm et la cobalt in vitro sur l’adhe ´ `a l’amphote ´ ricine B de Candida glabrata sensibilite P. Gupta a, S. Nath b, R.C. Meena b, N. Kumar a,* a
Department of Biotechnology, Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun, 248001 Uttarakhand, India b Department of Molecular Biology, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, 110054 Delhi, India Received 30 January 2014; received in revised form 26 June 2014; accepted 18 August 2014 Available online 22 October 2014
KEYWORDS Candida glabrata; Hypoxia; Cobalt chloride; Virulence; Biofilm; Adhesion; 1% O2; Amphotericin B
Summary Objectives. — Candida glabrata has emerged as potent pathogen in nosocomial infections. The objective of this study is to investigate the effects of hypoxia (an important host factor) and hypoxia mimetic cobalt chloride upon growth, in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata. Materials and methods. — Growth was checked by spotting assays. Expression of TDH3, a gene of glycolytic pathway was analyzed as intracellular hypoxia marker by reverse transcription PCR. In vitro adhesion, biofilm development and susceptibility of biofilm to amphotericin B were performed on polystyrene plates and quantified by XTT assay in RPMI 1640 and YNB media. Experiments were performed in triplicates and Student’s t-test was used for statistical analysis.
* Corresponding author. Tel.: +91 7417325585; +91 135 2642799; 91 135 2642727X217; fax: +91 135 26644025. E-mail addresses: [email protected], [email protected] (N. Kumar). http://dx.doi.org/10.1016/j.mycmed.2014.08.003 1156-5233/# 2014 Published by Elsevier Masson SAS.
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P. Gupta et al. Results. — Hypoxia did not compromise the growth of C. glabrata unlike CoCl2. Hypoxia and CoCl2 upregulated TDH3 expression. Adhesion was reduced upon exposure to hypoxia and CoCl2. Biofilm activity remained unchanged in the presence of CoCl2 in both media. In comparison to normoxia control, hypoxia increased biofilm activity to 259.33 22.05% in RPMI 1640, while hypoxia reduced it to 70.99 2.99% in YNB. Biofilm susceptibility to amphotericin B was significantly decreased in RPMI 1640 and remained unaffected in YNB in hypoxia. Conclusions. — C. glabrata grows well even under hypoxia but not upon CoCl2 exposures. CoCl2 mimics hypoxia like expression of TDH3 but affects the virulence properties unlike hypoxia. Both, hypoxia and CoCl2 affects adhesion adversely. Hypoxia increases biofilm development and reduces the susceptibility of biofilm to amp B in RPMI 1640 but not in YNB. # 2014 Published by Elsevier Masson SAS.
MOTS CLÉS Candida glabrata ; Hypoxie ; Chlorure de Cobalt ; Virulence ; Biofilm ; Adhérence ; Amphotéricine B
Re ´sume ´ Objectifs. — Candida glabrata a émergé comme un important agent pathogène, cause d’infections nosocomiales. L’objectif de cette étude était d’étudier les effets d’une hypoxie (un important facteur de l’hôte) et l’hypoxie induite par le chlorure de cobalt sur la croissance in vitro, sur l’adhérence, la formation de biofilm et la sensibilité à l’amphotéricine B de C. glabrata. ´thodes. — La croissance a été étudiée par un spotting test. L’expression de TDH3, Mate ´riels et me un gène de la voie glycolytique, a été analysée comme marqueur d’une hypoxie intracellulaire par transcription inverse-PCR. In vitro, l’adhérence, le développement de biofilm et la sensibilité des biofilms à l’amphotéricine B ont été étudiés sur plaques de polystyrène et quantifiés par dosage XTT en milieu RPMI 1640 et YNB. Des expériences ont été effectuées en triplicate et le test t de Student a été utilisé pour l’analyse statistique. Re ´sultats. — Hypoxie n’a pas compromis la croissance de C. glabrata contrairement à l’action du CoCl2. Hypoxie et CoCl2 exercent une régulation positive sur l’expression de TDH3. L’adhérence était réduite lors de l’exposition à l’hypoxie et au CoCl2. La production de biofilm est restée inchangée en présence de CoCl2 dans les deux milieux. En comparaison de contrôles en normoxie, l’hypoxie a augmenté la production de biofilm de 259,33 22,05 % en RPMI 1640, tandis qu’une hypoxie a réduit cette production de 70,99 2,99 % en YNB. La sensibilité du biofilm à l’amphotéricine B a été considérablement diminuée en RPMI 1640 et est restée inchangée en YNB en hypoxie. Conclusions. — C. glabrata pousse bien, même sous hypoxie mais pas en présence de CoCl2. Le CoCl2 imite une hypoxie pour l’expression de TDH3 mais affecte des propriétés de virulence contrairement à l’hypoxie. Hypoxie et CoCl2 affectent négativement l’adhérence. L’hypoxie augmente le développement de biofilm et réduit la sensibilité du biofilm à l’amphotéricine B en RPMI 1640 mais pas en YNB. # 2014 Publié par Elsevier Masson SAS.
Introduction Incidence of invasive infections due to Candida is increasing alarmingly worldwide, mainly due to medical device interventions and immunosuppressive states in different ailments like AIDS and cancer [6,11,30]. Candida albicans and Candida glabrata are two clinically important fungal pathogens of humans [3,29]. Both are opportunistic commensals found, especially, in oral cavity and gastrointestinal tract of healthy humans [9,42]. C. glabrata has become second most causative of bloodstream infections in human after C. albicans in the last two decades [31,32,37]. C. glabrata is a non-dimorphic, haploid yeast, which accounts for approximately 15% of all Candida infections particularly blood stream infections worldwide [33] and responsible for 11 to 37% candidaemia cases in the United States across the nine US Bureau of the Census Regions [30]. C. glabrata is more similar to Saccharomyces cerevisiae than C. albicans in several aspects like virulence mechanisms, genetic repertoire, life cycle, morphogenesis [2,20,39].
During colonization and infection in animal hosts, the pathogen faces environmental challenges that are crucial for their survival and virulence. One such challenge is hypoxia to which both pathogen and host tissue are exposed [21,25]. C. glabrata has different adaptations for its pathogenic mode in the host body like adaptation to hypoxia which is an important virulence characteristic [14,39]. Hypoxia mediated transcriptional regulations and its effect on virulence in C. albicans and other fungi have been investigated [4,8,14,44]. This regulation includes the increased expression of genes encoding enzymes of sterol, lipids and heme biosynthesis along with glycolytic pathways. Sterol regulatory element-binding proteins (SREBPs) are the regulators of hypoxic responses in mammals that regulate the expression of cholesterol synthesis genes [17]. Members of ascomycete (Schizosaccharomyces pombe) and basidiomycete (Crptococcus neoformans) have SREBP homologs to control the expression of genes involved in biosynthesis of lipid, ergosterol and heme [4]. But in some members of Saccharomycotina (S. cerevisiae, C. albicans, C. parapsilopsis), the role of
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride SREBPs is replaced by different regulator Upc2 and Ecm22, which binds sterol regulatory element in the promoters of target genes [47]. In C. albicans, Upc2 and Efg1 (transcription factors) regulate genes of ergosterol and fatty acid biosynthesis pathways respectively under hypoxic conditions, while Tye7 and Gal4 induce genes of glycolytic pathway in hypoxia [1,40,44]. C. parapsilosis has been studied for transcriptional profile of biofilm formation under hypoxic condition and the genes of glycolysis, fatty acid metabolism and ergosterol synthesis were found to be upregulated, suggesting interrelationship of biofilm development with hypoxia in other Candida species [38]. Cobalt chloride, in sublethal concentration mimics hypoxia like state in S. cerevisiae [13,23,27,46], C. neoformans [24] and mammalian systems [48]. In C. neoformans, both CoCl2 and low oxygen conditions affect the majority of genes of ergosterol pathway and iron/copper transport in similar manner. These common transcriptional responses are mediated through common Sre1p. pathway by which yeast cells sense and adapt to CoCl2 and oxygen levels [24]. Hypoxia is reported to be associated tightly with the virulence attributes especially adhesion, biofilm development and response to antifungal drugs in other fungal pathogens [4,5,38,49]. But the effect of hypoxia upon virulence characteristics mainly adhesion, biofilm formation and susceptibility to antifungal remain unaddressed for C. glabrata. It has also not been explored whether cobalt chloride (a hypoxia mimetic in transcriptional responses of genes of ergosterol biosynthesis, lipid biosynthesis and glycolytic pathways) has hypoxia like effects upon virulence characteristics. Therefore, we performed a comparative study to analyze the effects of hypoxia and cobalt chloride upon key virulence properties like growth, in vitro adhesion, biofilm formation and resistance to antifungal for C. glabrata.
Materials and methods Cultures, culture condition and chemicals C. glabrata (MTCC3019) strain was procured from the Institute of Microbial Technology (CSIR Laboratory), Chandigarh, India. Strain was routinely cultivated at 37 8C in YPD medium (1% yeast extract, 2% Bacto-peptone and 2% dextrose; BD) in orbital shaker at 120 rpm. Biofilm and adhesion assays were performed in RPMI 1640 (HiMedia) medium supplemented with 50 mM HEPES and L-Glutamine, pH 7.0 (referred as to RPMI 1640 henceforth) and YNB medium (0.67% yeast nitrogen base w/o amino acids with ammonium sulfate supplemented with 2% dextrose; BD; pH 7.0) on the surface of pre-sterilized, polystyrene, flat bottomed 96-well microtiter plates (HiMedia) [24,31]. Cobalt chloride (CoCl2; Sigma-Aldrich Chemicals Ltd.) at a concentration of 0.75 mM was used as hypoxia mimetic agent. All other routine chemicals of molecular grade were procured from Merck and plastic-wares from Tarson. Amphotericin B (amp B), Menadione and 2,3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were purchased from HiMedia. For exposure of cells to hypoxic conditions in solid media, cells were grown in YPD plates either containing 0.75 mM CoCl2 or the plates were incubated in a hypoxic chamber
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(multi-gas incubator, CO2/O2 incubator Model GA156, LEEC, UK) with 1% O2, 5% CO2 and 94% N2. Similarly, cells were grown on the surface of polystyrene, flat bottomed 96-well microtiter plates either containing 0.75 mM CoCl2 or the plates were incubated in a hypoxic chamber for exposures to hypoxia mimetic agent and hypoxia respectively for adhesion, biofilm and susceptibility of biofilm to antifungal assays.
Effect of hypoxia and CoCl2 on growth Growth was analyzed using plate spotting assay [23]. Log phase cells of C. glabrata (OD600nm: 0.6) were spotted in 10 fold serial dilutions (103, 102, 101, 100 cells) onto YPD agar plates with or without CoCl2. Different concentrations of CoCl2 (0.5 mM, 0.75 mM, 1.0 mM and 5 mM) were maintained on the agar plates. For normoxic control and hypoxia, YPD plates without cobalt chloride were spotted and incubated at respective conditions. Experiments were done in triplicates. Plates were incubated at 37 8C for 18 hours (h).
Effect of CoCl2 on colony forming unit (CFU) We followed Sousa et al., [43] to find out sub lethal concentration of CoCl2 for C. glabrata that do not cause significant lethality. We selected 0.75 mM and 1.0 mM CoCl2 because in other yeasts 0.75 mM has been used to mimic hypoxia; we also took 1.0 mM to see the effect of immediate higher concentration. Log phase cells (107cells/mL) of C. glabrata (grown in YPD) were exposed to CoCl2 in PBS for 2 h. Afterwards, cell suspensions having 100 cells were spread onto YPD plates without CoCl2. Plates were incubated at 37 8C for 18 h and colony forming units (CFU) were counted for each plate. Experiment was done in triplicate. Mean values SD of one of three independent assays are represented and Student’s t-test was applied to analyze the significant differences in the number of CFU.
Analysis of gene expression by semi-quantitative RT-PCR Semi-quantitative reverse transcription PCR was performed to analyze the expression of hypoxia marker gene upon exposure to normoxia, hypoxia and 0.75 mM CoCl2 in C. glabrata [15]. TDH3, a gene of glycolytic pathway was used as hypoxia marker [44] and ACT1 as control. Total RNA was isolated from the log phase cells exposed to hypoxia and 0.75 mM CoCl2 separately for 2 h along with unexposed control cells using RNeasy Kit from Qiagen. RNA was quantified and purity was checked using spectroscopic method [15] using Eppendorf Biophotometer Plus-1632. Reverse transcription (RT) was done using Fermentas First Strand cDNA Synthesis Kit (Cat. No. #K1611) followed by PCR using specific primers synthesized from Bioserve, Hyderabad (Table 1). Taq polymerase and dNTPs were from SigmaAldrich. PCR products were checked by running onto 1.2% agarose gel in TAE before being photographed by gel documentation system (Merck- GeNei). Band intensities of ACT1 and TDH3 expressed under different conditions were measured using Image J software in arbitrary units. Intensities of DNA bands of genes in three independent experiments of
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Table 1 Sequences of the primer used in this study. ´ dans cette ´etude. ´ quences du primer utilise Se Sr. No.
Name of primer
Sequence of primer (50 —30 )
1 2 3 4
ACT1F ACT1R TDH3F TDH3R
ATGGATTCTGAAGTTGCT TTAGAAACACTTGTGGTG ATGGTTAGAATTGCTATT TTAGTTCTTGGCAACGTG
agarose gel electrophoresis were used to calculate mean SD. Significant differences in gene expressions under normoxic and hypoxic conditions were analyzed as mentioned below in statistical analysis.
Effect on adhesion In vitro adhesion assays were performed to investigate the effect of hypoxia and 0.75 mM CoCl2 on adherence of C. glabrata cells in YNB and RPMI 1640 medium on the surface of pre-sterilized, polystyrene, flat bottomed 96-well microtiter plate [35]. One hundred mL of cell suspension (107 cells/ mL) in media without and with CoCl2 was added to the wells respectively and plates were incubated at 37 8C on normoxia for 90 minutes with shaking. For hypoxia, cell suspension in media (107cells/mL) was added to wells and plates were incubated at 37 8C for 90 minutes After incubation of 90 minutes, wells were washed twice with 200 mL of PBS to remove the unadhered cells. The density of adhered cells to each well was quantified using XTT reduction assay following Ramage et al., [34] with minor modifications. Absorbance was measured at 450 nm using ELISA Reader (Molecular Devices, SPECTRA max M2). Values are represented in terms of relative metabolic activity (RMA) in percentage, relative to the adhesion of respective normoxia control (Mean of metabolic activities of normoxia control is taken as 100%).
Effect on biofilm formation C. glabrata biofilm was developed on the surface of presterilized, polystyrene, flat bottomed 96-well microtiter plate [36]. Briefly, cell suspension was prepared in PBS at a concentration of 107 cells/mL and 100 mL of suspension was added to each well. Plates were incubated for 90 min at 37 8C on normoxic condition for adhesion phase. Adhesion was visualized under inverted light microscope (OLYMPUS). Wells were washed twice with 200 mL of PBS to remove nonadhered cells. For control and CoCl2 exposure, 200 mL of culture media without and with CoCl2 was added to the wells respectively and plates were incubated at 37 8C on normoxia for 60 h with shaking. For hypoxia, culture media was added to wells and plate was incubated in low oxygen chamber at 37 8C for 60 h. Afterwards, wells were again washed twice with 200 mL of PBS and biofilm was quantified by XTT reduction assay [34] at 450 nm using ELISA Reader (Molecular Devices, SPECTRA max M2). Biofilm developed is represented in terms of relative metabolic activity (RMA) in percentage, relative to the biofilm of respective normoxia control (Mean of metabolic activities of normoxia control is taken as 100%).
Effect on biofilm antifungal drug susceptibility Effect of hypoxia upon susceptibility of biofilm development to antifungal drug was analyzed in the presence of different concentrations of amp B (0, 0.0625, 0.125, 0.25 mg/mL) following the methodology as mentioned above by XTT reduction assay [34]. We investigated the susceptibility of C. glabrata biofilm development to amp B in both the media YNB and RPMI 1640 upon exposure to hypoxia in comparison to normoxia controls.
Statistical analysis All experiments were performed in triplicates and values presented are the mean with standard deviation, obtained from three different observations for XTT assays. Student’s t-test was used for statistical analysis and a value of P < 0.05 was considered statistically significant (*) and P < 0.001 as highly significant (**) for comparisons [17].
Results C. glabrata growth was not affected by hypoxia but by CoCl2 As shown in Fig. 1A, exposure to 1% O2 did not affect the growth of C. glabrata on YPD agar plate when compared with normoxia control. On the other hand, C. glabrata growth on CoCl2 containing YPD agar plates was found to be compromised in different magnitudes according to the concentrations of CoCl2 when compared with normoxia control (Fig. 1A). Cells were significantly sensitive when grown in the presence of 1 mM CoCl2, while CoCl2 at a concentration of 5 mM was found to be lethal in our experiments. We found 0.5 mM and 0.75 mM CoCl2 to be the sublethal concentrations for C. glabrata (Fig. 1A). Therefore, we used CoCl2 at a concentration of 0.75 mM in our further experiments as a hypoxia mimetic that has also been used for S. cerevisiae by other groups [26,30].
0.75 mM CoCl2 did not affect cell viability In our CFU counting experiments, CoCl2 at a concentration of 1 mM was found to be significantly lethal when compared with untreated cells (P < 0.05), while 0.75 mM of CoCl2 did not affect cell survival in comparison to untreated cells (P > 0.05) (Fig. 1B). Henceforth, CoCl2 at a concentration of 0.75 mM was used in this study.
Hypoxia and CoCl2 upregulated TDH3 expression TDH3, a gene of glycolytic pathway was used as marker for intracellular hypoxia along with a control ACT1 to compare the effects of 1% O2 and 0.75 mM CoCl2 on the expression of hypoxia marker gene along with normoxia control. As per the qualitative observation (Fig. 1C and D), expression of TDH3 was significantly upregulated in the log phase cells after exposure to 1% O2 and 0.75 mM CoCl2 for 2 h in C. glabrata, while expression of control gene ACT1 remained statistically unchanged under all conditions when compared to respective normoxic controls. Unlike ACT1, changes in the
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride
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Figure 1 Effect of hypoxia and cobalt chloride on growth and cell viability of C. glabrata. A. Growth of C. glabrata cells in spotting assays onto YPD agar plates upon exposure to different conditions; B. Cell viability of C. glabrata cells upon exposure to different concentrations of CoCl2 counted in form of CFU Mean values of three independent CFU experiments SD is shown and (*) represents statistically significant differences in CFU with respect to normoxia control (P < 0.05); C. Expression of TDH3 and ACT1 gene under normoxia, hypoxia and 0.75 mM CoCl2 conditions by semi-quantitative RT-PCR; D. Average of three band intensities SD of the TDH3 and ACT1 upon exposure to normoxia (Nor), hypoxia (Hyp) and 0.75 mM CoCl2 (CoCl2) is shown in form of bar diagram and (**) represents statistically highly significant differences (P < 0.001) in band intensity with respect to normoxia control of particular gene. ´ cellulaire de C. glabrata. A. La croissance de C. glabrata par Effet de l’hypoxie et du chlorure de cobalt sur la croissance et la viabilite ´ lose YPD lors de l’exposition `a des conditions diffe ´ rentes ; B. Viabilite ´ cellulaire de la technique du spottings sur des plaques de ge ´ rentes concentrations de CoCl2 exprime ´ e en cfu. Valeurs moyennes SD des cfu de trois C. glabrata lors de l’exposition `a diffe ´ pendantes (*) repre ´ sentent des diffe ´ rences statistiquement significatives des cfu `a l’e ´ gard du contro ˆ le normoxique experiences inde ` nes de TDH3 et ACT1 sous normoxie, hypoxie et 0,75 mm CoCl2 en RT-PCR semi-quantitative ; ( p < 0,05) ; C. Expression des ge ´ s de bande SD de TDH3 et ACTl1 lors de l’exposition `a la normoxie (Ni), `a l’hypoxie (Hyp) et `a 0,75 mM D. Moyenne de trois intensite ´ e sous forme de diagramme `a barres, (**) repre ´ sente des diffe ´ rences statistiquement tre ` s importantes CoCl2 (CoCl2) illustre ´ de bande compare ´ au ge ` ne contro ˆ le en normoxie. ( p < 0,001) en intensite
expression of TDH3 were statistically highly significant (P < 0.001) when exposed to 0.75 mM CoCl2 and 1% O2 Ratios of TDH3/ACT1 band intensities were 1.713 for normoxia, 2.482 for hypoxia and 2.281 for CoCl2 experiments. Therefore, it has been concluded that CoCl2 at a concentration of 0.75 mM is adequate to mimic hypoxia like intracellular gene expression without inducing lethality in C. glabrata.
Hypoxia and CoCl2 affected adhesion and biofilm differentially Since adhesion and biofilm formation by Candida are affected by several environmental conditions, therefore, we compared the effect of hypoxia and CoCl2 on adhesion and biofilm formation by C. glabrata in two commonly used media, namely RPMI 1640 and YNB.
In YNB medium, reduction in adhesion potential was reported to be significant upon exposure to CoCl2 (RMA: 77.19 4.3214%; P < 0.05) and highly significant upon exposure to hypoxia (RMA: 69.67 3.9825%; P < 0.001) (Fig. 2A). In RPMI 1640 medium, reduction in adhesion potential was reduced insignificantly upon exposure to CoCl2 (RMA: 90.16 3.9098%; P > 0.05) and significantly upon exposure to hypoxia (RMA: 70.84 4.4050%; P < 0.05) (Fig. 2A). In YNB medium, change in biofilm developed remained almost unaffected upon exposure to CoCl2 (RMA: 110 10.727%; P > 0.05), while biofilm activity was reduced significantly upon exposure to hypoxia (RMA: 70.99 2.9964%; P < 0.05) (Fig. 2B). Biofilm activity in RPMI 1640 was not affected in the presence of CoCl2 (RMA: 102.22 11.827%; P > 0.05) but increased to 259.33 22.053%) with highly significant difference (P < 0.001), when compared to normoxia control (Fig. 2B).
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Hypoxia reduced susceptibility of biofilm formation to antifungal Since hypoxia increased biofilm formation in RPMI 1640 medium, we anticipated the positive role of hypoxia in reducing the biofilm susceptibility to existing antifungal drug like amp B. In YNB medium, hypoxia could not drive the biofilm development significantly different from that of normoxic control in the presence of gradient of concentration of amp B (0, 0.0625, 0.125, 0.25 mg/mL) (Fig. 3A) and these differences in OD at 450 nm were not found to be significant statistically (P > 0.05, except for the point 0.125 mg/mL of amp B). Mean values of absorbance SD along with Student’s ttest values are shown in Table 2. Hypoxia in RPMI 1640 increased the biofilm development in the presence of amp B, when compared with normoxic biofilm activity to same antifungal and these differences were statistically significant (P < 0.05) (Fig. 3B). Thus, hypoxia reduced the susceptibility of biofilm development to the amp B in RPMI 1640 medium. Mean values of absorbance SD along with Student’s t-test values are shown in Table 3.
Discussion During pathogenesis, C. glabrata faces hypoxic microenvironment inside host tissues. Oxygen level in human tissues ranges 20—70 mmHg (2.5—9% O2) and in damaged tissue, it is less than 10 mm Hg (1% O2) [21,25]. Cobalt chloride has been widely used as a hypoxia mimetic for inducing low oxygen like conditions for biochemical and transcriptional responses in different models [13,48]. In yeast, cobalt is reported to target diiron-oxo enzymes involved in sterol pathway (Erg3p and Erg25p) and fatty acid pathway (Ole1p);
P. Gupta et al. similarly hypoxia affects sterol biosynthesis and fatty acid synthesis due to the requirement of molecular oxygen at various steps [13,18]. We studied the comparative effects of hypoxia and cobalt chloride on growth, in vitro adhesion, biofilm formation and anti fungal susceptibility of C. glabrata. In our experiments, moderate sensitivity of C. glabrata to CoCl2 might be due to inhibitory effect of cobalt on metabolic pathways as evidenced in earlier studies [13]. 0.75 mM CoCl2 was not found to cause lethality rather sufficient to induce transcriptional responses as evidenced by upregulation of TDH3 (a representative gene of glycolytic pathway) [44], similar to hypoxia (Fig. 1B and D). Hypoxia is reported to induce similar transcriptional response in C. parapsilopsis, C. albicans, and S. cerevisiae [38,44,46]. Our results confirmed that hypoxia has no adverse effect on the growth of C. glabrata (Fig. 1A), indicating the presence of necessary mechanisms for adaptation to hypoxia in the pathogen mediated through transcriptional responses. We found that hypoxia and CoCl2 had significantly reduced adhesion potential of C. glabrata in both media (except for CoCl2 exposure in RPMI 1640) (Fig. 2A), probably due to the effects of hypoxia and/or cobalt exposure on cell metabolism [13] and requirement of more time for transcriptional responses. EPA genes (a family of glycosylphosphatydylinnositol (GPI)-anchored cell wall protein genes) code for adhesins which are effectors of virulence properties (adhesion and biofilm) in C. glabrata [10,19,36]. Higher expression of EPA6 is reported in stationary phase culture of C. glabrata [19], while we used log phase cells for adhesion. Hypoxia is also reported to induce C. glabrata EPA6 expression even in the low density cultures but in the presence of paraben stress [28]. Previous reports also support our observation where cobalt was inhibitory for biofilm formation, if planktonic inoculums of C. albicans and C. tropicalis were pretreated
Figure 2 Effect of hypoxia and CoCl2 on in vitro adhesion and biofilm formation by C. glabrata. A. Comparative relative metabolic activity of in vitro adhesion of C.glabrata cells under different conditions in different media; B. Comparative relative metabolic activity of in vitro biofilm formation by C. glabrata cells under different conditions in different media. RMA is the % metabolic activity of adhesion and biofilm formed under specific conditions relative to normoxia control in that particular medium. Mean values of RMA SD of three independent experiments are shown; P < 0.05 is considered statistically significant and P < 0.001 highly significant and represented as (*) and (**) respectively. ´ rence et la formation de biofilm par C. glabrata. A. Comparaison in vitro de l’activite ´ Effet d’hypoxie et de CoCl2 in vitro sur l’adhe ´ rence de C. glabrata dans des conditions diffe ´ rentes et dans diffe ´ rents milieux ; B. Comparaison in vitro de ´ tabolique et de l’ adhe me ´ rentes et dans diffe ´ rents milieux. RMA ´ me ´ tabolique et de la formation de biofilm par C. glabrata dans des conditions diffe l’activite ´ me ´ tabolique, de l’adhe ´ rence et de biofilm forme ´ dans des conditions spe ´ cifiques relatives au contro ˆ le de la est le % d’activite ´ riences inde ´ pendantes sont indique ´ es ; p < 0,05 est normoxie dans ce milieu. Les valeurs moyennes de RMA SD de trois expe ´ sente ´ par (*) et (**) respectivement. ´ re ´ comme statistiquement significatif et p < 0,001 hautement significatif et est repre conside
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride
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Figure 3 Effect of hypoxia on susceptibility of in vitro biofilm development to Amphotericin B by C. glabrata. A. Susceptibility of in vitro biofilm development in YNB medium; B. Susceptibility of in vitro biofilm development in RPMI-1640 medium. Results were shown here as mean SD of A450 plotted on Y-axis against concentration of amp B on X- axis. Statistically significant differences in biofilm activities in presence of amp B between normoxia and hypoxia are shown as (*) (P < 0.05). ´ in vitro du de ´ veloppement de biofilm par C. glabrata sous l’amphote ´ ricine B. A. Sensibilite ´ in L’effet de l’hypoxie sur la sensibilite ´ veloppement du biofilm en milieu YNB ; B. Sensibilite ´ in vitro biofilm du de ´ veloppement du biofilm en milieu RPMI-1640. vitro du de ´ s sur l’axe des Y vis-a ` -vis des concentrations d’ampho B sur l’axe des X. Des diffe ´ rences statistiquement moyenne SD de 450 trace ´ biofilm en pre ´ sence d’ampho B entre normoxie et hypoxie sont indique ´ es (*) ( p < 0,05). significatives en activite
with 0.70 mM cobalt [16], indicating the adverse effect of cobalt on adhesion (phase earlier to biofilm formation). CoCl2 did not affect biofilm development significantly in either media (Fig. 2B; P > 0.05 for both media), probably due to sublethal concentration of CoCl2 and independency of virulence determinants (mainly adhesins) on CoCl2, which needs further studies to analyze the EPA expression in presence of CoCl2. In a study, MIC of biofilm was found to be around 4.2 mM for intermediate stage of biofilm formed by C. tropicalis [16], whereas we used 0.75 mM CoCl2. It was demonstrated previously that aerobic/anaerobic conditions led to differential biofilm development in different clinical
isolates of Candida; one of the C. glabrata isolates showed higher biofilm development under anaerobic/dynamic conditions similar to our results [45]. We found that hypoxia reduced biofilm significantly in YNB (P < 0.05) but increased biofilm strikingly in RPMI 1640 (P < 0.001) (Fig. 2B). Similarly in an earlier study, C. glabrata proliferated in adherent manner resulting in poor biofilm along with low level of EPA6 expression in YNB, in contrast to the thicker and multilayer biofilm formation accompanied by higher level of EPA6 expression in RPMI 1640 medium [22]. Oxygen deficient conditions in high density cultures are also reported to upregulate EPA6 expression
Table 2 Quantification of biofilm formation in the presence of ampho B under hypoxic and normoxic conditions in YNB medium. ´ sence d’ampho B sous conditions hypoxiques ou normoxiques en milieu YNB. Quantification de la formation de biofilm en pre Sr. No.
Concentration of amp B in mg/mL
Mean SD of OD 450 nm under normoxic condition
Mean SD of OD 450 nm under hypoxic condition
Values of P by Student’s t-test for given concentration of amp B in two conditions
1 2 3 4
0 0.0625 0.1250 0.2500
0.081 0.00529 0.067 0.00960 0.065 0.00862 0.062 0.00493
0.087 0.00529 0.085 0.00404 0.084 0.00550 0.058 0.00360
0.23724 0.06413 0.03549 0.72632
Table 3 Quantification of biofilm formation in the presence of ampho B under hypoxic and normoxic conditions in RPMI 1640 medium. ´ sence d’ampho B sous conditions hypoxiques ou normoxiques en milieu RPMI 1640. Quantification de la formation de biofilm en pre Sr. No.
Concentration of ampho B in mg/mL
Mean SD of OD 450 nm under normoxic condition
Mean SD of OD 450 nm under hypoxic condition
Values of P by Student’s t-test for given concentration of amp B in two conditions
1 2 3 4
0 0.0625 0.1250 0.2500
0.138 0.00655 0.126 0.00916 0.065 0.00888 0.061 0.00351
0.239 0.01379 0.151 0.00945 0.11 0.01050 0.078 0.00737
0.00128 0.02907 0.00505 0.03948
e176 in C. glabrata along with paraben stress [28]. In our results, abrupt increase in biofilm in RPMI1640 medium upon exposure to hypoxia may be due to induction of EPA genes due to the combined effect of hypoxia and RPMI1640 medium. Since hypoxia modulated biofilm, we further investigated the effect of hypoxia on biofilm development in the presence of amp B. We found that hypoxia has significantly increased biofilm formation by C. glabrata upon exposure to hypoxia in RPMI 1640 (Fig. 3B). These results could be explained in the light of following reasoning. Polyene antifungals target ergosterol of the fungal cell membrane to generate pores and causing leakage of cytoplasmic content [12,41] and hypoxia lowers ergosterol synthesis in yeast by affecting O2 mediated steps [13] which might be responsible for developing resistance to amp B. Hypoxia affects different steps of ergosterol biosynthesis involving oxygen dependent enzymes Erg1p. Erg3p, Erg5p, Erg11p and Erg25 and reduces the level of ergosterol in yeast [13]. Polyene resistance in Candida species results when ergosterol content lowers, probably due to mutation in ERG3 (gene that produces d 5,6-sterol desaturase) and ERG11 (gene that produces lanosterol 14a-demethylase) [7,26]. RPMI1640 is reported to induce C. glabrata biofilm development and expression of adhesins better than that in YNB [22]. Hypoxia also induces expression of adhesins (EPA6) in saturated cultures [28]. Hypoxia might also upregulate the expression of ABC transporters (CDR1, PDR16 and CDR2) in C. glabrata, as happened in the case of azole resistance [2,20]. To our knowledge, this is the first direct study on the comparative effects of hypoxia and hypoxia mimetic CoCl2 upon the virulence properties of C. glabrata in RPMI 1640 and YNB. Further studies on C. glabrata are being undertaken by our group for exploring the role of hypoxia in C. glabrata pathogenesis to find out potential molecular targets for the novel drug development.
Conclusion This study demonstrated that hypoxia augments biofilm formation by C. glabrata in RPMI1640 medium. We found that susceptibility of C. glabrata biofilm to amphotericin B decreases upon exposure to hypoxia in RPMI1640. Both, hypoxia and CoCl2 affects adhesion adversely. We found that CoCl2 mimics hypoxia in upregulating the expression of a gene of glycolytic pathway (TDH3), but appears not to cause hypoxia like effects upon growth and biofilm formation by C. glabrata.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement We thank Dr. Shashi Bala Singh Director, Defence Institute of Physiology and Allied Sciences, Delhi for giving us permission to perform experiments related to hypoxia chamber in DIPAS. We are grateful to Dr. Amitabha Chakrabarti, DIPAS
P. Gupta et al. for allowing us to use his laboratory for hypoxic exposures. PG is supported by the INSPIRE fellowship from Department of Science and Technology, Government of India and SN is supported by fellowship from Council of Scientific and Industrial Research, Government of India. We are also thankful to Dr. Ashish Thapliyal, Dr. Nishant Rai and Dr. Pankaj Gautam from Graphic Era University for their support during course of this study.
References [1] Askew C, Sellam A, Epp E, Hogues H, Mullick A, Nantel A, et al. Transcriptional regulation of carbohydrate metabolism in the human pathogen Candida albicans. Plos Pathog 2009;5: e1000612. [2] Bialkova A, Subik J. Biology of the pathogenic Yeast Candida glabrata. Folia Microbiol 2006;51:3—20. [3] Brunke S, Hube B. Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol 2013;15:701—8. [4] Butler G. Hypoxia and gene expression in eukaryotic microbes. Annu Rev Microbiol 2013;67:291—312. [5] Chung D, Haas H, Cramer RA. Coordination of hypoxia adaptation and iron homeostasis in human pathogenic fungi. Front Microbiol 2012;3:1—13. [6] Delaloye J, Calandra T. Invasive candidiasis as a cause of sepsis in the critically ill patient. Virulence 2014;5:1—9. [7] Dick JD, Merz WG, Saral R. Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob Agents Chemother 1980;18:158—63. [8] Ernst JF, Tielker D. Responses to hypoxia in fungal pathogens. Cell Microbiol 2009;11:183—90. [9] Fidel PL, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 1999;12:80—96. [10] Gabaldon T, Martin T, Marchet-Houben M, Durrens P, BolotinFukuhara M, Lespinet O, et al. Comparative genomics of emerging pathogens in the Candida glabrata clade. BMC Genomics 2013;14:623—39. [11] Galle F, Catania MR, Liguori G. Nosocomial Candida infections: epidemiology of candidaemia. J Prev Med Hyg 2006;47:119—26. [12] Ghannoum MA, Rice LB. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12:501—17. [13] Gleason JE, Corrigan DJ, Cox JE, Reddi AR, McGinnis LA, Culotta VC. Analysis of hypoxia and hypoxia-like states through metabolite profiling. PLoS One 2011;6:e24741. [14] Grahl N, Shepardson KM, Chung D, Cramer RA. Hypoxia and fungal pathogenesis: to air ot not to air? Eukaryot Cell 2012;11:560—71. [15] Guzman-Gonzalez KD, Parra-Ortega B, Martinez-Rivera MA, Hernandez-Rodriguez C, Perez NO, Rodriguez-Tovar AV. Investigating the role of Candida glabrata SLN1 gene in stress adaptation: in silico and molecular analysis. Afr J Microbiol Res 2013;7:460—70. [16] Harrison JJ, Ceri H, Yerly J, Rabiei M, Hu Y, Martinuzzi R, et al. Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms. Appl Environ Microbiol 2007;73:4940—9. [17] He M, Du M, Fan M, Bian Z. In vitro activity of eugenol against Candida albicans biofilms. Mycopathologia 2007;163:137—43. [18] Horton JD, Goldstein JL, Brown MS. SREBPs: activation of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125—31. [19] Iraqui I, Garcia-Sanchez S, Aubert S, Dromer F, Ghigo JM, d’Enfert C, et al. The Yak1p kinase controls expression of
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
adhesions and biofilm formation in Candida glabrata in a Sir4pdependent pathway. Mol Microbiol 2005;55:1259—71. Kaur R, Domergue R, Zupancic ML, Cormack BP. A yeast by any other name: Candida glabrata and its interaction with the host. Curr Opin Microbiol 2005;8:378—84. Kessler M, Höper J, Schäfer D, Starlinger H. In: Mikrozirkulation, Ahnefeld FW, Burri C, Dick W, Halmágyi M, editors. Sauerstofftransport im Gewebe. Berlin, Heidelberg, New York: Springer Verlag; 1974. p. 36—52. Kucharikova S, Tournu H, Lagrou K, Dijck PV, Bujdakova H. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J Med Microbiol 2011;60: 1261—9. Kumar N, Meena RC, Chakrabarti A. Over-expression of YLR162W in Saccharomyces cerevisiae inhibits cell proliferation and renders cell susceptible to the hypoxic conditions induced by CoCl2. Indian J of Microbiol 2011;51:206—11. Lee H, Bien CM, Hughes AL, Espenshade PJ, Kwon-Chung KJ, Chang YC. Cobalt chloride, a hypoxia-mimicking agent, targets sterol synthesis in the pathogenic fungus Cryptococcus neoformans. Mol Microbiol 2007;65:1018—33. Lewis JS, Lee JA, Underwood JCE, Harris AL, Lewis CE. Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol 1999;66:889—900. Lupeti A, Danesi R, Campa M, Tacca MD, Kelly S. Molecular basis of resistance to azole antifungals. Trends Mol Med 2002;8: 76—81. Meena RC, Kumar N, Nath S, Chakrabarti A. Homologous Recombination is activated at early time points following exposure to cobalt chloride induced hypoxic conditions in Saccharomyces cerevisiae. Indian J Microbiol 2012;52:209—23. Mundy RD, Cormack B. Expression of Candida glabrata adhesions after exposure to chemical preservatives. J Infect Dis 2009;199:1891—9. Papon N, Courdavault V, Clastre M, Bennett RJ. Emerging and emerged pathogenic Candida species: beyond the Candida albicans paradigm. PLOS Pathog 2013;9:e1003550. Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. Global surveillance of in vitro activity of micafungin against Candida: a comparison with caspofungin by CLSI-recommended methods. J Clin Microbiol 2006;44:3533—8. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Ellis D, Tullio V, et al. 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 2010;48:1366—77. Pfaller MA, Diekema DJ, International Fungal Surveillance Participant Group. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Clin Microbiol Infect 2004;1:11—23. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbio Rev 2007;20:133—63. Ramage G, Walle KV, Wickes BL, Lopez-Ribot JL. Standardized method for in vitro antifungal susceptibility testing of Candida
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43]
[44]
[45]
[46]
[47]
[48]
[49]
e177
albicans biofilm. Antimicrob Agent Chemother 2001;45: 2475—9. Raut JS, Chauhan NM, Shinde RB, Karuppayil SM. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by Candida albicans. Biofouling 2013;29:87—96. Riera M, Mogensen E, Enfert C, Janbon G. New regulators of biofilm development in Candida glabrata. Res Microbiol 2012;163:297—307. Rodrigues CF, Silva S, Henriques M. Candida glabrata: a review of its features and resistance. Eur J Clin Microbiol Infect Dis 2013. http://dx.doi.org/10.1007/s10096-013-2009-3 [Online link: http://www.link.springer.com/article/10.1007%2Fs10096-0132009-3]. Rossignol T, Ding C, Guida A, d’Enfert C, Higgins DG, Butler G. Correlation between biofilm formation and the hypoxic response in Candida parapsilosis. Eukaryot Cell 2009;8:550—9. Rotzer A, Gabaldon T, Schuller C. From Saccharomyces cerevisiae to Candida glabrata in a few easy steps: important adaptations for an opportunistic pathogen. FEMS Microbiol Lett 2011;314:1—9. Setadi ER, Doedi T, Cottier F, Noffz C, Ernst JF. Transcriptional response of Candida albicans to hypoxia: linkage of oxygen sensing and Efg1p-regulatory networks. J Mol Biol 2006;361:399—411. Silva S, Negri M, Henriques M, Oliveria R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36:288—305. Soll DR, Lockhart SR, Zhao R. Relationship between switching and mating in Candida albicans. Eukaryot Cell 2003;2:390—7. Sousa M, Duarte AM, Fernandes TR, Chaves SR, Pacheco A, Leao C, et al. Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced programmed cell death in Saccharomyces cerevisiae. BMC Genomics 2013;14:838—53. Synnott JM, Guida A, Mulhern-Haughey S, Higgins DG, Butler G. Regulation of the hypoxic response in Candida albicans. Eukaryot Cell 2010;9:1734—46. Thein ZM, Samaranayake YH, Samaranayake LP. In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions. Arch Oral Biol 2007;52:761—8. Vasconcelles MJ, Kevin YJ. Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. J Biol Chem 2001;276:14374—8. Vik A, Rine J. Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cereviseae. Mol Cell Biol 2001;21:6395—405. Wang GL, Semenza GL. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 1993;82:3610—5. Warn PA, Sharp A, Guinea J, Denning DW. Effect of hypoxic conditions on in vitro susceptibility testing of amphotericin B, itraconazole and micafungin against Aspergillus and Candida. J Antimicrob Chemother 2004;53:743—9.
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ORIGINAL ARTICLE/ARTICLE ORIGINAL
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride on in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata ´ s d’une hypoxie et d’une hypoxie au chlorure de Effets compare ´ rence, la formation de biofilm et la cobalt in vitro sur l’adhe ´ `a l’amphote ´ ricine B de Candida glabrata sensibilite P. Gupta a, S. Nath b, R.C. Meena b, N. Kumar a,* a
Department of Biotechnology, Graphic Era University, 566/6, Bell Road, Clement Town, Dehradun, 248001 Uttarakhand, India b Department of Molecular Biology, Defence Institute of Physiology and Allied Sciences, Lucknow Road, Timarpur, 110054 Delhi, India Received 30 January 2014; received in revised form 26 June 2014; accepted 18 August 2014 Available online 22 October 2014
KEYWORDS Candida glabrata; Hypoxia; Cobalt chloride; Virulence; Biofilm; Adhesion; 1% O2; Amphotericin B
Summary Objectives. — Candida glabrata has emerged as potent pathogen in nosocomial infections. The objective of this study is to investigate the effects of hypoxia (an important host factor) and hypoxia mimetic cobalt chloride upon growth, in vitro adhesion, biofilm formation and susceptibility to amphotericin B of Candida glabrata. Materials and methods. — Growth was checked by spotting assays. Expression of TDH3, a gene of glycolytic pathway was analyzed as intracellular hypoxia marker by reverse transcription PCR. In vitro adhesion, biofilm development and susceptibility of biofilm to amphotericin B were performed on polystyrene plates and quantified by XTT assay in RPMI 1640 and YNB media. Experiments were performed in triplicates and Student’s t-test was used for statistical analysis.
* Corresponding author. Tel.: +91 7417325585; +91 135 2642799; 91 135 2642727X217; fax: +91 135 26644025. E-mail addresses: [email protected], [email protected] (N. Kumar). http://dx.doi.org/10.1016/j.mycmed.2014.08.003 1156-5233/# 2014 Published by Elsevier Masson SAS.
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P. Gupta et al. Results. — Hypoxia did not compromise the growth of C. glabrata unlike CoCl2. Hypoxia and CoCl2 upregulated TDH3 expression. Adhesion was reduced upon exposure to hypoxia and CoCl2. Biofilm activity remained unchanged in the presence of CoCl2 in both media. In comparison to normoxia control, hypoxia increased biofilm activity to 259.33 22.05% in RPMI 1640, while hypoxia reduced it to 70.99 2.99% in YNB. Biofilm susceptibility to amphotericin B was significantly decreased in RPMI 1640 and remained unaffected in YNB in hypoxia. Conclusions. — C. glabrata grows well even under hypoxia but not upon CoCl2 exposures. CoCl2 mimics hypoxia like expression of TDH3 but affects the virulence properties unlike hypoxia. Both, hypoxia and CoCl2 affects adhesion adversely. Hypoxia increases biofilm development and reduces the susceptibility of biofilm to amp B in RPMI 1640 but not in YNB. # 2014 Published by Elsevier Masson SAS.
MOTS CLÉS Candida glabrata ; Hypoxie ; Chlorure de Cobalt ; Virulence ; Biofilm ; Adhérence ; Amphotéricine B
Re ´sume ´ Objectifs. — Candida glabrata a émergé comme un important agent pathogène, cause d’infections nosocomiales. L’objectif de cette étude était d’étudier les effets d’une hypoxie (un important facteur de l’hôte) et l’hypoxie induite par le chlorure de cobalt sur la croissance in vitro, sur l’adhérence, la formation de biofilm et la sensibilité à l’amphotéricine B de C. glabrata. ´thodes. — La croissance a été étudiée par un spotting test. L’expression de TDH3, Mate ´riels et me un gène de la voie glycolytique, a été analysée comme marqueur d’une hypoxie intracellulaire par transcription inverse-PCR. In vitro, l’adhérence, le développement de biofilm et la sensibilité des biofilms à l’amphotéricine B ont été étudiés sur plaques de polystyrène et quantifiés par dosage XTT en milieu RPMI 1640 et YNB. Des expériences ont été effectuées en triplicate et le test t de Student a été utilisé pour l’analyse statistique. Re ´sultats. — Hypoxie n’a pas compromis la croissance de C. glabrata contrairement à l’action du CoCl2. Hypoxie et CoCl2 exercent une régulation positive sur l’expression de TDH3. L’adhérence était réduite lors de l’exposition à l’hypoxie et au CoCl2. La production de biofilm est restée inchangée en présence de CoCl2 dans les deux milieux. En comparaison de contrôles en normoxie, l’hypoxie a augmenté la production de biofilm de 259,33 22,05 % en RPMI 1640, tandis qu’une hypoxie a réduit cette production de 70,99 2,99 % en YNB. La sensibilité du biofilm à l’amphotéricine B a été considérablement diminuée en RPMI 1640 et est restée inchangée en YNB en hypoxie. Conclusions. — C. glabrata pousse bien, même sous hypoxie mais pas en présence de CoCl2. Le CoCl2 imite une hypoxie pour l’expression de TDH3 mais affecte des propriétés de virulence contrairement à l’hypoxie. Hypoxie et CoCl2 affectent négativement l’adhérence. L’hypoxie augmente le développement de biofilm et réduit la sensibilité du biofilm à l’amphotéricine B en RPMI 1640 mais pas en YNB. # 2014 Publié par Elsevier Masson SAS.
Introduction Incidence of invasive infections due to Candida is increasing alarmingly worldwide, mainly due to medical device interventions and immunosuppressive states in different ailments like AIDS and cancer [6,11,30]. Candida albicans and Candida glabrata are two clinically important fungal pathogens of humans [3,29]. Both are opportunistic commensals found, especially, in oral cavity and gastrointestinal tract of healthy humans [9,42]. C. glabrata has become second most causative of bloodstream infections in human after C. albicans in the last two decades [31,32,37]. C. glabrata is a non-dimorphic, haploid yeast, which accounts for approximately 15% of all Candida infections particularly blood stream infections worldwide [33] and responsible for 11 to 37% candidaemia cases in the United States across the nine US Bureau of the Census Regions [30]. C. glabrata is more similar to Saccharomyces cerevisiae than C. albicans in several aspects like virulence mechanisms, genetic repertoire, life cycle, morphogenesis [2,20,39].
During colonization and infection in animal hosts, the pathogen faces environmental challenges that are crucial for their survival and virulence. One such challenge is hypoxia to which both pathogen and host tissue are exposed [21,25]. C. glabrata has different adaptations for its pathogenic mode in the host body like adaptation to hypoxia which is an important virulence characteristic [14,39]. Hypoxia mediated transcriptional regulations and its effect on virulence in C. albicans and other fungi have been investigated [4,8,14,44]. This regulation includes the increased expression of genes encoding enzymes of sterol, lipids and heme biosynthesis along with glycolytic pathways. Sterol regulatory element-binding proteins (SREBPs) are the regulators of hypoxic responses in mammals that regulate the expression of cholesterol synthesis genes [17]. Members of ascomycete (Schizosaccharomyces pombe) and basidiomycete (Crptococcus neoformans) have SREBP homologs to control the expression of genes involved in biosynthesis of lipid, ergosterol and heme [4]. But in some members of Saccharomycotina (S. cerevisiae, C. albicans, C. parapsilopsis), the role of
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride SREBPs is replaced by different regulator Upc2 and Ecm22, which binds sterol regulatory element in the promoters of target genes [47]. In C. albicans, Upc2 and Efg1 (transcription factors) regulate genes of ergosterol and fatty acid biosynthesis pathways respectively under hypoxic conditions, while Tye7 and Gal4 induce genes of glycolytic pathway in hypoxia [1,40,44]. C. parapsilosis has been studied for transcriptional profile of biofilm formation under hypoxic condition and the genes of glycolysis, fatty acid metabolism and ergosterol synthesis were found to be upregulated, suggesting interrelationship of biofilm development with hypoxia in other Candida species [38]. Cobalt chloride, in sublethal concentration mimics hypoxia like state in S. cerevisiae [13,23,27,46], C. neoformans [24] and mammalian systems [48]. In C. neoformans, both CoCl2 and low oxygen conditions affect the majority of genes of ergosterol pathway and iron/copper transport in similar manner. These common transcriptional responses are mediated through common Sre1p. pathway by which yeast cells sense and adapt to CoCl2 and oxygen levels [24]. Hypoxia is reported to be associated tightly with the virulence attributes especially adhesion, biofilm development and response to antifungal drugs in other fungal pathogens [4,5,38,49]. But the effect of hypoxia upon virulence characteristics mainly adhesion, biofilm formation and susceptibility to antifungal remain unaddressed for C. glabrata. It has also not been explored whether cobalt chloride (a hypoxia mimetic in transcriptional responses of genes of ergosterol biosynthesis, lipid biosynthesis and glycolytic pathways) has hypoxia like effects upon virulence characteristics. Therefore, we performed a comparative study to analyze the effects of hypoxia and cobalt chloride upon key virulence properties like growth, in vitro adhesion, biofilm formation and resistance to antifungal for C. glabrata.
Materials and methods Cultures, culture condition and chemicals C. glabrata (MTCC3019) strain was procured from the Institute of Microbial Technology (CSIR Laboratory), Chandigarh, India. Strain was routinely cultivated at 37 8C in YPD medium (1% yeast extract, 2% Bacto-peptone and 2% dextrose; BD) in orbital shaker at 120 rpm. Biofilm and adhesion assays were performed in RPMI 1640 (HiMedia) medium supplemented with 50 mM HEPES and L-Glutamine, pH 7.0 (referred as to RPMI 1640 henceforth) and YNB medium (0.67% yeast nitrogen base w/o amino acids with ammonium sulfate supplemented with 2% dextrose; BD; pH 7.0) on the surface of pre-sterilized, polystyrene, flat bottomed 96-well microtiter plates (HiMedia) [24,31]. Cobalt chloride (CoCl2; Sigma-Aldrich Chemicals Ltd.) at a concentration of 0.75 mM was used as hypoxia mimetic agent. All other routine chemicals of molecular grade were procured from Merck and plastic-wares from Tarson. Amphotericin B (amp B), Menadione and 2,3-bis (2-methoxy-4-nitro-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) were purchased from HiMedia. For exposure of cells to hypoxic conditions in solid media, cells were grown in YPD plates either containing 0.75 mM CoCl2 or the plates were incubated in a hypoxic chamber
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(multi-gas incubator, CO2/O2 incubator Model GA156, LEEC, UK) with 1% O2, 5% CO2 and 94% N2. Similarly, cells were grown on the surface of polystyrene, flat bottomed 96-well microtiter plates either containing 0.75 mM CoCl2 or the plates were incubated in a hypoxic chamber for exposures to hypoxia mimetic agent and hypoxia respectively for adhesion, biofilm and susceptibility of biofilm to antifungal assays.
Effect of hypoxia and CoCl2 on growth Growth was analyzed using plate spotting assay [23]. Log phase cells of C. glabrata (OD600nm: 0.6) were spotted in 10 fold serial dilutions (103, 102, 101, 100 cells) onto YPD agar plates with or without CoCl2. Different concentrations of CoCl2 (0.5 mM, 0.75 mM, 1.0 mM and 5 mM) were maintained on the agar plates. For normoxic control and hypoxia, YPD plates without cobalt chloride were spotted and incubated at respective conditions. Experiments were done in triplicates. Plates were incubated at 37 8C for 18 hours (h).
Effect of CoCl2 on colony forming unit (CFU) We followed Sousa et al., [43] to find out sub lethal concentration of CoCl2 for C. glabrata that do not cause significant lethality. We selected 0.75 mM and 1.0 mM CoCl2 because in other yeasts 0.75 mM has been used to mimic hypoxia; we also took 1.0 mM to see the effect of immediate higher concentration. Log phase cells (107cells/mL) of C. glabrata (grown in YPD) were exposed to CoCl2 in PBS for 2 h. Afterwards, cell suspensions having 100 cells were spread onto YPD plates without CoCl2. Plates were incubated at 37 8C for 18 h and colony forming units (CFU) were counted for each plate. Experiment was done in triplicate. Mean values SD of one of three independent assays are represented and Student’s t-test was applied to analyze the significant differences in the number of CFU.
Analysis of gene expression by semi-quantitative RT-PCR Semi-quantitative reverse transcription PCR was performed to analyze the expression of hypoxia marker gene upon exposure to normoxia, hypoxia and 0.75 mM CoCl2 in C. glabrata [15]. TDH3, a gene of glycolytic pathway was used as hypoxia marker [44] and ACT1 as control. Total RNA was isolated from the log phase cells exposed to hypoxia and 0.75 mM CoCl2 separately for 2 h along with unexposed control cells using RNeasy Kit from Qiagen. RNA was quantified and purity was checked using spectroscopic method [15] using Eppendorf Biophotometer Plus-1632. Reverse transcription (RT) was done using Fermentas First Strand cDNA Synthesis Kit (Cat. No. #K1611) followed by PCR using specific primers synthesized from Bioserve, Hyderabad (Table 1). Taq polymerase and dNTPs were from SigmaAldrich. PCR products were checked by running onto 1.2% agarose gel in TAE before being photographed by gel documentation system (Merck- GeNei). Band intensities of ACT1 and TDH3 expressed under different conditions were measured using Image J software in arbitrary units. Intensities of DNA bands of genes in three independent experiments of
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Table 1 Sequences of the primer used in this study. ´ dans cette ´etude. ´ quences du primer utilise Se Sr. No.
Name of primer
Sequence of primer (50 —30 )
1 2 3 4
ACT1F ACT1R TDH3F TDH3R
ATGGATTCTGAAGTTGCT TTAGAAACACTTGTGGTG ATGGTTAGAATTGCTATT TTAGTTCTTGGCAACGTG
agarose gel electrophoresis were used to calculate mean SD. Significant differences in gene expressions under normoxic and hypoxic conditions were analyzed as mentioned below in statistical analysis.
Effect on adhesion In vitro adhesion assays were performed to investigate the effect of hypoxia and 0.75 mM CoCl2 on adherence of C. glabrata cells in YNB and RPMI 1640 medium on the surface of pre-sterilized, polystyrene, flat bottomed 96-well microtiter plate [35]. One hundred mL of cell suspension (107 cells/ mL) in media without and with CoCl2 was added to the wells respectively and plates were incubated at 37 8C on normoxia for 90 minutes with shaking. For hypoxia, cell suspension in media (107cells/mL) was added to wells and plates were incubated at 37 8C for 90 minutes After incubation of 90 minutes, wells were washed twice with 200 mL of PBS to remove the unadhered cells. The density of adhered cells to each well was quantified using XTT reduction assay following Ramage et al., [34] with minor modifications. Absorbance was measured at 450 nm using ELISA Reader (Molecular Devices, SPECTRA max M2). Values are represented in terms of relative metabolic activity (RMA) in percentage, relative to the adhesion of respective normoxia control (Mean of metabolic activities of normoxia control is taken as 100%).
Effect on biofilm formation C. glabrata biofilm was developed on the surface of presterilized, polystyrene, flat bottomed 96-well microtiter plate [36]. Briefly, cell suspension was prepared in PBS at a concentration of 107 cells/mL and 100 mL of suspension was added to each well. Plates were incubated for 90 min at 37 8C on normoxic condition for adhesion phase. Adhesion was visualized under inverted light microscope (OLYMPUS). Wells were washed twice with 200 mL of PBS to remove nonadhered cells. For control and CoCl2 exposure, 200 mL of culture media without and with CoCl2 was added to the wells respectively and plates were incubated at 37 8C on normoxia for 60 h with shaking. For hypoxia, culture media was added to wells and plate was incubated in low oxygen chamber at 37 8C for 60 h. Afterwards, wells were again washed twice with 200 mL of PBS and biofilm was quantified by XTT reduction assay [34] at 450 nm using ELISA Reader (Molecular Devices, SPECTRA max M2). Biofilm developed is represented in terms of relative metabolic activity (RMA) in percentage, relative to the biofilm of respective normoxia control (Mean of metabolic activities of normoxia control is taken as 100%).
Effect on biofilm antifungal drug susceptibility Effect of hypoxia upon susceptibility of biofilm development to antifungal drug was analyzed in the presence of different concentrations of amp B (0, 0.0625, 0.125, 0.25 mg/mL) following the methodology as mentioned above by XTT reduction assay [34]. We investigated the susceptibility of C. glabrata biofilm development to amp B in both the media YNB and RPMI 1640 upon exposure to hypoxia in comparison to normoxia controls.
Statistical analysis All experiments were performed in triplicates and values presented are the mean with standard deviation, obtained from three different observations for XTT assays. Student’s t-test was used for statistical analysis and a value of P < 0.05 was considered statistically significant (*) and P < 0.001 as highly significant (**) for comparisons [17].
Results C. glabrata growth was not affected by hypoxia but by CoCl2 As shown in Fig. 1A, exposure to 1% O2 did not affect the growth of C. glabrata on YPD agar plate when compared with normoxia control. On the other hand, C. glabrata growth on CoCl2 containing YPD agar plates was found to be compromised in different magnitudes according to the concentrations of CoCl2 when compared with normoxia control (Fig. 1A). Cells were significantly sensitive when grown in the presence of 1 mM CoCl2, while CoCl2 at a concentration of 5 mM was found to be lethal in our experiments. We found 0.5 mM and 0.75 mM CoCl2 to be the sublethal concentrations for C. glabrata (Fig. 1A). Therefore, we used CoCl2 at a concentration of 0.75 mM in our further experiments as a hypoxia mimetic that has also been used for S. cerevisiae by other groups [26,30].
0.75 mM CoCl2 did not affect cell viability In our CFU counting experiments, CoCl2 at a concentration of 1 mM was found to be significantly lethal when compared with untreated cells (P < 0.05), while 0.75 mM of CoCl2 did not affect cell survival in comparison to untreated cells (P > 0.05) (Fig. 1B). Henceforth, CoCl2 at a concentration of 0.75 mM was used in this study.
Hypoxia and CoCl2 upregulated TDH3 expression TDH3, a gene of glycolytic pathway was used as marker for intracellular hypoxia along with a control ACT1 to compare the effects of 1% O2 and 0.75 mM CoCl2 on the expression of hypoxia marker gene along with normoxia control. As per the qualitative observation (Fig. 1C and D), expression of TDH3 was significantly upregulated in the log phase cells after exposure to 1% O2 and 0.75 mM CoCl2 for 2 h in C. glabrata, while expression of control gene ACT1 remained statistically unchanged under all conditions when compared to respective normoxic controls. Unlike ACT1, changes in the
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Figure 1 Effect of hypoxia and cobalt chloride on growth and cell viability of C. glabrata. A. Growth of C. glabrata cells in spotting assays onto YPD agar plates upon exposure to different conditions; B. Cell viability of C. glabrata cells upon exposure to different concentrations of CoCl2 counted in form of CFU Mean values of three independent CFU experiments SD is shown and (*) represents statistically significant differences in CFU with respect to normoxia control (P < 0.05); C. Expression of TDH3 and ACT1 gene under normoxia, hypoxia and 0.75 mM CoCl2 conditions by semi-quantitative RT-PCR; D. Average of three band intensities SD of the TDH3 and ACT1 upon exposure to normoxia (Nor), hypoxia (Hyp) and 0.75 mM CoCl2 (CoCl2) is shown in form of bar diagram and (**) represents statistically highly significant differences (P < 0.001) in band intensity with respect to normoxia control of particular gene. ´ cellulaire de C. glabrata. A. La croissance de C. glabrata par Effet de l’hypoxie et du chlorure de cobalt sur la croissance et la viabilite ´ lose YPD lors de l’exposition `a des conditions diffe ´ rentes ; B. Viabilite ´ cellulaire de la technique du spottings sur des plaques de ge ´ rentes concentrations de CoCl2 exprime ´ e en cfu. Valeurs moyennes SD des cfu de trois C. glabrata lors de l’exposition `a diffe ´ pendantes (*) repre ´ sentent des diffe ´ rences statistiquement significatives des cfu `a l’e ´ gard du contro ˆ le normoxique experiences inde ` nes de TDH3 et ACT1 sous normoxie, hypoxie et 0,75 mm CoCl2 en RT-PCR semi-quantitative ; ( p < 0,05) ; C. Expression des ge ´ s de bande SD de TDH3 et ACTl1 lors de l’exposition `a la normoxie (Ni), `a l’hypoxie (Hyp) et `a 0,75 mM D. Moyenne de trois intensite ´ e sous forme de diagramme `a barres, (**) repre ´ sente des diffe ´ rences statistiquement tre ` s importantes CoCl2 (CoCl2) illustre ´ de bande compare ´ au ge ` ne contro ˆ le en normoxie. ( p < 0,001) en intensite
expression of TDH3 were statistically highly significant (P < 0.001) when exposed to 0.75 mM CoCl2 and 1% O2 Ratios of TDH3/ACT1 band intensities were 1.713 for normoxia, 2.482 for hypoxia and 2.281 for CoCl2 experiments. Therefore, it has been concluded that CoCl2 at a concentration of 0.75 mM is adequate to mimic hypoxia like intracellular gene expression without inducing lethality in C. glabrata.
Hypoxia and CoCl2 affected adhesion and biofilm differentially Since adhesion and biofilm formation by Candida are affected by several environmental conditions, therefore, we compared the effect of hypoxia and CoCl2 on adhesion and biofilm formation by C. glabrata in two commonly used media, namely RPMI 1640 and YNB.
In YNB medium, reduction in adhesion potential was reported to be significant upon exposure to CoCl2 (RMA: 77.19 4.3214%; P < 0.05) and highly significant upon exposure to hypoxia (RMA: 69.67 3.9825%; P < 0.001) (Fig. 2A). In RPMI 1640 medium, reduction in adhesion potential was reduced insignificantly upon exposure to CoCl2 (RMA: 90.16 3.9098%; P > 0.05) and significantly upon exposure to hypoxia (RMA: 70.84 4.4050%; P < 0.05) (Fig. 2A). In YNB medium, change in biofilm developed remained almost unaffected upon exposure to CoCl2 (RMA: 110 10.727%; P > 0.05), while biofilm activity was reduced significantly upon exposure to hypoxia (RMA: 70.99 2.9964%; P < 0.05) (Fig. 2B). Biofilm activity in RPMI 1640 was not affected in the presence of CoCl2 (RMA: 102.22 11.827%; P > 0.05) but increased to 259.33 22.053%) with highly significant difference (P < 0.001), when compared to normoxia control (Fig. 2B).
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Hypoxia reduced susceptibility of biofilm formation to antifungal Since hypoxia increased biofilm formation in RPMI 1640 medium, we anticipated the positive role of hypoxia in reducing the biofilm susceptibility to existing antifungal drug like amp B. In YNB medium, hypoxia could not drive the biofilm development significantly different from that of normoxic control in the presence of gradient of concentration of amp B (0, 0.0625, 0.125, 0.25 mg/mL) (Fig. 3A) and these differences in OD at 450 nm were not found to be significant statistically (P > 0.05, except for the point 0.125 mg/mL of amp B). Mean values of absorbance SD along with Student’s ttest values are shown in Table 2. Hypoxia in RPMI 1640 increased the biofilm development in the presence of amp B, when compared with normoxic biofilm activity to same antifungal and these differences were statistically significant (P < 0.05) (Fig. 3B). Thus, hypoxia reduced the susceptibility of biofilm development to the amp B in RPMI 1640 medium. Mean values of absorbance SD along with Student’s t-test values are shown in Table 3.
Discussion During pathogenesis, C. glabrata faces hypoxic microenvironment inside host tissues. Oxygen level in human tissues ranges 20—70 mmHg (2.5—9% O2) and in damaged tissue, it is less than 10 mm Hg (1% O2) [21,25]. Cobalt chloride has been widely used as a hypoxia mimetic for inducing low oxygen like conditions for biochemical and transcriptional responses in different models [13,48]. In yeast, cobalt is reported to target diiron-oxo enzymes involved in sterol pathway (Erg3p and Erg25p) and fatty acid pathway (Ole1p);
P. Gupta et al. similarly hypoxia affects sterol biosynthesis and fatty acid synthesis due to the requirement of molecular oxygen at various steps [13,18]. We studied the comparative effects of hypoxia and cobalt chloride on growth, in vitro adhesion, biofilm formation and anti fungal susceptibility of C. glabrata. In our experiments, moderate sensitivity of C. glabrata to CoCl2 might be due to inhibitory effect of cobalt on metabolic pathways as evidenced in earlier studies [13]. 0.75 mM CoCl2 was not found to cause lethality rather sufficient to induce transcriptional responses as evidenced by upregulation of TDH3 (a representative gene of glycolytic pathway) [44], similar to hypoxia (Fig. 1B and D). Hypoxia is reported to induce similar transcriptional response in C. parapsilopsis, C. albicans, and S. cerevisiae [38,44,46]. Our results confirmed that hypoxia has no adverse effect on the growth of C. glabrata (Fig. 1A), indicating the presence of necessary mechanisms for adaptation to hypoxia in the pathogen mediated through transcriptional responses. We found that hypoxia and CoCl2 had significantly reduced adhesion potential of C. glabrata in both media (except for CoCl2 exposure in RPMI 1640) (Fig. 2A), probably due to the effects of hypoxia and/or cobalt exposure on cell metabolism [13] and requirement of more time for transcriptional responses. EPA genes (a family of glycosylphosphatydylinnositol (GPI)-anchored cell wall protein genes) code for adhesins which are effectors of virulence properties (adhesion and biofilm) in C. glabrata [10,19,36]. Higher expression of EPA6 is reported in stationary phase culture of C. glabrata [19], while we used log phase cells for adhesion. Hypoxia is also reported to induce C. glabrata EPA6 expression even in the low density cultures but in the presence of paraben stress [28]. Previous reports also support our observation where cobalt was inhibitory for biofilm formation, if planktonic inoculums of C. albicans and C. tropicalis were pretreated
Figure 2 Effect of hypoxia and CoCl2 on in vitro adhesion and biofilm formation by C. glabrata. A. Comparative relative metabolic activity of in vitro adhesion of C.glabrata cells under different conditions in different media; B. Comparative relative metabolic activity of in vitro biofilm formation by C. glabrata cells under different conditions in different media. RMA is the % metabolic activity of adhesion and biofilm formed under specific conditions relative to normoxia control in that particular medium. Mean values of RMA SD of three independent experiments are shown; P < 0.05 is considered statistically significant and P < 0.001 highly significant and represented as (*) and (**) respectively. ´ rence et la formation de biofilm par C. glabrata. A. Comparaison in vitro de l’activite ´ Effet d’hypoxie et de CoCl2 in vitro sur l’adhe ´ rence de C. glabrata dans des conditions diffe ´ rentes et dans diffe ´ rents milieux ; B. Comparaison in vitro de ´ tabolique et de l’ adhe me ´ rentes et dans diffe ´ rents milieux. RMA ´ me ´ tabolique et de la formation de biofilm par C. glabrata dans des conditions diffe l’activite ´ me ´ tabolique, de l’adhe ´ rence et de biofilm forme ´ dans des conditions spe ´ cifiques relatives au contro ˆ le de la est le % d’activite ´ riences inde ´ pendantes sont indique ´ es ; p < 0,05 est normoxie dans ce milieu. Les valeurs moyennes de RMA SD de trois expe ´ sente ´ par (*) et (**) respectivement. ´ re ´ comme statistiquement significatif et p < 0,001 hautement significatif et est repre conside
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride
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Figure 3 Effect of hypoxia on susceptibility of in vitro biofilm development to Amphotericin B by C. glabrata. A. Susceptibility of in vitro biofilm development in YNB medium; B. Susceptibility of in vitro biofilm development in RPMI-1640 medium. Results were shown here as mean SD of A450 plotted on Y-axis against concentration of amp B on X- axis. Statistically significant differences in biofilm activities in presence of amp B between normoxia and hypoxia are shown as (*) (P < 0.05). ´ in vitro du de ´ veloppement de biofilm par C. glabrata sous l’amphote ´ ricine B. A. Sensibilite ´ in L’effet de l’hypoxie sur la sensibilite ´ veloppement du biofilm en milieu YNB ; B. Sensibilite ´ in vitro biofilm du de ´ veloppement du biofilm en milieu RPMI-1640. vitro du de ´ s sur l’axe des Y vis-a ` -vis des concentrations d’ampho B sur l’axe des X. Des diffe ´ rences statistiquement moyenne SD de 450 trace ´ biofilm en pre ´ sence d’ampho B entre normoxie et hypoxie sont indique ´ es (*) ( p < 0,05). significatives en activite
with 0.70 mM cobalt [16], indicating the adverse effect of cobalt on adhesion (phase earlier to biofilm formation). CoCl2 did not affect biofilm development significantly in either media (Fig. 2B; P > 0.05 for both media), probably due to sublethal concentration of CoCl2 and independency of virulence determinants (mainly adhesins) on CoCl2, which needs further studies to analyze the EPA expression in presence of CoCl2. In a study, MIC of biofilm was found to be around 4.2 mM for intermediate stage of biofilm formed by C. tropicalis [16], whereas we used 0.75 mM CoCl2. It was demonstrated previously that aerobic/anaerobic conditions led to differential biofilm development in different clinical
isolates of Candida; one of the C. glabrata isolates showed higher biofilm development under anaerobic/dynamic conditions similar to our results [45]. We found that hypoxia reduced biofilm significantly in YNB (P < 0.05) but increased biofilm strikingly in RPMI 1640 (P < 0.001) (Fig. 2B). Similarly in an earlier study, C. glabrata proliferated in adherent manner resulting in poor biofilm along with low level of EPA6 expression in YNB, in contrast to the thicker and multilayer biofilm formation accompanied by higher level of EPA6 expression in RPMI 1640 medium [22]. Oxygen deficient conditions in high density cultures are also reported to upregulate EPA6 expression
Table 2 Quantification of biofilm formation in the presence of ampho B under hypoxic and normoxic conditions in YNB medium. ´ sence d’ampho B sous conditions hypoxiques ou normoxiques en milieu YNB. Quantification de la formation de biofilm en pre Sr. No.
Concentration of amp B in mg/mL
Mean SD of OD 450 nm under normoxic condition
Mean SD of OD 450 nm under hypoxic condition
Values of P by Student’s t-test for given concentration of amp B in two conditions
1 2 3 4
0 0.0625 0.1250 0.2500
0.081 0.00529 0.067 0.00960 0.065 0.00862 0.062 0.00493
0.087 0.00529 0.085 0.00404 0.084 0.00550 0.058 0.00360
0.23724 0.06413 0.03549 0.72632
Table 3 Quantification of biofilm formation in the presence of ampho B under hypoxic and normoxic conditions in RPMI 1640 medium. ´ sence d’ampho B sous conditions hypoxiques ou normoxiques en milieu RPMI 1640. Quantification de la formation de biofilm en pre Sr. No.
Concentration of ampho B in mg/mL
Mean SD of OD 450 nm under normoxic condition
Mean SD of OD 450 nm under hypoxic condition
Values of P by Student’s t-test for given concentration of amp B in two conditions
1 2 3 4
0 0.0625 0.1250 0.2500
0.138 0.00655 0.126 0.00916 0.065 0.00888 0.061 0.00351
0.239 0.01379 0.151 0.00945 0.11 0.01050 0.078 0.00737
0.00128 0.02907 0.00505 0.03948
e176 in C. glabrata along with paraben stress [28]. In our results, abrupt increase in biofilm in RPMI1640 medium upon exposure to hypoxia may be due to induction of EPA genes due to the combined effect of hypoxia and RPMI1640 medium. Since hypoxia modulated biofilm, we further investigated the effect of hypoxia on biofilm development in the presence of amp B. We found that hypoxia has significantly increased biofilm formation by C. glabrata upon exposure to hypoxia in RPMI 1640 (Fig. 3B). These results could be explained in the light of following reasoning. Polyene antifungals target ergosterol of the fungal cell membrane to generate pores and causing leakage of cytoplasmic content [12,41] and hypoxia lowers ergosterol synthesis in yeast by affecting O2 mediated steps [13] which might be responsible for developing resistance to amp B. Hypoxia affects different steps of ergosterol biosynthesis involving oxygen dependent enzymes Erg1p. Erg3p, Erg5p, Erg11p and Erg25 and reduces the level of ergosterol in yeast [13]. Polyene resistance in Candida species results when ergosterol content lowers, probably due to mutation in ERG3 (gene that produces d 5,6-sterol desaturase) and ERG11 (gene that produces lanosterol 14a-demethylase) [7,26]. RPMI1640 is reported to induce C. glabrata biofilm development and expression of adhesins better than that in YNB [22]. Hypoxia also induces expression of adhesins (EPA6) in saturated cultures [28]. Hypoxia might also upregulate the expression of ABC transporters (CDR1, PDR16 and CDR2) in C. glabrata, as happened in the case of azole resistance [2,20]. To our knowledge, this is the first direct study on the comparative effects of hypoxia and hypoxia mimetic CoCl2 upon the virulence properties of C. glabrata in RPMI 1640 and YNB. Further studies on C. glabrata are being undertaken by our group for exploring the role of hypoxia in C. glabrata pathogenesis to find out potential molecular targets for the novel drug development.
Conclusion This study demonstrated that hypoxia augments biofilm formation by C. glabrata in RPMI1640 medium. We found that susceptibility of C. glabrata biofilm to amphotericin B decreases upon exposure to hypoxia in RPMI1640. Both, hypoxia and CoCl2 affects adhesion adversely. We found that CoCl2 mimics hypoxia in upregulating the expression of a gene of glycolytic pathway (TDH3), but appears not to cause hypoxia like effects upon growth and biofilm formation by C. glabrata.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgement We thank Dr. Shashi Bala Singh Director, Defence Institute of Physiology and Allied Sciences, Delhi for giving us permission to perform experiments related to hypoxia chamber in DIPAS. We are grateful to Dr. Amitabha Chakrabarti, DIPAS
P. Gupta et al. for allowing us to use his laboratory for hypoxic exposures. PG is supported by the INSPIRE fellowship from Department of Science and Technology, Government of India and SN is supported by fellowship from Council of Scientific and Industrial Research, Government of India. We are also thankful to Dr. Ashish Thapliyal, Dr. Nishant Rai and Dr. Pankaj Gautam from Graphic Era University for their support during course of this study.
References [1] Askew C, Sellam A, Epp E, Hogues H, Mullick A, Nantel A, et al. Transcriptional regulation of carbohydrate metabolism in the human pathogen Candida albicans. Plos Pathog 2009;5: e1000612. [2] Bialkova A, Subik J. Biology of the pathogenic Yeast Candida glabrata. Folia Microbiol 2006;51:3—20. [3] Brunke S, Hube B. Two unlike cousins: Candida albicans and C. glabrata infection strategies. Cell Microbiol 2013;15:701—8. [4] Butler G. Hypoxia and gene expression in eukaryotic microbes. Annu Rev Microbiol 2013;67:291—312. [5] Chung D, Haas H, Cramer RA. Coordination of hypoxia adaptation and iron homeostasis in human pathogenic fungi. Front Microbiol 2012;3:1—13. [6] Delaloye J, Calandra T. Invasive candidiasis as a cause of sepsis in the critically ill patient. Virulence 2014;5:1—9. [7] Dick JD, Merz WG, Saral R. Incidence of polyene-resistant yeasts recovered from clinical specimens. Antimicrob Agents Chemother 1980;18:158—63. [8] Ernst JF, Tielker D. Responses to hypoxia in fungal pathogens. Cell Microbiol 2009;11:183—90. [9] Fidel PL, Vazquez JA, Sobel JD. Candida glabrata: review of epidemiology, pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev 1999;12:80—96. [10] Gabaldon T, Martin T, Marchet-Houben M, Durrens P, BolotinFukuhara M, Lespinet O, et al. Comparative genomics of emerging pathogens in the Candida glabrata clade. BMC Genomics 2013;14:623—39. [11] Galle F, Catania MR, Liguori G. Nosocomial Candida infections: epidemiology of candidaemia. J Prev Med Hyg 2006;47:119—26. [12] Ghannoum MA, Rice LB. Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 1999;12:501—17. [13] Gleason JE, Corrigan DJ, Cox JE, Reddi AR, McGinnis LA, Culotta VC. Analysis of hypoxia and hypoxia-like states through metabolite profiling. PLoS One 2011;6:e24741. [14] Grahl N, Shepardson KM, Chung D, Cramer RA. Hypoxia and fungal pathogenesis: to air ot not to air? Eukaryot Cell 2012;11:560—71. [15] Guzman-Gonzalez KD, Parra-Ortega B, Martinez-Rivera MA, Hernandez-Rodriguez C, Perez NO, Rodriguez-Tovar AV. Investigating the role of Candida glabrata SLN1 gene in stress adaptation: in silico and molecular analysis. Afr J Microbiol Res 2013;7:460—70. [16] Harrison JJ, Ceri H, Yerly J, Rabiei M, Hu Y, Martinuzzi R, et al. Metal ions may suppress or enhance cellular differentiation in Candida albicans and Candida tropicalis biofilms. Appl Environ Microbiol 2007;73:4940—9. [17] He M, Du M, Fan M, Bian Z. In vitro activity of eugenol against Candida albicans biofilms. Mycopathologia 2007;163:137—43. [18] Horton JD, Goldstein JL, Brown MS. SREBPs: activation of the complete program of cholesterol and fatty acid synthesis in the liver. J Clin Invest 2002;109:1125—31. [19] Iraqui I, Garcia-Sanchez S, Aubert S, Dromer F, Ghigo JM, d’Enfert C, et al. The Yak1p kinase controls expression of
Comparative effects of hypoxia and hypoxia mimetic cobalt chloride
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
adhesions and biofilm formation in Candida glabrata in a Sir4pdependent pathway. Mol Microbiol 2005;55:1259—71. Kaur R, Domergue R, Zupancic ML, Cormack BP. A yeast by any other name: Candida glabrata and its interaction with the host. Curr Opin Microbiol 2005;8:378—84. Kessler M, Höper J, Schäfer D, Starlinger H. In: Mikrozirkulation, Ahnefeld FW, Burri C, Dick W, Halmágyi M, editors. Sauerstofftransport im Gewebe. Berlin, Heidelberg, New York: Springer Verlag; 1974. p. 36—52. Kucharikova S, Tournu H, Lagrou K, Dijck PV, Bujdakova H. Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J Med Microbiol 2011;60: 1261—9. Kumar N, Meena RC, Chakrabarti A. Over-expression of YLR162W in Saccharomyces cerevisiae inhibits cell proliferation and renders cell susceptible to the hypoxic conditions induced by CoCl2. Indian J of Microbiol 2011;51:206—11. Lee H, Bien CM, Hughes AL, Espenshade PJ, Kwon-Chung KJ, Chang YC. Cobalt chloride, a hypoxia-mimicking agent, targets sterol synthesis in the pathogenic fungus Cryptococcus neoformans. Mol Microbiol 2007;65:1018—33. Lewis JS, Lee JA, Underwood JCE, Harris AL, Lewis CE. Macrophage responses to hypoxia: relevance to disease mechanisms. J Leukoc Biol 1999;66:889—900. Lupeti A, Danesi R, Campa M, Tacca MD, Kelly S. Molecular basis of resistance to azole antifungals. Trends Mol Med 2002;8: 76—81. Meena RC, Kumar N, Nath S, Chakrabarti A. Homologous Recombination is activated at early time points following exposure to cobalt chloride induced hypoxic conditions in Saccharomyces cerevisiae. Indian J Microbiol 2012;52:209—23. Mundy RD, Cormack B. Expression of Candida glabrata adhesions after exposure to chemical preservatives. J Infect Dis 2009;199:1891—9. Papon N, Courdavault V, Clastre M, Bennett RJ. Emerging and emerged pathogenic Candida species: beyond the Candida albicans paradigm. PLOS Pathog 2013;9:e1003550. Pfaller MA, Boyken L, Hollis RJ, Messer SA, Tendolkar S, Diekema DJ. Global surveillance of in vitro activity of micafungin against Candida: a comparison with caspofungin by CLSI-recommended methods. J Clin Microbiol 2006;44:3533—8. Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Ellis D, Tullio V, et al. 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 2010;48:1366—77. Pfaller MA, Diekema DJ, International Fungal Surveillance Participant Group. Twelve years of fluconazole in clinical practice: global trends in species distribution and fluconazole susceptibility of bloodstream isolates of Candida. Clin Microbiol Infect 2004;1:11—23. Pfaller MA, Diekema DJ. Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbio Rev 2007;20:133—63. Ramage G, Walle KV, Wickes BL, Lopez-Ribot JL. Standardized method for in vitro antifungal susceptibility testing of Candida
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43]
[44]
[45]
[46]
[47]
[48]
[49]
e177
albicans biofilm. Antimicrob Agent Chemother 2001;45: 2475—9. Raut JS, Chauhan NM, Shinde RB, Karuppayil SM. Terpenoids of plant origin inhibit morphogenesis, adhesion, and biofilm formation by Candida albicans. Biofouling 2013;29:87—96. Riera M, Mogensen E, Enfert C, Janbon G. New regulators of biofilm development in Candida glabrata. Res Microbiol 2012;163:297—307. Rodrigues CF, Silva S, Henriques M. Candida glabrata: a review of its features and resistance. Eur J Clin Microbiol Infect Dis 2013. http://dx.doi.org/10.1007/s10096-013-2009-3 [Online link: http://www.link.springer.com/article/10.1007%2Fs10096-0132009-3]. Rossignol T, Ding C, Guida A, d’Enfert C, Higgins DG, Butler G. Correlation between biofilm formation and the hypoxic response in Candida parapsilosis. Eukaryot Cell 2009;8:550—9. Rotzer A, Gabaldon T, Schuller C. From Saccharomyces cerevisiae to Candida glabrata in a few easy steps: important adaptations for an opportunistic pathogen. FEMS Microbiol Lett 2011;314:1—9. Setadi ER, Doedi T, Cottier F, Noffz C, Ernst JF. Transcriptional response of Candida albicans to hypoxia: linkage of oxygen sensing and Efg1p-regulatory networks. J Mol Biol 2006;361:399—411. Silva S, Negri M, Henriques M, Oliveria R, Williams DW, Azeredo J. Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 2012;36:288—305. Soll DR, Lockhart SR, Zhao R. Relationship between switching and mating in Candida albicans. Eukaryot Cell 2003;2:390—7. Sousa M, Duarte AM, Fernandes TR, Chaves SR, Pacheco A, Leao C, et al. Genome-wide identification of genes involved in the positive and negative regulation of acetic acid-induced programmed cell death in Saccharomyces cerevisiae. BMC Genomics 2013;14:838—53. Synnott JM, Guida A, Mulhern-Haughey S, Higgins DG, Butler G. Regulation of the hypoxic response in Candida albicans. Eukaryot Cell 2010;9:1734—46. Thein ZM, Samaranayake YH, Samaranayake LP. In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions. Arch Oral Biol 2007;52:761—8. Vasconcelles MJ, Kevin YJ. Identification and characterization of a low oxygen response element involved in the hypoxic induction of a family of Saccharomyces cerevisiae genes. J Biol Chem 2001;276:14374—8. Vik A, Rine J. Upc2p and Ecm22p, dual regulators of sterol biosynthesis in Saccharomyces cereviseae. Mol Cell Biol 2001;21:6395—405. Wang GL, Semenza GL. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 1993;82:3610—5. Warn PA, Sharp A, Guinea J, Denning DW. Effect of hypoxic conditions on in vitro susceptibility testing of amphotericin B, itraconazole and micafungin against Aspergillus and Candida. J Antimicrob Chemother 2004;53:743—9.
