1 Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
2 Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, OH 44106, USA
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
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ABSTRACT |
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INTRODUCTION |
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Although we have used Northern blots for ALS gene expression analyses (Hoyer et al., 1995, 1998a
, b
), our focus shifted toward development of an RT-PCR assay for analysis of specimens from disease models and human clinical material where C. albicans cell numbers are more limited than from a culture flask. Such assays have been developed and used widely for analysis of gene expression for other C. albicans gene families (Schaller et al., 1998
, 2003
; Hube et al., 2000
; Ripeau et al., 2002
; Naglik et al., 1999
, 2003
; Schofield et al., 2003
). This paper describes development of an ALS gene RT-PCR assay and its application to cells from the reconstituted human buccal epithelium (RHE) disease model, which was originally introduced into the C. albicans literature by Schaller et al. (1998)
. While working with these specimens, we noted that the C. albicans cells inoculated onto the RHE surface formed a biofilm-like structure over the epithelial layer. Consequently, we expanded our analysis of ALS gene expression to include model denture and catheter biofilms. Our results show that expression of each ALS gene can be detected by RT-PCR and validate use of this method for analysis of ALS gene expression in disease models.
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METHODS |
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Inoculum preparation.
Methods for inoculum preparation largely followed those described by Schaller et al. (1998, 1999)
. A starter culture of C. albicans cells was inoculated from a single colony on a fresh YPD plate into 10 ml liquid YPD medium and incubated in a 37 °C water bath, with 200 r.p.m. orbital shaking. Approximately 24 h later, cells from the entire culture were collected by centrifugation, washed three times in PBS and counted. Four million cells were inoculated into 10 ml fresh YPD and incubated in a 37 °C water bath with 200 r.p.m. orbital shaking. At approximately 24 h, cells were collected, washed three times in PBS and counted in triplicate. An inoculum stock of 4x107 cells ml-1 was prepared for use in the RHE model (see below). Pelleted cells from the remainder of the washed inoculum culture were flash-frozen in a dry ice/ethanol bath and stored at -80 °C for subsequent RT-PCR analysis. Cells that had not been washed in PBS were also pelleted and flash-frozen for subsequent analysis.
RHE model.
Reconstituted human epithelium (RHE) is a product of SkinEthic Laboratories (Nice, France). The product consists of human epithelial cell lines cultured on polycarbonate filters in vitro at the airliquid interface in a serum-free chemically defined medium. The experiments here used oral RHE (derived from the TR146 cell line) or oesophageal RHE (from the Kyse 510 cell line) in maintenance medium without antimicrobials. The maintenance medium is based on the MCDB-153 of Clonetics and contains 5 µg insulin ml-1.
RHE was inoculated by pipetting 50 µl C. albicans/PBS suspension (2x106 cells total) onto the surface of the tissue. Samples were incubated at 37 °C with 5 % CO2 and saturated humidity. Maintenance medium was changed every 24 h. At specified time points, tissues were harvested and processed for microscopy as described below, or flash-frozen and stored at -80 °C for RT-PCR analysis. Samples were run in duplicate.
ALS family PCR primers.
PCR primers specific for each ALS gene (Table 1) were designed using Primer3 software (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Preference was given to primer pairs with a Tm between 58 and 60 °C and with products in the 150400 bp size range. To validate the specificity of each primer pair for its corresponding ALS gene, PCR products were amplified from SC5314 genomic DNA. PCR reactions contained 1 µM of each primer, 1 mM MgCl2, 1x Invitrogen Taq polymerase buffer, 0·75 units Taq polymerase (Invitrogen) and 200 ng genomic DNA. PCR reactions were denatured for 5 min at 94 °C and subjected to 40 cycles of 94 °C (30 s), 58 °C (30 s) and 72 °C (30 s). A final 7 min extension at 72 °C completed the reaction. PCR products were resolved on 2 % agarose/TAE gels or 8 % acrylamide/TBE gels and visualized by staining with ethidium bromide. PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega) and the DNA sequence determined (Elim Biopharmaceuticals). The derived DNA sequences for each gene-specific primer pair matched exactly with the predicted sequence and validated the specificity of the primers against the C. albicans genome. ALS primers were also validated using all eight cloned ALS genes as PCR templates. Each primer pair only amplified its specific gene and no cross-reactivity was observed, further demonstrating the specificity of primer pairs across the ALS gene family.
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The SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen) was used to synthesize cDNA according to the kit instructions. Random hexamers were used to prime the cDNA synthesis reaction. RNA concentration was measured spectrophotometrically and 2 µg added to the cDNA synthesis reaction. The 2 µg RNA represented nearly the entire sample recovered using this method. In a limited number of cases, less than 2 µg RNA was recovered. In these situations, the entire RNA sample was added to the cDNA synthesis reaction; this quantity was at least 1·5 µg. Samples with less than 2 µg RNA yielded results identical to replicate samples with the full 2 µg RNA (see below). One-tenth volume of the final cDNA product (2 µl per reaction) was added to PCR reactions specific for each gene in the analysed family. PCR tubes specific for amplification of individual ALS genes, containing all reaction components except template, were prepared in advance and frozen at -20 °C until used. Preparation of larger stocks of PCR tubes allowed quality control that was not possible on the level of an individual reaction. Randomly selected PCR tubes from each batch were run as positive and negative controls to ensure the validity of the results.
Specimen preparation and microscopy.
RHE specimens for light microscopy were removed from the maintenance medium and placed into 24-well plates containing 1 ml Karnovsky's fixative (2 % glutaraldehyde, 2·5 % paraformaldehyde). Karnovsky's fixative was also added to cover the top of the RHE specimen. Following 1 h incubation at room temperature, the samples were transferred to 4 °C overnight. Samples were embedded in epoxy using the rapid microwave fixation method optimized at the University of Illinois Center for Microscopic Imaging (http://treefrog.cvm.uiuc.edu/meth_stdMW.html). This method involved microwaving the samples in Karnovsky's solution for the primary fixation followed by washing in cacodylate buffer. Osmium tetroxide was used for the secondary fixation step. Samples were then dehydrated in a series of ethanol/acetonitrile. Embedding was carried out in increasing concentrations of epoxy, using the resin LX112. Blocks were sectioned and stained with toluidine blue. Specimens were examined using a Nikon Eclipse E600 microscope fitted with a Spot camera (Diagnostic Instruments). Images were collected using Metamorph software (Universal Imaging Corporation) and processed with Adobe Photoshop.
Model biofilm growth and RT-PCR analysis.
Model biofilms were grown on denture acrylic (Chandra et al., 2001) or silicone elastomer catheter material (Kuhn et al., 2002
) as described previously. Biofilms for RT-PCR analysis were flash-frozen in liquid nitrogen and stored at -80 °C until used. Biofilm RNA was prepared using a hot phenol extraction method (Collart & Oliviero, 1993
) and further purified using an RNeasy kit (Qiagen). DNase treatment and verification that genomic DNA was removed followed protocols listed above. RT-PCR analysis was conducted as described above with 2 µg total RNA added to the cDNA synthesis reaction and 0·1 vol. of the resulting product used for each ALS-specific PCR reaction. A negative control containing the equivalent amount of RNA added to the cDNA reaction was run in parallel to provide secondary assurance that the specimens lacked detectable genomic DNA. PCR products were resolved and visualized as described above.
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RESULTS |
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A more limited analysis was conducted with other C. albicans strains to determine whether there was strain variability in gene expression and whether differences due to the effect of RHE type could be detected (Fig. 4). These comparisons showed that gene expression patterns for other C. albicans strains were similar to those observed for SC5314 (Fig. 4a
). For example, ALS6 fell below the limit of detection of the assay more readily than the other genes when strain GDH2346 was inoculated onto buccal RHE (Fig. 4b
). Strain B311 failed to produce an ALS5-specific RT-PCR product (Fig. 4c
). In this case, however, the negative result was due to the fact that the ALS5 coding region is not present in this strain (Hoyer & Hecht, 2001
). The few samples of oesophageal RHE that were tested showed a trend similar to that observed for the buccal RHE. Fig. 4(d)
shows a typical result where ALS7 was not detectable when strain SC5314 was inoculated onto oesophageal RHE. In order to conclude that the two types of RHE produce identical ALS gene expression results, however, a more extensive analysis is required.
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Since C. albicans appears to form a biofilm on RHE, RT-PCR analysis of ALS gene expression was conducted on RNA isolated from model biofilm specimens (Table 3). For this analysis, semi-quantitative judgements were made about the relative abundance of RT-PCR product for each assay. These estimates were more justified for this analysis than for the RHE model because biofilm specimens only contained C. albicans RNA. A denture biofilm model (Chandra et al., 2001
) and a catheter biofilm model (Kuhn et al., 2002
) were analysed. Samples included matched sets of planktonic cells and biofilm specimens across a 48 h time-course.
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DISCUSSION |
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Development of the RT-PCR assay provides a tool for analysis of ALS gene expression that is more sensitive than previous detection studies using Northern blots of total RNA. This improved detection limit is inherent in the amplification-based nature of the RT-PCR method. In addition, positive signals can be derived by RT-PCR from a specific transcript that is not full-length. An improved limit of detection is required for gene expression analysis of specimens where C. albicans cell numbers are not as abundant as in liquid cultures. ALS gene expression patterns observed with the RT-PCR assay, in most cases, match those observed on Northern blots (Hoyer et al., 1995, 1998a
, b
). Previous Northern analysis suggested that ALS3 is expressed in the hyphal form of the organism, yet a positive signal was derived from the YPD-grown culture of yeast forms used to inoculate the RHE model. This result is probably attributable to a very low number of germ tubes in the culture. Previous Northern blot results for ALS3 expression in strain SC5314 showed detectable transcripts in cultures where the abundance of germ tubes was below 0·1 % (Hoyer et al., 1998b
). The improved limit of detection of the RT-PCR assay should be able to detect ALS3-specific signals from even fewer germ tube forms.
Perhaps the largest divergence between previous Northern results and the RT-PCR analysis is for detection of ALS2. In the RT-PCR assay, ALS2 appeared as one of the two strongest signals, whereas the transcript eluded previous attempts at detection on Northern blots (Hoyer et al., 1998b). Many factors may contribute to this disparity of results. Alleles of all ALS genes are known to vary in size mainly due to a central domain consisting entirely of a 108 bp tandemly repeated sequence (Hoyer, 2001
). Among the genes in the ALS family, ALS2 is one of the largest, with an average tandem-repeat domain containing over 30 copies of the 108 bp sequence (J. A. Nuessen & L. L. Hoyer, unpublished results). This number of repeat copies results in an average ALS2 allele size of nearly 6 kb. Transcripts of this size may be extracted less efficiently from the C. albicans cell. In Northern blot analysis of disparate-sized ALS alleles from the same C. albicans strain, a less intense signal is frequently observed for the larger allele (Hoyer et al., 1995
, 1998a
, b
). Transcripts of this large size may also be degraded more readily than those for smaller genes. Considering these variables, it is expected that RT-PCR would be more successful at detection of the ALS2 transcript than Northern blotting. Additional difficulties in detection of ALS2 on Northern blots arise because of its sequence similarity to other ALS genes, particularly ALS4. Specific detection of ALS2 transcripts requires an end-labelled oligonucleotide probe that results in a reduced Northern blot signal compared to what is observed when hybridizing with a larger DNA probe (Hoyer et al., 1998b
). Hybridization of Northern blots with a larger probe that recognizes both ALS2 and ALS4 improves the signal strength, but ALS2 and ALS4 alleles tend to migrate in the same size range and obscure each other on Northern blots. For example, in strain SC5314, the alleles of ALS4 are approximately 4 and 6 kb while both alleles of ALS2 are approximately 6 kb (Hoyer et al., 1998b
; X. Zhao & L. L. Hoyer, unpublished data). As other reagents are developed, the nature of the ALS2 expression will become clear. Recent construction of an ALS2-GFP reporter strain and demonstration of its obvious fluorescence when cultured under the same conditions utilized for the RT-PCR assays described here (C. B. Green & L. L. Hoyer, unpublished results) substantiates the RT-PCR results and suggests that these data accurately reflect ALS2 expression patterns.
The observation that ALS7 transcription more readily falls below the RT-PCR assay detection limit concurs with recently published observations by Zhang et al. (2003). In that study, 20 µg poly(A) RNA was required to detect ALS7 transcripts by Northern blot analysis. The authors estimated the abundance of ALS7 transcript to be approximately 12 % that of actin mRNA. Growth conditions used in their report are similar to those used for our RT-PCR analysis of RNA from cultured C. albicans cells. Currently, it is not known how much transcript is required to produce mature, active Als proteins. It is possible that the ALS7 transcript is more difficult to observe on Northern blots for reasons similar to the ALS2 size and stability arguments outlined above. However, it is also possible that only a low level of ALS7 transcript is required for production of sufficient quantities of Als7p. Understanding the relationship between ALS transcript abundance and the presence of functional protein on the C. albicans cell surface is a priority for future investigations. It is likely that these parameters vary for each gene in the ALS family.
Another motivation for our studies of ALS gene expression in disease models was to compare general themes for the ALS family with those from other C. albicans gene families, most notably the SAP (secreted aspartyl proteinase) genes. Many authors have described the use of RT-PCR to study SAP gene expression in various disease models and human clinical specimens (Schaller et al., 1998, 2003
; Naglik et al., 1999
, 2003
; Ripeau et al., 2002
; Schofield et al., 2003
). Meaningful comparisons between the various SAP analyses and our ALS analysis are complicated by the varying detection limits for each assay. The detection limit for any of the RT-PCR assays is due to choices made for the overall assay design as well as the relative efficiency of amplification from the specific pairs of primers. For example, incorporation of radionucleotides into the RT-PCR products serves to increase limit of detection (Naglik et al., 1999
; 2003
); limit of detection is also enhanced by Southern blotting of the RT-PCR products (Ripeau et al., 2002
). Design of primers to amplify smaller and similarly sized PCR products increases detection limit and consistency among the results for each gene (Naglik et al., 1999
; 2003
; Schofield et al., 2003
). The overall body of work on RT-PCR analysis of SAP gene expression suggests differential expression across the family. However, detection of expression from all SAP genes in specific samples is being reported with increased frequency as RT-PCR methodology becomes more refined (Naglik et al., 2003
; Schofield et al., 2003
). The use of quantitative PCR methods would provide more precise estimates of transcript abundance for various genes within the SAP family and for our ALS analysis. However, a quantitative method would still not address the central need to understand the relationship between transcript abundance and protein production, which may differ for each gene in a family. Northern blots are useful for detecting large increases and decreases in transcript abundance and these changes are likely to have biological meaning. However, it is more complicated to use Northern blots to interpret the profile of expression for a gene that may never be abundantly expressed. In theory, protein may be present whenever gene-specific transcript is detected. For the ALS genes, many levels of regulation are encountered during the production of a heavily glycosylated cell wall protein from an mRNA molecule. Additionally, the fate of the Als proteins once they are localized on the C. albicans cell surface is still unknown. Other mechanisms that regulate the profile of Als proteins on the cell surface may be operational in C. albicans. The data presented here highlight the importance of these questions in our ongoing efforts to characterize the ALS family and the utility of our RT-PCR assay in addressing these relationships.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Chandra, J., Mukherjee, P. K., Leidich, S. D., Faddoul, F. F., Hoyer, L. L., Douglas, L. J. & Ghannoum, M. A. (2001). Antifungal resistance of candidal biofilms formed on denture acrylic in vitro. J Dent Res 80, 903908.[Abstract]
Collart, M. A. & Oliviero, S. (1993). Preparation of yeast RNA. In Current Protocols in Molecular Biology, vol. 2, pp. 13.12.113.12.5. Edited by F. M. Ausubel. New York: Wiley.
De Bernardis, F., Sullivan, P. A. & Cassone, A. (2001). Aspartyl proteinases of Candida albicans and their role in pathogenicity. Med Mycol 39, 303313.[Medline]
Fu, Y., Ibrahim, A. S., Sheppard, D. C., Chen, Y. C., French, S. W., Cutler, J. E., Filler, S. G. & Edwards, J. E., Jr (2002). Candida albicans Als1p: an adhesin that is a downstream effector of the EFG1 filamentation pathway. Mol Microbiol 44, 6172.[CrossRef][Medline]
Gaur, N. K. & Klotz, S. A. (1997). Expression, cloning, and characterization of a Candida albicans gene, ALA1, that confers adherence properties upon Saccharomyces cerevisiae for extracellular matrix proteins. Infect Immun 65, 52895294.[Abstract]
Gillum, A. M., Tsay, E. Y. H. & Kirsch, D. R. (1984). Isolation of the Candida albicans genes for orotidine-5'-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations. Mol Gen Genet 198, 179182.[Medline]
Hoyer, L. L. (2001). The ALS gene family of Candida albicans. Trends Microbiol 9, 176180.[CrossRef][Medline]
Hoyer, L. L. & Hecht, J. E. (2001). The ALS5 gene of Candida albicans and analysis of the Als5p N-terminal domain. Yeast 18, 4960.[CrossRef][Medline]
Hoyer, L. L., Scherer, S., Shatzman, A. R. & Livi, G. P. (1995). Candida albicans ALS1: domains related to a Saccharomyces cerevisiae sexual agglutinin separated by a repeating motif. Mol Microbiol 15, 3954.[Medline]
Hoyer, L. L., Payne, T. L., Bell, M., Myers, A. M. & Scherer, S. (1998a). Candida albicans ALS3 and insights into the nature of the ALS gene family. Curr Genet 33, 451459.[CrossRef][Medline]
Hoyer, L. L., Payne, T. L. & Hecht, J. E. (1998b). Identification of Candida albicans ALS2 and ALS4 and localization of Als proteins to the fungal cell surface. J Bacteriol 180, 53345343.
Hube, B. & Naglik, J. (2001). Candida albicans proteinases: resolving the mystery of a gene family. Microbiology 147, 19972005.
Hube, B., Stehr, F., Bossenz, M., Mazur, A., Kretschmar, M. & Schafer, W. (2000). Secreted lipases of Candida albicans: cloning, characterisation and expression analysis of a new gene family with at least ten members. Arch Microbiol 174, 362374.[CrossRef][Medline]
Kuhn, D. M., Chandra, J., Mukherjee, P. K. & Ghannoum, M. A. (2002). Comparison of biofilms formed by Candida albicans and Candida parapsilosis on bioprosthetic surfaces. Infect Immun 70, 878888.
Monod, M. & Borg-von Zepelin, M. (2002). Secreted aspartic proteases as virulence factors of Candida species. Biol Chem 383, 10871093.[Medline]
Naglik, J. R., Newport, G., White, T. C., Fernandes-Naglik, L. L., Greenspan, J. S., Greenspan, D., Sweet, S., Challacombe, S. J. & Agabian, N. (1999). In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis. Infect Immun 67, 24822490.
Naglik, J. R., Rodgers, C. A., Shirlaw, P. J., Dobbie, J. L., Fernandes-Naglik, L. L., Greenspan, D., Agabian, N. & Challacombe, S. J. (2003). Differential expression of Candida albicans secreted aspartyl proteinase and phospholipase B genes in humans correlates with active oral and vaginal infections. J Infect Dis 188, 469479.[CrossRef][Medline]
Odds, F. C. (1988). Candida and Candidosis. A Review and Bibliography, 2nd edn. London: Baillière Tindall.
Ripeau, J.-S., Fiorillo, M., Aumont, F., Belhumeur, P. & de Repentigny, L. (2002). Evidence for differential expression of Candida albicans virulence genes during oral infection in intact and human immunodeficiency virus type 1-transgenic mice. J Infect Dis 185, 10941102.[CrossRef][Medline]
Schaller, M., Schafer, W., Korting, H. C. & Hube, B. (1998). Differential expression of secreted aspartyl proteinases in a model of human oral candidosis and in patient samples from the oral cavity. Mol Microbiol 29, 605615.[CrossRef][Medline]
Schaller, M., Korting, H. C., Schafer, W., Bastert, J., Chen, W.-C. & Hube, B. (1999). Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol Microbiol 34, 169180.[CrossRef][Medline]
Schaller, M., Bein, M., Korting, H. C., Baur, S., Hamm, G., Monod, M., Beinhauer, S. & Hube, B. (2003). The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an in vitro model of vaginal candidiasis based on reconstituted human vaginal epithelium. Infect Immun 71, 32273234.
Schofield, D. A., Westwater, C., Warner, T., Nicholas, P. J., Paulling, E. E. & Balish, E. (2003). Hydrolytic gene expression during oroesophageal and gastric candidiasis in immunocompetent and immunodeficient gnotobiotic mice. J Infect Dis 188, 591599.[CrossRef][Medline]
Zhang, N., Harrex, A. L., Holland, B. R., Fenton, L. E., Cannon, R. & Schmid, J. (2003). Sixty alleles of the ALS7 open reading frame in Candida albicans: ALS7 is a hypermutable contingency locus. Genome Res 13, 20052017.
Received 12 August 2003;
revised 23 October 2003;
accepted 27 October 2003.