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Antimicrobial Agents and Chemotherapy, August 2007, p. 2793-2800, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00094-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Differentially Expressed Proteins in Derivatives of Candida albicans Displaying a Stable Histatin 3-Resistant Phenotype{triangledown}

Deirdre H. Fitzgerald-Hughes,1* David C. Coleman,1 and Brian C. O’ Connell2

Microbiology Research Unit, Division of Oral Biosciences,1 Division of Restorative Dentistry and Periodontology, School of Dental Science and Dublin Dental Hospital, University of Dublin, Trinity College Dublin, Dublin 2, Republic of Ireland2

Received 22 January 2007/ Returned for modification 1 March 2007/ Accepted 25 April 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histatin-resistant derivatives of Candida albicans strain 132A, generated by successive exposure to increasing concentrations of histatin 3, were previously reported to be similar to the parent strain in their histatin binding, internalization, oxygen consumption, ATP efflux, and histatin degradation. Proteomic analysis of further histatin-resistant secondary derivatives of this series revealed that 59 proteins were differentially expressed compared to the parental strain. Of these 59 proteins, 3 were absent in histatin-resistant secondary derivatives and 11 were absent in the parent strain. Of the proteins absent in the histatin-resistant derivatives, the most notable was elongation factor 2, a target for the natural antifungal sordarin. Of the proteins absent in the parent strain but present in histatin-resistant derivatives, those identified included isocitrate lyase (Icl1p), fructose biphosphate aldolase (Fba1p), pyruvate decarboxylase (Pdc2p), and ketol-acid reductoisomerase (Ilv5p). The present secondary derivatives showed significantly decreased rates of oxygen consumption and histatin 3-mediated ATP release compared to the parent strain and also showed stability of the histatin-resistant phenotype. A significant (twofold) decrease in transcript levels of the potassium transporter encoded by TRK1, a critical mediator of histatin killing, was found in only one of the secondary histatin-resistant derivatives compared to the parent strain. The sequential exposure of C. albicans to histatin 3 described here resulted in the induction or selection of a phenotype with impaired metabolic function. The results support an important role for metabolic pathways in the histatin resistance mechanism and suggest that there may be several intracellular targets for histatin 3 in C. albicans.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oral candidiasis is a common infection caused predominantly by the opportunistic yeast pathogen Candida albicans, which is frequently a commensal of the oral cavity. The human innate immune system provides the first line of defense against infection with oral Candida species, and the secreted antimicrobial proteins responsible for combating oral yeasts include the ß-defensins, lactoferrin, lysozyme, and the salivary histatins. The histatins have potential as novel therapeutic antifungal agents, and it has been known for some time that they differ in their mechanism of antifungal activity compared to the azole antifungal drugs, such as fluconazole and itraconazole, most commonly used therapeutically to treat Candida infection. The mechanism of action of histatin involves a multistep process initiated by binding to a yeast cell surface receptor, identified as heat shock protein Ssa2p (17, 18). This is followed by internalization and interaction with intracellular targets, such as the mitochondrion (8, 10). Yeast cell killing in vitro correlates with the release of ATP and potassium and magnesium ions, mediated by the potassium transporter Trk1p (2). We previously reported that histatin-resistant derivatives of C. albicans 132A (termed CAHR1 and CAHR4), generated by sequential exposure to increasing concentrations of histatin 3, were similar to their parental strain in their histatin binding, internalization, and degradation and showed no difference in ATP efflux or oxygen consumption rates (4). Since no difference in the histatin killing mechanism of the histatin-resistant derivatives could be inferred from our previous study, the present study was undertaken in order to globally assess proteomic alterations between newly generated histatin-resistant derivatives and the histatin-susceptible parent to identify other potential C. albicans targets that may play a role in histatin-mediated killing. For the purpose of this study we were interested specifically in changes that occurred at the level of protein expression or posttranslational changes, since changes at these levels are more meaningful than changes at the gene or transcript level, in which single base changes resulting in alterations at the protein level may not be detected.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Candida strains, growth conditions, and generation of histatin-resistant derivatives. C. albicans strain CA132A (5) was the histatin-susceptible parent strain used in this study. The C. albicans respiratory-deficient mutant strain MMU11 (6) was provided by Kevin Kavanagh, National University of Ireland, Maynooth, Co. Kildare, Ireland. Two histatin-resistant derivatives of CA132A generated in a previous study (CAHR1 and CAHR4) by sequential exposure to histatin 3 (4) lost the histatin-resistant phenotype (killing activity at 0.2 mg/ml histatin 3: CAHR1, 86.4% ± 9.4%; CAHR4, 94.4% ± 4.6% [mean ± standard error of the mean, SEM]) after storage at –80°C for 1 year (reactivated by growth on potato dextrose agar [PDA] at 37°C for 48 h). One of these primary derivatives, CAHR4, was therefore exposed to further sequential rounds of histatin 3 exposure as described previously (4) to regenerate the histatin-resistant phenotype. Briefly, single colonies of CAHR4 were grown overnight (18 h) at 37°C in 10 ml yeast extract-peptone-dextrose medium in a shaking incubator at 200 rpm. Cells (1 x 105) were incubated with histatin 3 (0.4 to 0.8 mg/ml) and 10 mM potassium phosphate buffer, pH 7.4, in a total volume of 100 µl, for 1 h at 37°C before adding 900 µl 0.9% (wt/vol) NaCl. A 100-µl aliquot was spread onto PDA (Oxoid, United Kingdom) plates and grown at 37°C for 48 h, and at least two surviving colonies were selected and replated onto PDA and, independently of each other, were subsequently subjected to successive rounds of histatin 3 exposure as described above. The history of histatin exposure for the secondary histatin-resistant derivatives generated in this way is summarized in Table 1. The secondary derivatives CAHR04A and CAHR04B were generated from CAHR4 following the exposure regimen outlined. Secondary derivative CAHR04U was generated from CAHR04A following a further four consecutive rounds of histatin 3 exposure. Cells were counted using a hemocytometer, and cell numbers were adjusted by dilution.


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  		TABLE 1. Histatin exposure regimen used to generate histatin-resistant derivatives of C. albicans strain CA132Aa

 
Candidacidal assay of histatin 3. Histatin 3 (DSH/AKR/HHG/YKR/KFH/EKH/HSH/RGY/RSN/YLY/DN) was synthesized commercially by JPT Peptide Technologies (Invalidenstrasse 130, 10115 Berlin, Germany). Candidacidal assays were carried out on the C. albicans parental strain CA132A and histatin-resistant derivatives as described previously (4). Briefly, killing assays were performed in 1.5-ml microtubes in 100 µl of 10 mM potassium phosphate buffer, pH 7.4, and contained 1 x 10 5 cells and 0 to 0.4 mg/ml histatin 3 (this range spans the physiological concentration of histatin reported in saliva [16]). The assay mixture was incubated for 1 h at 37°C with vigorous mixing before adding 900 µl of 0.9% (wt/vol) NaCl, and 100 µl of this dilution was spread onto PDA plates, followed by incubation at 37°C for 48 h. Killing activity was calculated as the number of CFU on test plates as a percentage of CFU on plates incubated in the absence of histatin 3.

Colony morphology and growth characteristics. To determine their respiratory status, the parental strain CA132A, the three secondary histatin-resistant derivatives CAHR04A, CAHR04B, and CAHR04U, and the classic respiratory-deficient mutant MMU11 (used as a respiratory-incompetent reference strain) were grown on yeast extract-peptone agar containing glycerol as the carbon source (38 ml/liter). The method described by Gyurko et al. (8) was used for color-based differentiation between the histatin-resistant secondary derivatives and classic respiration-deficient mutant MMU11. Colonies were grown on indicator plates containing (per liter) 1.5 g of KH2PO4, 1.5 g of (NH4)2SO4, 1 g of MgSO4·H2O, 1.5 g of peptone, 1.5 g of yeast extract, 20 g of glucose, 0.01 g of eosin Y, 0.01 g of trypan blue, and 15 g of agar.

Growth rate determinations for CA132A and histatin-resistant derivatives. Two of the three secondary histatin-resistant derivatives were selected for growth rate determinations. Overnight cultures of CA132A and secondary histatin-resistant derivatives CAHR04A and CAHR04B were adjusted to 1 x 105 cells/ml, and 200-µl aliquots were added to 96-well plates with flat-bottomed wells (Greiner Bio-One, Frickenhausen, Germany). Increasing cell density was measured with time (20 h) as an increase in absorbance at 595 nm using a GENios model Tecan spectrophotometer (Crailsheim, Germany). The plates were set to shake continuously at normal intensity (instrument setting).

Stability of the histatin-resistant phenotype in derivatives. Two of the histatin-resistant derivatives, CAHR04A and CAHR04B, were subjected to 10 consecutive subcultures on histatin 3-free PDA. Following subculture, the derivatives recovered were designated CAHR04sub10A and CAHR04sub10B, and their histatin 3 susceptibility and growth rate were determined as described above.

Oxygen consumption measurement. Endogenous oxygen consumption rates were measured using a Clark-type biological oxygen electrode (Rank Brothers, Cambridge, United Kingdom). Rates were measured at room temperature for 1 x 10 8 cells/ml in 10 mM potassium phosphate buffer, pH 7.4. An unpaired Student's t test was used to compare sets of data. A P value of less than 0.05 indicated a significant difference between sets of data. The free online statistical calculator provided by the Department of Statistics, UCLA (http://calculators.stat.ucla.edu/) was used to calculate P values.

Measurement of histatin-mediated ATP release. The method of Ansehn and Nielson (1) was used to measure the effect of histatin 3 on the release of ATP from overnight cultures of C. albicans CA132A, CAHR04A, CAHR04B, and CAHR04U and was the same method used previously by us on the primary histatin-resistant derivatives (4). Briefly, C. albicans cells (1 x 10 6) were incubated with histatin 3 (0.2 mg/ml) for 45 min. The reaction mixture was centrifuged at 5,000 x g for 5 min. A 25-µl volume of supernatant was added to 210 µl boiling TE buffer (50 mM Tris, 2 mM EDTA, pH 7.8), boiled for a further 4 min, and stored on ice until ATP measurement. ATP levels were measured by luminometry using an ATP assay kit (Sigma-Aldrich, Ireland Ltd.). The luciferin-luciferase assay mix (2.5 mg/ml; 50 µl) was added to 20 µl of the extracellular material, and the light emission was measured in a TD20/20 luminometer (Turner Designs, Sunnyvale, CA). Relative light units were expressed as pmol ATP by reference to ATP standard curves.

Detection and quantification of TRK1 transcript in histatin-resistant derivatives. Total RNA was extracted from C. albicans cultures grown to mid-exponential phase (optical density at 600 nm, 0.6) in 50-ml volumes of YPD broth at 37°C with shaking at 200 rpm in an orbital incubator (Gallenkamp). Extractions were carried out by the glass bead disruption method described by Hube et al. (12). RNA was DNase treated (Promega, United Kingdom) before being reverse transcribed using the SuperScript first-strand synthesis kit (Invitrogen, Paisley, United Kingdom) according to the manufacturer's instructions. Primer pairs used to detect TRK1 were 5'-GGTAAGAGATTGAAGGAGCAG-3' (trkF) and 5'-ATAGACATCCCCACCGTACCAT-3' (trkR). The TRK1 primer set was designed on the basis of the sequence of the C. albicans TRK1 gene using PrimerExpress software (Applied Biosystems, Foster City, CA) and amplified a 92-bp fragment. The EFB1 primers, 5'-CCACTGAAGTCAAGTCCGTTG-3' (ef1F) and 5'-CACCTTCAGCCAATTGTTCGT-3' (ef1R), described by Green et al. (7) were used as an internal control. Real-time PCR was performed using the Quantitect SYBR green PCR kit (QIAGEN, West Sussex, United Kingdom) on an ABI Prism sequence detection system (model 7700; PE Applied Biosystems, Foster City, CA) using 40 amplification cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. The threshold cycle values were calculated for each sample and used to calculate the comparative expression levels of TRK 1 in histatin-resistant derivatives relative to the parental strain CA132A using the calculation described in the ABI user's manual. The copy numbers for the TRK1 target were first normalized to the copy numbers obtained for EFB1.

2D gel electrophoresis. Two-dimensional (2D) gel electrophoresis and image analyses were undertaken commercially by the Proteome Factory (Dorotheenstr. 94, D-10117 Berlin, Germany). Triplicate whole-cell lysates from parental strain CA132A and secondary histatin-resistant derivatives CAHR04A, CAHR04B, and CAHR04U were prepared according to the Proteome Factory protocol. Briefly, six volumes of buffer containing 9 M urea, 70 mM dithiothreitol, and 2% ampholytes (2-4) were added to samples after disruption using a mortar and pestle. The lysates were frozen in liquid nitrogen, thawed five times, and then sonicated three times for 15 s. Following incubation for 30 min the samples were centrifuged for 45 min at 15,000 x g and the supernatant was frozen at –80°C. Two-dimensional gel electrophoresis was performed according to the Proteome Factory 2D electrophoresis protocol based on the method of Klose and Kobalz (14). After electrophoresis, gels were silver stained for detection of protein spots using the method described by Heukeshoven and Dernick (11).

Image analysis. Two-dimensional image analysis was provided commercially by the Proteome Factory (Dorotheenstr. 94, D-10117 Berlin, Germany). The 2D electrophoresis gels used for comparison analysis were digitized at a resolution of 150 dots per in. using a PowerLook 2100XL scanner with transparency adapter. Two-dimensional image analysis was performed using the Proteomweaver software (Definiens AG, Munich, Germany). Spot volumes were normalized against total spot volume and total spot area after background subtraction. Features that displayed statistically significant changes in mean normalized spot volume on all three replicate gels (P ≤ 0.05 by Student's t test) between histatin-resistant derivatives and the parent strain were deemed significantly altered.

Identification of differentially expressed proteins. Proteins that were completely absent in the histatin-resistant derivatives but present in the parent (3 proteins) or absent in the parent strain but present in the three histatin-resistant derivatives (11 proteins) were selected for identification. Identification of these protein spots was undertaken commercially by the Proteome Factory (Dorotheenstr. 94, D-10117 Berlin, Germany). Analysis of protein spots excised from 2D gels was performed by in-gel tryptic digestion. The generated peptides were applied to an Agilent 1100 nano-LC system with a trap column online coupled to an ion trap mass spectrophotometer (Esquire 3000plus; Bruker Biosciences Corporation, MA). The tandem mass spectrometry data were searched against the NCBInr protein database (http://www.ncbi.nlm.nih.gov/) using the Mascot search engine.

DNA fingerprinting using the Ca3 probe. The Ca3 DNA fingerprinting probe is a moderately repetitive C. albicans-specific DNA sequence used specifically to assess the genetic relatedness of C. albicans strains based on the number and size of Ca3-specific bands occurring in genomic DNA (9, 19, 22). Fingerprinting analysis yields identical or similar banding patterns for closely related strains, and this probe was used to confirm that both primary and secondary histatin-resistant derivatives were clonally related to each other and to the histatin-susceptible parent. Southern blot hybridization of endonuclease-digested DNA was carried out as described previously (23). Genomic DNA from the C. albicans parental strain CA132A, its histatin-resistant derivatives CAHR1 and CAHR4 (primary derivatives) and CAHR04A, CAHR04B, and CAHR04U (secondary derivatives) were digested with EcoR1 before electrophoresis through a 0.8% (wt/vol) agarose gel for 20 h at 40 V and transferred by capillary blotting to a nylon membrane (Biobond Plus; Sigma-Aldrich, United Kingdom) as described previously (23). Hybridization reactions were carried out under high-stringency conditions with 32P-labeled Ca3 probe (19) and random primer labeling. The relatedness of secondary histatin-resistant derivatives to the parent strain, CA132A, was computed using the Dendron software package, version 2 (Solltech, Iowa City, IA). This package computes a similarity coefficient (SAB) for relatedness between two isolates (A and B) based on the number of changes in Ca3 probe-specific bands. A similarity coefficient of 0.0 indicates unrelated isolates, and 1.0 indicates identical isolates.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DNA fingerprinting and phenotypic features of histatin-resistant derivatives. The Ca3 probe is a moderately repetitive sequence used specifically to determine the clonal relationship between C. albicans isolates. It is routinely used for this purpose, as it gives an accurate measure of the genetic distance between strains based on the hypervariability of genomic sequences homologous to the C fragment of the probe (19). The parental strain, CA132A, the histatin-resistant secondary derivatives generated during this study (CAHR04A, CAHR04B, and CAHR04U), and the primary derivatives from our previous study (CAHR1 and CAHR4) (4) yielded identical fingerprint patterns which differed by a single band from the parental strain fingerprint pattern, indicating their close clonal relationship (fingerprint not shown; similarity coefficient SAB of 0.9 to 1.0; computed with the Dendron software package, version 2, Solltech, Iowa City, IA) and providing convincing evidence that the histatin-resistant derivatives did not arise from contamination during their generation.

Although the secondary histatin-resistant derivatives CAHR04A, CAHR04B, and CAHR04U generated in this study produced smooth round white colonies similar to CA132A on PDA plates, they were approximately one-third smaller compared to colonies of the parental strain, and this reduced colony size was retained on repeated subculture. Unlike CA132A, which produced colonies on medium containing glycerol as a carbon source, and the classic petite mutant strain MMU11, which was incapable of growth on this medium, the secondary histatin-resistant derivatives grew very poorly on glycerol-limited medium (produced colonies approximately three times smaller than those of CA132A and which were visible only after 4 days at 37°C [data not shown]), indicating that respiration was compromised compared to CA132A. Furthermore, the histatin-resistant derivatives were shown to be distinct from a classical respiration-deficient strain (MMU11) by producing pinkish colonies similar in color to the parental strain CA132A on medium supplemented with trypan blue and eosin Y (Fig. 1). The respiration-deficient strain MMU11 produced blue colonies on this medium.


Figure 1
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  		FIG. 1. Color differentiation of histatin-resistant secondary derivatives, CA132A, and MMU11 on indicator plates containing eosin Y and trypan blue. On this medium, CA132A and the histatin-resistant derivatives form pink colonies, while the respiration-deficient strain MMU11 grows as blue colonies.

 
Two of the three histatin-resistant secondary derivatives (CAHR04A and CAHR04B) were selected for growth rate determinations, and as shown in Fig. 2C, both derivatives had a slower growth rate than the parental strain (t1/2 values: CA132A, 5 h; CAHR04A, 7.3 h; CAHR04B, 7.4 h). The susceptibility of histatin-resistant secondary derivatives CAHR04A, CAHR04B, and CAHR04U to histatin 3 was compared to the parental strain. At each concentration of histatin 3 studied, the derivatives were significantly less susceptible to histatin 3 than their parent strain, CA132A. At 0.2 mg/ml histatin, which caused 91.7% killing of CA132A, only 17.9 to 31.9% killing activity was found for the histatin-resistant secondary derivatives (Fig. 2A).


Figure 2
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  		FIG. 2. Histatin susceptibility and stability of the histatin-resistant phenotype of resistant derivatives. A. Candidacidal activity was determined for the histatin-resistant derivatives CAHR04A, CAHR04B, and CAHR04U, compared to the parental strain CA132A. B. The histatin susceptibility of histatin-resistant derivatives CAHR04A and CAHR04B was determined following 10 consecutive subcultures on histatin 3-free PDA (CAHR04Asub10 and CAHR04Bsub10). For panels A and B, results are expressed as the mean ± SEM for three separate determinations. C. The growth rate was determined (see Materials and Methods) for histatin-resistant derivatives that had been subcultured 10 times onto fresh histatin 3-free PDA plates and compared to those that had not been subcultured. The data point symbols for CAHR04B are masked by the symbols of the other series.

 
The histatin-resistant phenotype of the primary derivatives CAHR1 and CAHR4 generated previously in this laboratory (4) was transient, and colonies reverted to the histatin-susceptible phenotype after reactivation from frozen storage (killing activity at 0.2 mg/ml histatin 3: CAHR1, 86.4% ± 9.4%; CAHR4, 94.4% ± 4.6%) or when subcultured in the absence of histatin up to three times (pre-cryopreservation). Two of the secondary histatin-resistant derivatives (CAHR04A and CAHR04B) were selected for determination of the stability of the histatin-resistant phenotype. Both CAHR04A and CAHR04B, generated from the primary histatin-resistant derivative CAHR4, showed stability of the histatin-resistant phenotype even after 10 consecutive subcultures on histatin 3-free PDA (Fig. 2B). Furthermore, the growth rate of the secondary derivatives, CAHR04A and CAHR04B, which was reduced compared to the parental strain, CA132A, did not change after subculturing 10 times onto fresh PDA (Fig. 2C).

Oxygen consumption measurement. The rates of oxygen consumption of histatin-resistant primary and secondary derivatives and the parent strain CA132A are shown in Table 2. Unlike the primary derivatives CAHR1 and CAHR4 (4) that exhibited an unstable histatin-resistant phenotype and showed no significant alteration in oxygen consumption rates compared to the parent strain, the oxygen consumption rates of all three secondary histatin-resistant derivatives (CAHR04A, CAHR04B, and CAHR04U) were significantly reduced (17.1 ± 0.9%, 27.0 ± 0.5%, and 34.6 ± 0.4%, respectively) compared to the parent strain (P < 0.001).


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  		TABLE 2. Oxygen consumption measurements for strains and derivatives

 
Histatin-mediated ATP release and TRK1 transcript levels. As shown in Fig. 3B, the rate of histatin-mediated ATP release from the secondary histatin-resistant derivatives CAHR04A, CAHR04B and CAHR04U was significantly reduced compared to that found for the parental strain, CA132A, after 45 min of incubation with histatin 3. The reduction in ATP release was accompanied by a 2-fold and 1.6-fold reduction in expression of the potassium transporter transcript TRK1 for CAHR04A and CAHR04B, respectively, but no significant alteration in TRK1 expression was found for CAHR04U as shown by quantitative real-time PCR (Fig. 3A).


Figure 3
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  		FIG. 3. Expression levels of TRK1 transcript and histatin 3-mediated release of ATP. A. Quantitative real-time PCR from total RNA samples prepared from C. albicans CA132A and histatin-resistant derivatives CAHR04A, CAHR04B, and CAHR04U, using specific primers for the potassium transporter gene TRK1 and elongation factor 1 gene EFB1. Comparative expression levels were calculated as described in Materials and Methods. The data presented are the means ± standard deviations for two separate reactions each carried out in triplicate. B. ATP levels were measured following incubation with 50 µM histatin (filled bars) or with buffer (open bars) for 45 min at 37°C. Values shown are the mean ± SEM of four determinations.

 
Comparative proteomic analysis of CA132A and histatin-resistant derivatives. Comparative 2D electrophoretic analysis of the proteome of the parental strain, CA132A, with those of the secondary histatin-resistant derivatives, CAHR04A, CAHR04B, and CAHR04U, showed that 20 proteins were significantly down-regulated and 39 proteins were significantly up-regulated (P ≤ 0.05) in all three histatin-resistant derivatives relative to the parental strain. The total numbers (mean ± SEM) of protein spots detected by this method were as follows: 1,631 ± 53 (CA132A), 1,565 ± 51 (CAHR04A), 1,484 ± 16 (CAHR04B), and 1,579 ± 71 (CAHR04U). On closer inspection of the 20 down-regulated protein spots detected by 2D gel analysis, it was noted that 3 of these proteins were completely absent in the histatin-resistant secondary derivatives, and of the 39 up-regulated proteins, 11 of these represented proteins that were absent in the parent strain but present in all the secondary derivatives. Only this subset of differentially expressed proteins (i.e., those present in the parent strain but absent in the secondary histatin-resistant derivatives and those absent in the parent strain but present in the secondary histatin-resistant derivatives) was subsequently identified by mass spectrometry. These proteins were chosen for identification because they represented the most dramatic alterations in protein expression between the parent and the histatin-resistant derivatives. For 2 of the 11 proteins absent in the parental strain, CA132A, identification showed that they represented two different isoforms of the same protein (i.e., two isoforms of Fba1p and two isoforms of Pdc2p). Therefore, in total, 12 proteins were identified (3 down-regulated and 9 up-regulated), and the available information on these proteins is summarized in Table 3.


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  		TABLE 3. Differentially expressed proteins from secondary histatin-resistant derivatives identified by mass spectrometry of protein spots from 2D gel electrophoresis

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The generation and characterization of the primary histatin-resistant derivatives CAHR1 and CAHR4 were described in our previous paper on this subject (4). The earlier study showed that there was no difference in the ability of these derivatives to bind and internalize histatin 3, to degrade histatin 3, or to release intracellular ATP, compared to the parent strain. On reactivation of these derivatives following storage at –80°C for 1 year, the histatin-resistant phenotype was found to have been lost and therefore the secondary histatin-resistant derivatives described here were generated from the previously studied primary derivatives by further exposure to histatin 3. It is unclear why the primary histatin-resistant derivatives lost this phenotype after cryopreservation. Before cryopreservation, the primary derivatives also reverted to the histatin-susceptible phenotype after three successive subcultures on PDA (data not shown). The histatin-resistant phenotype of the secondary histatin-resistant derivatives was stable, even when they were frozen for 2 months and then thawed and plated onto PDA (data not shown). It is possible that further rounds of exposure to histatin 3 (made necessary by the transience of the histatin-resistant phenotype in primary derivatives) may have introduced/selected for additional genetic alterations in the secondary histatin-resistant derivatives that contributed to their stability even after cryopreservation or subculturing. The biochemical alterations leading to the histatin-resistant phenotype were investigated in secondary derivatives. In the present study a proteome-wide approach was employed to identify differentially expressed proteins in secondary histatin-resistant derivatives compared to the parent CA132A.

Analysis of the three independent histatin-resistant secondary derivatives (CAHR04A, CAHR04B, and CAHR04U) by 2D gel electrophoresis revealed 59 variant spots (from a possible 1,400 to 1,600) common to all three derivatives (20 down-regulated and 39 up-regulated). Identification of 12 of these differentially regulated proteins (those that were absent in either the parent CA132A strain or absent in all three histatin-resistant secondary derivatives) revealed a number of interesting proteins, the regulation of which may contribute to the reduced susceptibility of strains CAHR04A, CAHR04B, and CAHR04U to histatin 3. The nature of the regulation of these proteins cannot be inferred from the proteomic data alone. To determine whether these proteins are regulated at the level of transcription or posttranslationally would require gene/transcript analysis. Future work in this area will involve transcript analysis using C. albicans-specific DNA microarray analysis, and the rationale for these studies will be guided by the data reported in the present study, such as specific lesions in metabolic pathways.

Significance of proteins present in parent strain CA132A but absent from histatin-resistant secondary derivatives. Elongation factor 2 (Ef2) was absent in all three histatin-resistant derivatives. This protein has been described as an essential protein that catalyzes ribosomal translocation during protein synthesis. Ef2 is a specific target for the naturally occurring antifungal agent sordarin, produced by species of the fungal genus Sordaria. The antifungal activity of sordarin involves specific binding to the yeast Ef2-ribosome complex and inhibition of the release of Ef2 from the posttranslocational ribosome. Mutations in the EFT2 gene have been shown to result in sordarin resistance (13). The absence of Ef2 protein in the histatin-resistant secondary derivatives may represent an adaptive response to the altered environment, and in this regard it is significant that Ef2 is also among several translation elongation factors that are repressed in response to phagocytosis of C. albicans by macrophages (20). Further investigation of the significance of this protein in histatin resistance and in the histatin killing mechanism would be important, as it may represent a potential antifungal drug target.

An uncharacterized enzyme regulator activity (ID ORF19.1467), localized to mitochondrial complex IV (cytochrome oxidase complex), was also absent in the histatin-resistant secondary derivatives. This respiratory chain complex catalyzes the oxidation of reduced cytochrome c by dioxygen. The absence of this mitochondrial regulator protein in histatin-resistant derivatives may also be of significance in histatin-mediated candidacidal activity, since in petite mutants (now more commonly referred to as respiration-deficient derivatives), disruptions in cytochrome oxidase activity reflected by reduced levels of cytochrome c, b, and aa3 also contribute to the histatin-resistant phenotype (8).

Significance of proteins absent from the parent CA132A but present in histatin-resistant secondary derivatives. The protein spots identified in histatin-resistant secondary derivatives but absent from the parental strain included the characterized proteins isocitrate lyase (Icl1p), fructose biphosphate aldolase (Fba1p), pyruvate decarboxylase (Pdc2p), and ketol-acid reductoisomerase (Ilv5p). Isocitrate lyase is a key component of the glyoxylate pathway that allows the conservation of carbon by bypassing three Kreb's cycle reactions, and induction of ICL1 transcription has been reported to be a virulence mechanism in C. albicans upon internalization by macrophages (20). Fructose biphosphate aldolase is a cytoplasmic enzyme required for glycolysis and gluconeogenesis, catalyzing the conversion of fructose-1,6-bisphosphate into two three-carbon products: glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. This protein was one of two represented twice in the up-regulated protein spots of the histatin-resistant derivatives, and both were absent in the parent, CA132A. Since FBA1 is considered an essential gene, it is possible that Fba1p is present in an active form in the parent CA132A and that the two proteins identified in histatin-resistant derivatives represent phosphorylated isoforms of Fba1p, suggesting that this enzyme activity may be regulated by posttranslational modification that may disrupt glycolytic and gluconeogenic pathways for energy production. It has been suggested that C. albicans may protect itself against the first line of innate antimicrobial defenses, such as macrophages and neutrophils, by facilitating anabolic reactions by up-regulation of glyoxylate and gluconeogenic pathways (3). The up-regulation of these processes in response to histatin 3 suggests that this mechanism may also protect against this aspect of the host innate immune response. Ketol-acid reductoisomerase and pyruvate decarboxylase are also involved in carbohydrate metabolism. Interestingly, three of these four metabolic activities are among those regulated in response to amino acid starvation in C. albicans and Saccharomyces cerevisiae that is mediated through the Gcn4p transcriptional regulator (24). It is possible that C. albicans may adapt to repeated exposure to histatin 3 by a similar mechanism of alteration in a key transcriptional regulator. Such Gcn4-like effects may allow this yeast to employ a variety of mechanisms to compensate for deficiencies in its central metabolism. Although such a transcriptional activator was not among those absent in the secondary histatin-resistant derivatives identified in this study, we cannot exclude the possibility that such a regulator may be among the 20 proteins which were down-regulated in histatin-resistant secondary derivatives but were not identified by mass spectrometry. The use of the alternative glyoxylate cycle and enhancement of central metabolic enzyme activities, as found in this study, may occur due to genetic or biochemical alterations following repeated exposure to histatin 3. Genetic analysis of the histatin-resistant derivatives would help to establish whether changes also occur at the genetic level for the proteins identified or if transcriptional regulators may be involved. Alternatively, repeated in vitro exposure to histatin 3 may select for a subpopulation of cells that have inherent reduced metabolic activity, which may provide a growth advantage in the presence of histatin 3. Respiration-deficient mutants, or petites, of C. albicans have previously been found to be less susceptible to histatin than the wild type. The secondary histatin-resistant derivatives generated by repeated exposure to histatin 3, although compromised metabolically, are distinct from classical respiration-deficient C. albicans generated by exposure to ethidium bromide (strain MMU11), as demonstrated by their colony color on plates supplemented with trypan blue and eosin Y (Fig. 1). On this medium, the respiratory MMU11 mutants appeared blue/violet, whereas the histatin-resistant derivatives and the parent strain CA132A appeared pink on this medium. The abilities of secondary histatin-resistant derivatives to grow, albeit poorly, on glycerol-limited medium and to consume oxygen at significantly reduced rates suggest that, although these derivatives appear to be metabolically compromised with lesions in metabolically important proteins, they retain some mitochondrial function.

Effect of prolonged exposure to histatin 3 on metabolic activity. The histatin-mediated killing mechanism is thought to involve disruption of cellular metabolic processes, since the peptide itself is targeted to the mitochondrion (10) and specific inhibitors of respiration and petite mutation protect C. albicans from the candidacidal effects of histatin (8). Sequential exposure of C. albicans to histatin 3, described here, results in the induction or selection of derivatives with impaired metabolic function. In support of this theory, analysis of the information available for the differentially expressed proteins identified in the histatin-resistant secondary derivatives CAHR04A, CAHR04B, and CAHR04U showed a preponderance of enzyme activities involved in either central or peripheral metabolic processes. Furthermore, functional analysis revealed that the metabolic activity of the histatin-resistant secondary derivatives was markedly affected, with oxygen consumption rates reduced to 17.1 to 34.6% of that of the parent CA132A. Previously, we found no difference in oxygen consumption rates in the histatin-resistant primary derivatives CAHR1 and CAHR4 compared to the parent strain, CA132A (4), and this was confirmed in the present study (Table 2). Furthermore, unlike the primary histatin-resistant derivatives (4), secondary histatin-resistant derivatives did not demonstrate significant histatin 3-mediated release of intracellular ATP, which is characteristic of histatin-mediated candidacidal activity (15). Loss of histatin-mediated ATP efflux was accompanied by a modest reduction (twofold compared to the parental strain) in TRK1 expression in only one of the histatin-resistant secondary derivatives (CAHR04A). TRK1 is an important mediator of histatin toxicity and has been reported to be the critical pathway for histatin-mediated loss of ATP (2). However, the absence of a significant correlation between loss of ATP efflux in histatin-resistant secondary derivatives and levels of TRK1 transcript suggests that attenuation of ATP release may involve TRK1-independent mechanisms. However, we cannot exclude the possibility that diminished ATP synthesis as a result of mitochondrial impairment may contribute to the low rate of ATP efflux in secondary histatin-resistant derivatives. Stability of the histatin-resistant phenotype, as demonstrated by the secondary derivatives but not by their predecessors, appears to coincide with a reduction in oxygen consumption and ATP release. Analysis of the differentially expressed proteins also suggested that despite disruption of central metabolism, activation of alternative metabolic pathways during prolonged histatin exposure may somewhat compensate for disruption to cell bioenergetics.

Although alterations in metabolism in the secondary histatin-resistant derivatives were evident based on differential expression levels of metabolic proteins, reduced oxygen consumption, and ATP release, the nature of the metabolic disturbance was difficult to characterize. Lack of growth on glycerol-limited medium indicates the lack of mitochondrial DNA. We found that the histatin-resistant secondary derivatives grew poorly on this medium (colonies visible after 4 days at 37°C). Furthermore, the histatin-resistant secondary derivatives produced pale pink colonies similar in color but smaller than parent strain CA132A on differential medium (medium supplemented with trypan blue and eosin Y) used to distinguish between respiratory-competent and respiratory mutant yeast strains. On this medium, the C. albicans respiratory mutant reference strain MMU11 produced blue colonies (Fig. 1). The physiological basis of color differentiation on this medium is poorly understood, but respiratory mutants appear blue/violet on this medium (21). These growth characteristics (on glycerol-limited and respiratory mutant differential medium) suggest that the secondary histatin-resistant derivatives, although compromised metabolically, are distinct from the classical petite mutants that lack mitochondrial DNA due to treatment with chemical mutagens, such as ethidium bromide. These phenotypic findings are supported by the proteomic data, which showed that mitochondrially expressed proteins were not among those absent in secondary histatin-resistant derivatives, and functional assays that indicate that derivatives show a limited ability to consume oxygen.

Although resistance to naturally occurring antifungal peptides is not widely reported to occur in vivo, the present study demonstrates that repeated in vitro exposure of C. albicans to histatin 3 results in the generation or selection of stable histatin-resistant derivatives with compromised metabolic activity. An important consideration in the development potential of histatin as a therapeutic agent would be the determination of the in vivo mechanism, occurrence, and significance of resistance to this peptide.


   ACKNOWLEDGMENTS
 
This work was supported by the Irish Health Research Board, grant no. RP 119-2002-2005.

We thank Christian Scheler, The Proteome Factory, Dorotheenstr. 94, D-10117 Berlin, Germany, for proteome analysis and protein identification.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Clinical Microbiology, RCSI Education and Research Centre, Smurfit Building, Beaumont Hospital, P.O. Box 9063, Dublin 9, Ireland. Phone: 353-1-809-3711. Fax: 353-1-809-3709. E-mail: dfitzgeraldhughes{at}rcsi.ie Back

{triangledown} Published ahead of print on 7 May 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Antimicrobial Agents and Chemotherapy, August 2007, p. 2793-2800, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.00094-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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