JAC Advance Access originally published online on November 22, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):315-322; doi:10.1093/jac/dkm456
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Original research |
Azole antifungals induce up-regulation of SAP4, SAP5 and SAP6 secreted proteinase genes in filamentous Candida albicans cells in vitro and in vivo
Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, UK
* Corresponding author. Tel/Fax: +44-1224-555828; E-mail: f.odds{at}abdn.ac.uk
Received 9 August 2007; returned 25 September 2007; revised 22 October 2007; accepted 29 October 2007
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Abstract |
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Objectives: Expression of fungal virulence factors can be influenced by exposure to antifungal agents. To test this hypothesis, we determined the effects of subinhibitory concentrations of three antifungal agents on expression of three secreted proteinase genes associated with virulence in filamentous forms of Candida albicans.
Methods: GFP-SAP promoter constructs and fluorescence measurement, transcript profiling and RT–PCR in vitro and in an animal model of disseminated Candida infection.
Results: Exposure of C. albicans to subinhibitory concentrations of fluconazole in RPMI 1640 in the absence of serum led to up-regulation of the virulence-associated genes SAP4, SAP5 and SAP6 in hyphae and long pseudohyphae. Measurements with green fluorescent protein (GFP)-tagged promoters showed that the fluorescence of SAP4 and SAP6 under these conditions was strongest in the apical tip compartments of these filamentous cells and declined in compartments more proximal to the parent yeast cell. By contrast, SAP5-GFP fluorescence was expressed at similar levels in all cell compartments. Exposure to fluconazole led to significant increases in GFP-SAP4 and -SAP6 fluorescence in the filaments; itraconazole exposure also significantly increased GFP-SAP4 fluorescence, whereas flucytosine had no effect on any of the constructs. In experimentally infected animals, fluorescence of the GFP-SAP promoter fungal cells in kidney tissues was greater than that was seen in vitro for all four SAP constructs: treatment of animals with fluconazole did not significantly increase SAP promoter expression as measured by GFP fluorescence.
Conclusions: Azole antifungal agents stimulated up-regulation of SAP4 and SAP6 genes in filamentous C. albicans cells in vitro and may therefore influence virulence as well as growth of the fungus. However, such effects appear to be transient in vivo.
Keywords: fluconazole , flucytosine , itraconazole , virulence genes , C. albicans
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Introduction |
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Collateral effects of antifungal agents on Candida albicans at or below the MIC include interference with gross processes such as adhesion, resistance to phagocytosis and expression of immunomodulatory substances,1–4 and also have an impact on expression of molecular virulence attributes of the fungus.5–8 As part of an ongoing investigation of antifungal effects on virulence in C. albicans, we showed previously that the specific activity of aspartyl proteinase enzyme Sap2, secreted by C. albicans in a medium containing albumin as sole nitrogen source, was elevated in most isolates exposed to subinhibitory concentrations of antifungals belonging to the azole, echinocandin and pyrimidine analogue classes, and that expression of the SAP2 gene was up-regulated in fluconazole-exposed yeast cells.8 This work extended a previous observation of increased proteinase activity in the filtrates of C. albicans cells grown in the presence of fluconazole.6 However, the conditions of all these experiments favoured growth of C. albicans in the yeast morphological form. In infected tissues, hyphal and pseudohyphal forms typically predominate yet the members of the SAP gene family expressed in such filamentous cells differ from those in yeast cells.9
To determine if expression of the three members of the SAP gene family associated mainly with the hyphal form of the fungus, SAP4, SAP5 and SAP6,9 might be similarly influenced by subinhibitory antifungal exposure as was SAP2 in yeast-form cells, we undertook microarray profiling with wild-type C. albicans exposed to fluconazole in vitro and green fluorescent protein (GFP) fluorescence measurements in vitro and in vivo with strains in which codon-optimized GFP was tagged to SAP promoters. We used RPMI 1640 medium without addition of a protein nitrogen source for the work in vitro. This choice was based on two criteria: RPMI 1640 is specified in the CLSI reference method for antifungal susceptibility testing with yeasts,10 and we also wished to determine if the filamentous growth of C. albicans in this medium involved up-regulation of SAP promoters in the absence of a high-molecular weight proteinase inducer, as appeared likely in our pilot experimentation.
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Strains of C. albicans
C. albicans SC5314, originally isolated from a patient with generalized Candida infection, was the wild-type strain used in this study. Mutants of SC5314 were transformed with plasmids containing GFP fusions with SAP and control promoters by the same basic methods as previously described for pSAP2-GFP8 and control strains.11 pGFP contained codon-modified yEGFP12 cloned into the C. albicans CIp10 integrating vector.13 Details of the six strains used in this study and the primers used for PCR amplification of the appropriate SAP promoter regions are shown in Table 1.
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Antifungal agents
Itraconazole pure substance was the gift of Janssen Pharmaceutica, Belgium. Fluconazole and flucytosine were purchased from Sigma.
Transcript profiling with C. albicans microarrays
For measurement of differential gene expression, C. albicans SC5314 was grown at 37°C for 6 h in RPMI 1640 then for 2 h with and without addition of fluconazole at 0.25 mg/L. Pilot experiments showed that cultures exposed to this concentration of fluconazole were inhibited to 70% to 90% of control growth. The cells were flash-frozen in liquid nitrogen and the RNA extracted as previously described.8 Expression profiles were determined in two, independent triplicate experiments with Eurogentec® Microarray slides as previously described.8 Combined, Loewess-normalized data were processed by Statistical Analysis of Microarray (SAM) software in a blocking design to allow inclusion of all information from the two experiments. Gene expression that was up- or down-regulated by a factor of 2-fold or more was regarded as significant. Gene annotation was done by reference to the Candida Genome Database (http://www.candidagenome.org) on 24 June 2007.
Growth conditions for SAP-GFP constructs in vitro
For all strains, inocula were prepared by overnight growth in NGY broth at 30°C.14 Samples of NGY cultures (0.5 mL) were inoculated into 25 mL volumes of the culture media described below and incubated with gyratory shaking at 200 rpm for 4 h. At this point, fluconazole was added to test cultures to a final concentration of 0.5 mg/L, and growth was continued for a further 4 h. Pilot experiments showed that cultures treated with this concentration of fluconazole were inhibited to 60% to 80% of control growth after 24 h at 37°C.
For preparation of yeast-form cells, the strains were grown in Yeast Carbon Base (YCB; Difco) + 0.5% bovine serum albumin fraction V (BSA; Sigma) at 30°C, as before.8 For preparation of pseudohyphal cells, high-phosphate glucose-phosphate-proline medium (GPP15) was used; incubation was at 37°C. Long pseudohyphal and true hyphal cells were grown in three different media, all incubated at 37°C. RPMI 1640 medium with glutamine, without bicarbonate, was buffered at pH 7.0 with 0.165 M MOPS and contained added glucose to a final concentration of 20 g/L. YPD + serum medium contained 10 g/L yeast extract (Difco), 20 g/L mycological peptone (Oxoid), 20 g/L glucose and 20% (v/v) fetal calf serum. Hyphal cells were also generated by a starvation/N-acetyl glucosamine induction procedure as follows. A yeast inoculum grown for 24 h in Lee's medium pH 4.516 at 25°C was added (0.5 mL per 25 mL) to Lee's medium pH 4.5 and incubated for a further 24 h at 37°C. Cells were harvested by centrifugation, washed in sterile water, resuspended in 25 mL of sterile water and starved by incubation at 37°C for a further 24 h. Growth was again harvested, washed in basal salt solution [0.5% NaCl, 0.5% (NH4)2SO4 and 0.02% MgSO4·7H2O] and resuspended at 2 x 106 cells/mL in the same medium plus 4 mM N-acetyl-D-glucosamine and 0.001% biotin then incubated for 4 h at 30°C before addition of fluconazole and a further 4 h of incubation, as before.
To determine the effects of fluconazole treatment on expression of SAP genes in vivo, pairs of mice were challenged intravenously with mutant strains bearing SAP-GFP promoter fusions and control fusions. The challenge inoculum, grown overnight at 30°C in 0.1% neopeptone/0.4% glucose/0.1% yeast extract, was 2 x 105 cells/g body weight. One day after challenge, one member of each pair of mice was dosed intraperitoneally with fluconazole, 10 mg/kg, or the same volume of saline as placebo. After a further 24 h, the mice were humanely terminated and kidneys removed with aseptic precautions and cryosectioned. The sections were analysed for levels of fluorescence in hyphal/pseudohyphal tip compartments. All animal experimentation conformed with the regulations and requirements of the UK Home Office.
For visualization of GFP in vitro, cells from 1 mL culture samples were harvested by centrifugation, washed with phosphate-buffered saline (PBS) and resuspended for 4 min in 200 µL of 4% paraformaldehyde. The cells were recovered, washed twice in PBS and stored at 4°C protected from light. Acridine orange staining for DNA and RNA was done by mixture of a washed cell suspension with 2 volumes of a solution containing 0.1% Triton X-100, 0.8 M HCl and 0.15 M NaCl. After 15 s, 4 volumes of ice-cold acridine orange solution were added (acridine orange, 6 mg/L, 1 mM Na EDTA, 0.2 M sodium phosphate and 0.1 M citrate, pH 6.0). The mixture was allowed to stand for 15 min then observed immediately.
An Axioplan 2 microscope (Carl Zeiss Ltd, UK) with filter sets XF 66, XF 67 and XF 77 (Omega Optical Inc., Brattleboro, VT, USA) was used for fluorescence microscopy. Images were generated with a Hamamatsu CCD camera and analysed with Openlab 3.0.9 software (Improvision Ltd, Coventry, UK). Mean fluorescence intensities (±SD) were then calculated for at least 50 individual cells based on region of interest (ROI) measurements.17
Fluorescence intensities were quantified with arbitrary units by image analysis of intensities measured in cell unit areas outlined by hand and corrected by subtraction for background fluorescence measured for adjacent areas containing no fungal elements. Preliminary observations of long pseudohyphae/hyphae exposed to fluconazole showed that the fluorescence intensity in some SAP-GFP constructs differed between cell compartments. For quantification of fluorescence in filamentous cells, the following approach was therefore used. Four adjacent filament compartments, with the distal, apical tip compartment recorded as compartment no. 1, were averaged separately for fluorescence intensity. Variations in intensity measurements between runs were minimized by calibration of readings for each experiment relative to means for Ca6 (ACT1-GFP positive control) and Ca62 (pGFP transformant negative control).
Statistical significance of the effects of variables on ROI fluorescence intensities was evaluated by ANOVA with the General Linear Model facility in the SPSS Statistical package, version 14.0. Non-parametric statistics were also calculated with the aid of SPSS 14.0. Student's t-tests were done with Microsoft Excel 2003. Because fluorescence intensities for individual ROIs varied considerably, and because many ANOVA calculations were done, increasing the risk of type I errors, a result was regarded as significant only when the probability, P, that it arose by chance was <0.01.
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Expression profile of C. albicans SC5314 exposed to fluconazole
The profiling data showed that the three SAP genes with expression patterns related to filamentous growth forms of C. albicans were up-regulated in cells exposed to fluconazole for 2 h at 0.25 mg/L in RPMI 1640 medium. SAP4, SAP5 and SAP6 were up-regulated, respectively, by an average of 5.2-fold, 3.5-fold and 4.9-fold. The growth forms under these conditions were predominantly filamentous and comprised pseudo- and true hyphae. SAP2 expression was unchanged from control values. rtPCR for SAPs 4–6 confirmed higher levels of expression of these genes in the presence of fluconazole after 2 and 4 h of exposure (data not shown). These results show that, by comparison with growth in YCB + BSA, in which expression of SAP2 but not of other SAPs was found to be up-regulated,8 growth in RPMI 1640 led to up-regulation of SAPs 4–6 but not of SAP2. SAP9, which encodes a cell surface protein that is not secreted like the other Saps,18 was also up-regulated 2.5-fold under the conditions of these experiments.
Among other genes up-regulated by fluconazole exposure, 12 encoding enzymes in the ergosterol biosynthesis pathway, ERG2, ERG3, ERG4, ERG5, ERG6, ERG10, ERG11, ERG13, ERG24, ERG25, ERG26 and ERG251 were up-regulated by factors of 2.1–3.3-fold, a predictable result from the inhibition of Erg11 function by fluconazole. Only a small number of other genes showed greater up-regulation of expression than the three SAP genes, namely PGA31 (57-fold), PHO100 (6.3-fold), HGT7 (4.8-fold), HGT12 (3.9-fold), PGA7 (3.7-fold), RBT5 (3.6-fold) and orf19.2369 (4.5-fold). With the exception of the latter, these genes all encode cell surface proteins or glucose transporters. Only two genes were down-regulated by 2-fold or more when cells were exposed to fluconazole. ARO10, encoding a pyruvate decarboxylase, was down-regulated 2.1-fold, and PHO84, encoding a protein similar to high-affinity phosphate transporters, was down-regulated 2.2-fold (the same figure was obtained for the two fragments representing PHO84 on the Eurogentec arrays).
A confirmatory expression profile was done with triplicate arrays for RNA extracted from cells grown for 4 instead of 2 h in the presence of fluconazole at 0.25 mg/L. Unfortunately, the batch of arrays used for this experiment was missing the SAP4, SAP5 and SAP6 genes, so the up-regulation of the SAPs could not be confirmed. For most of the other up-regulated genes, the results after 4 h of exposure were similar to those after 2 h of exposure.
Expression of SAP-GFP promoter fusions in C. albicans cells of different morphologies
Since expression of SAP4, SAP5 and SAP6 was up-regulated in filamentous C. albicans cells grown in RPMI 1640 and exposed to subinhibitory concentrations of fluconazole, we sought to investigate the relationships between morphology, fluconazole exposure and SAP expression in cells induced to grow in different morphologies. Use of our SAP promoter-GFP constructs for these experiments allowed us to profile gene expression in single cells, which was a finer-scale approach to assessment of expression than gross measurements of SAP RNAs in C. albicans populations.
We previously showed that C. albicans cells containing the SAP2-GFP construct grown in the yeast form in YCB + BSA and exposed to fluconazole at 1 mg/L fluoresced five times as strongly after 72 h as cells grown in the absence of fluconazole, confirming that fluconazole exposure led to up-regulation of SAP2 under these conditions.8 However, for the purposes of the present experiments, in which pseudohyphal and hyphal forms of various SAP-GFP constructs were to be compared, such a long growth period led to formation of clumps of filamentous cells in which it was impossible to determine levels of fluorescence in individual cells by image analysis. We therefore repeated the experiment in which the SAP2-GFP construct was grown in YCB + BSA but under the conditions used throughout the present study, i.e. 4 h growth in the absence of fluconazole followed by a further 4 h with fluconazole added at 0.5 mg/L.
In the absence and presence of fluconazole, respectively, Ca62, the negative control strain, gave mean ± SD fluorescence values of 239 ± 80 and 232 ± 98 arbitrary units, indicating no influence of fluconazole on basal fluorescence levels. For Ca106 cells, containing the SAP2-GFP construct, the mean fluorescence intensity was 300 ± 76 U (n =72) in the absence of fluconazole and 386 ± 96 U (n = 54) after 4 h growth in fluconazole at 0.5 mg/L (P < 0.00001 by Student's t-test and Wilcoxon U-test). The up-regulation of the SAP2 promoter in the presence of fluconazole was thus confirmed under short-term growth experimental conditions as it had been previously in older cultures.
Yeast-form growth experiments in YCB + BSA ± fluconazole were repeated under the same conditions for the constructs with GFP tagged to promoters of SAP4, SAP5 and SAP6. Mean ± SD fluorescence values (arbitrary units) for these three strains in the absence of fluconazole were 208 ± 101, 181 ± 85 and 229 ± 69, respectively. In the presence of fluconazole (0.5 mg/L for 4 h), the corresponding values were 233 ± 93, 218 ± 101 and 214 ± 66. None of these differences reached the criterion for statistical significance within each strain (P > 0.01). Numbers of individual yeast cells analysed ranged from 82 to 136 per strain/condition. All of the mean fluorescence values for SAP4–6-GFP strains grown in YCB + BSA were significantly lower than those for SAP2-GFP (Student's t-test and Wilcoxon U-test). Univariate ANOVA for the four SAP-GFP constructs, with fluorescence set as the dependent variable and strain and fluconazole exposure as fixed factors, confirmed significant influences of both factors on fluorescence measurements (P < 0.0001). Neither factor emerged as significant (P =0.03) when the data for the SAP2-GFP strain were omitted from the analysis. We interpret these results as evidence that SAP2-GFP expression was up-regulated in yeast-form cells of C. albicans grown in YCB + BSA and was further up-regulated by fluconazole exposure. By contrast, none of the constructs with GFP tagged to SAP4, SAP5 or SAP6 promoters showed significant up-regulation during yeast-form growth in YCB + BSA, nor was expression altered by addition of fluconazole to the medium. These data are consistent with the transcript profiling results.
Fluorescence of SAP4–6-GFP in filamentous cells
Fluorescence microscopy of hyphal or long pseudohyphal forms of strains carrying SAP-GFP promoter fusions grown in RPMI 1640 showed a consistent differential pattern of GFP staining between Ca188 (SAP5-GFP) and the other SAP-GFP constructs (Figure 1). Fluorescence intensity in each filamentous cell compartment was approximately the same for the SAP5-GFP strain Ca188 (Figure 1h), whereas for the strains (Ca70 and Ca86) with GFP tagged to the SAP4 (Figure 1f) and SAP6 (Figure 1j) promoters the most intense fluorescence appeared in the apical cell compartment and intensity decreased in a stepwise fashion in the two compartments located more proximally to the parent yeast cell. The negative control strain with pGFP, Ca62, showed only background fluorescence (Figure 1b), whereas the positive control strain Ca6, with GFP tagged to ACT1, fluoresced brightly along the length of the filament (Figure 1d). Cultures of Ca6 consistently contained a higher proportion of free yeast forms than the other strains when grown in RPMI 1640: the yeast cells also fluoresced brightly (Figure 1d).
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These differences in fluorescence intensity are quantified in Figure 2(a). The curves confirmed the differential expression between filament compartments of SAP-4-GFP and SAP6-GFP but not of SAP5-GFP or the positive control construct ACT1-GFP, when these strains were grown in RPMI 1640. Fluorescence in SAP4-GFP filaments also differed between compartments in a similar way when the cells were grown in GlcNAc or YPD-serum (Figure 2a). However, the fluorescence of fungal compartments of the SAP6-GFP construct grown in these two media did not show the same differential as in RPMI 1640. Fluorescence in the four filament compartments measured for the SAP5-GFP strain was similar in all media. The general linear ANOVA model of fluorescence data by compartment for the different strains grown in RPMI 1640 showed a highly significant compartmental variation for Ca70 (SAP4-GFP; P < 0.0001) and Ca86 (SAP6-GFP; P < 0.0001) but not for Ca62 (no GFP; P = 0.16), Ca6 (ACT1-GFP; P = 0.99) or Ca88 (SAP5-GFP; P = 0.89).
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To compare the compartmentalization of SAP-GFP expression with overall levels of gene expression in filament compartments, the fluorescence intensity of RNA was measured in cells stained with acridine orange and imaged with the nucleus excluded. The total RNA fluorescence intensity was an average of 1.7–3.0-fold higher in apical compartments than in other proximal compartments for all strains. The higher expression of the SAP4-GFP and SAP6-GFP in apical compartments of filamentous cells was therefore of a similar order as the higher levels of RNA in these compartments. By contrast, the fluorescence of actin and of SAP5-GFP in different compartments did not show the same correlation with overall RNA levels.
Effect of antifungal agents on SAP4–6-GFP expression in vitro
Exposure of the C. albicans mutants to fluconazole at 0.5 mg/L, a concentration that reduced growth to 60% to 80% of control by 24 h, increased the fluorescence intensity of the GFP, particularly in the apical compartments in hyphae/long pseudohyphae of SAP4-GFP and SAP6-GFP (Figure 2b). A similar effect was seen in cells treated with itraconazole, but the curves for flucytosine-treated cells matched those of controls (Figure 2b). The fluorescence data in Figure 2(b) were analysed statistically stratified by filament compartment and by drug exposure as fixed factors in the general linear ANOVA model, with results summarized in Table 2. Only in the SAP4-GFP and SAP6-GFP constructs exposed to fluconazole were both factors statistically significant contributors to fluorescence. Itraconazole exposure significantly increased fluorescence in the SAP4-GFP strain but the difference did not reach the P < 0.01 significance level in the SAP6-GFP construct. None of the cells exposed to flucytosine showed any significant fluorescence increase related to the drug. In the SAP5-GFP mutant, fluconazole exposure contributed more than filament compartment to fluorescence intensity differences, but neither factor reached the level of statistical significance as defined in this study. Fluorescence intensity in the negative and positive control strains showed no significant difference by compartment or fluconazole exposure.
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Effect of fluconazole on SAP2-GFP and SAP4–6-GFP expression in vivo
Quantification of fungal fluorescence for samples of kidney was technically difficult and required re-calibration of background fluorescence adjacent to almost every sample with a visible hyphal or long pseudohyphal tip. From 3 to 14 fungal samples were measured per mouse and relative fluorescence averaged. The results (Table 3) showed that, as in vitro, the lowest mean fluorescence was seen for the negative control strain and the highest for the ACT1-GFP construct. Levels of fluorescence for all four SAP-GFP constructs were, however, higher than measured in vitro relative to the positive and negative controls, but were not significantly different between animals that did and did not receive fluconazole treatment.
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This study has shown that a 4 h of exposure of filamentous C. albicans cells to subinhibitory concentrations of fluconazole in RPMI 1640 medium, in the absence of serum, causes a significant increase in expression of the SAP4 and SAP6 promoters as measured by the relative fluorescence intensity of GFP encoded from the promoter tags. Itraconazole, but not flucytosine, tended to produce a similar effect. The expression profiling and promoter-GFP results were consistent in indicating that SAP2 was not expressed at all in RPMI 1640, as would be predicted from previous data indicating that this enzyme is secreted predominantly by yeast-form cells.9 SAP4, SAP5 and SAP6 were all up-regulated in the transcript profiles and their promoter-GFP constructs fluoresced when grown in RPMI 1640 (Figure 1), consistent with their known association with hyphal growth.9 By contrast, the SAP2 promoter-GFP strain fluoresced brightly when yeast cells were grown in YCB + BSA medium, while negligible fluorescence was seen for the equivalent SAP4-6 promoter-GFP strains. We have referred to the filamentous growth in RPMI 1640 as ‘filamentous’ or ‘hyphal/long pseudohyphal’ because not all the cells were fully parallel-sided as would be expected of true hyphae, nor was it possible in every case to determine the location of the first-formed septum in the filaments.19
Because the fungus grew predominantly in a filamentous morphology in RPMI 1640, experimental conditions that had been used previously to test SAP expression8 had to be modified to include shorter incubation times. With filamentous growth compartments thus more readily visualized, it became evident that GFP fluorescence in the SAP4-GFP and SAP6-GFP strains was not equally bright in all cell compartments, but was strongest in the apical compartment and fell off gradually in compartments more proximal to the parent blastospore (Figure 1f and j). By contrast, fluorescence in the SAP5-GFP strain was both of lower intensity than in the SAP4 and SAP6 constructs, and seemed equally intense among all filament compartments. Staining of RNA in the cells indicated, as would be expected, that most RNA was produced in the growing apical tip compartment. The results for the SAP5-GFP strain show lower overall levels of GFP expression for this construct. Our data suggest that SAP4/6 and SAP5 are differentially regulated with regard to spatial expression patterns in the elongating hypha. This raises the possibility that these genes might contribute differentially to tissue invasion.
The compartmentalization of fluorescence added an extra complication to evaluation of the effects of antifungal drugs on SAP-GFP expression. Because of the fluorescence differences between compartments, we used univariate ANOVA to differentiate the contributions of compartmentalization from the contribution of antifungal treatment to the ROI fluorescence intensity. The results showed that fluconazole, and to a lesser extent itraconazole, exposures increased the overall level of fluorescence expressed in the SAP4–6-GFP strains (Figure 2 and Table 2), but the effects were particularly pronounced with the SAP4-GFP and SAP6-GFP strains. These observations extend our previous findings that several antifungal agents stimulate increased expression of SAP2 for C. albicans grown in the yeast form in YCB + BSA medium at 25°C.8 We speculate that the regulation of SAP gene expression by Stp1, shown for SAP2 under yeast-form-inducing conditions,20 may be a general control mechanism for expression of other SAP genes. The SAP expression is then up-regulated by Stp1 under indirect influence of stress responses induced by exposure of the cells to azole antifungal agents.
Since RPMI 1640 is both a tissue culture medium and the medium recommended for routine yeast susceptibility testing in vitro,10 the finding that the three SAP genes expressed in this medium at 37°C are up-regulated in this medium by exposure to subinhibitory levels of two azole antifungal agents suggests that a similar phenomenon may be seen in vivo. However, results from the single-cell profiling experiments in vivo did not show SAP up-regulation by antifungals to the extent seen in vitro. Within the kidney tissues, the fluorescence of the GFP-tagged SAP2 strain, as well as that of the SAP4, SAP5 and SAP6 constructs, all showed a general increase, relative to the fluorescence of the positive and negative controls, suggesting that all four genes were being expressed. Previous studies showed that, while these four SAPs (2, 4, 5 and 6) were up-regulated in infected kidney tissues,21 the up-regulation of SAP2 occurred later in the course of infection than that of the other three SAPs.22 The time after infectious challenge, as well as the timing after fluconazole treatment, all probably contribute to GFP fluorescence measurements within kidney tissues. Our results with a minimum number of animals (Table 2) are compatible with the fluconazole-induced SAP up-regulation data in vitro but suggest that the impact of fluconazole treatment on expression of these virulence factors in the whole animal may be transient, in comparison with the effects seen in vitro where the antifungal exposure was continuous through the culture period.
Although the main focus of this study was on expression of SAP4–6, the expression profiling results provided other findings of interest. Previous investigators have found up-regulation of ergosterol biosynthesis genes in C. albicans cells exposed to fluconazole and other azole antifungals under conditions different from those in the present study.23–26 Several of these publications focused on gene expression in relation to azole resistance development, whereas our work utilized the azole-susceptible strain SC5314. Our findings of fluconazole-induced up-regulation of genes encoding cell surface proteins and transporters of sugars and amino acids are consistent with other investigations done under other conditions. The picture that clearly emerges from expression profiling of azole-exposed C. albicans cells is of pathway-level up-regulation of genes involved in ergosterol biosynthesis, together with genes whose transporter products may facilitate import of small-molecular-weight nutrients to cells at risk of growth inhibition by an antifungal agent. We did not find up-regulation of any of the genes encoding the fluconazole efflux pumps CDR1, CDR2 or MDR1 under the conditions of our experiments.
The finding that at least four of the SAP genes normally involved in degradation of extracellular proteins for nutrients can be up-regulated by exposure to antifungal agents under conditions favouring different cell morphologies remains intriguing. The virulence role of the Sap enzymes is presumably related to their function as relatively non-specific degraders of host proteins.27 A similar tendency of SAP genes to respond to fluconazole exposure suggests their induction may be a part of a stress-related defence mechanism in C. albicans, yet the up-regulation of different SAPs in yeast and filamentous morphologies indicates that SAP expression is under different controls than more ubiquitous stress response genes.28,29
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This study was supported by a grant from the British Society for Antimicrobial Chemotherapy.
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F. C. O. holds shares in Johnson & Johnson, has received grant funding from Italfarmaco SpA, Syngenta Ltd, Pfizer Research Ltd and Merck Sharp & Dohme, Ltd, and has received honoraria as consultant or speaker from Astellas Pharma, Gilead Sciences, Merck, Sharp & Dohme, Pfizer and Schering-Plough. N. A. R. G. has received grant funding and honoraria from Gilead Sciences, Inc.
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Acknowledgements |
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We acknowledge the technical assistance of Mark J. Fordyce.
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References |
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