Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802, USA
Correspondence
Lois L. Hoyer
lhoyer{at}uiuc.edu
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ABSTRACT |
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INTRODUCTION |
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Another approach to studying ALS gene expression in vitro involves the construction of reporter strains. This manuscript describes use of green fluorescent protein (GFP) as a reporter of ALS promoter activity and flow-cytometry measurement of the resulting fluorescent signal in cultured C. albicans cells. Fusions of GFP to inducible promoters in C. albicans showed rapid production of GFP under inducing conditions and quick protein decay under repressing conditions, and demonstrated its effectiveness as a reporter of gene regulation (Barelle et al., 2004). Gene expression patterns of the PALS-GFP strains described here are verified against those in wild-type C. albicans using a newly developed real-time RT-PCR assay for the ALS family. The PALS-GFP clones provide a dynamic analysis of ALS gene expression that is quick and inexpensive compared to RT-PCR methods. This method provides a quantitative evaluation of fluorescence for comparison of the strength of the various ALS promoters. Culture conditions examined include those used to study ALS gene expression in previous work. Data derived from these studies show that some ALS genes are regulated by large increases in transcription while others have more low-level and subtle regulatory changes. Data presented here also identify in vitro conditions that can be used for phenotypic analysis of als/als mutant strains.
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METHODS |
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Construction of C. albicans PALS-GFP reporter strains.
All experiments used yeast-enhanced GFP (Cormack et al., 1997). GFP reporter constructs targeted for integration into specific ALS loci were generated using the method of Gerami-Nejad et al. (2001)
. Plasmid pGFP-URA3 was a generous gift from Cheryl Gale (University of Minnesota, Minneapolis, MN, USA). Plasmid pGFP-URA3 contains a reporter cassette with the GFP gene and the URA3 selectable marker, and was used as the PCR template to generate integration cassettes. General guidelines were used to design each set of PCR amplification primers for the eight ALS genes (Table 1
). Forward primers included 57 nt of sequence upstream of the ALS gene, followed by an ATG and 20 nt of plasmid pGFP-URA3 sequence (5'-ATG TCT AAA GGT GAA GAA TTA TT-3'). Reverse primers included 57 nt downstream of the ALS gene and 23 nt of plasmid pGFP-URA3 sequence (5'-TCT AGA AGG ACC ACC TTT GAT TG-3'). Each cassette was designed so that it could be knocked into the correct ALS locus, thereby generating ALS heterozygotes where one ALS allele was replaced with the GFP-URA3 cassette, while maintaining the native promoter sequence. PCR products were amplified using Taq polymerase (Invitrogen) and transformed into C. albicans CAI4 (Fonzi & Irwin, 1993
) using previously described methods (Zhao et al., 2004
). CAI4 was a generous gift from William Fonzi (Georgetown University, Washington, DC, USA). Transformants were selected on synthetic complete medium without uridine (SCUri; Hicks & Herskowitz, 1976
) containing 1 M sorbitol.
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RT-PCR analysis of GFP expression in PALS-GFP strains.
The PALS-GFP reporter strains were monitored under in vitro conditions in order to correlate GFP mRNA expression of each reporter strain with published RT-PCR data for the ALS family (Green et al., 2004). RNA was isolated from 1 ml of 16 h YPD cultures using a hot phenol extraction method (Collart & Oliviero, 1993
). RT-PCR was performed as previously described (Green et al., 2004
), but using GFP-specific primers GFPRTF and GFPRTR (Table 1
).
Real-time RT-PCR analysis of ALS gene expression.
Real-time RT-PCR primers were designed using PRIMEREXPRESS software (version 2.0; Applied Biosystems) to have a Tm between 59 and 60 °C and an amplicon size of 50100 bp (Table 3). DNA sequencing of the PCR product amplified from C. albicans genomic DNA verified primer specificity. C. albicans CAI12 was grown as for flow cytometry analysis (see below) for 16 h in YPD or in RPMI for 30 min, both at 37 °C and 200 r.p.m. shaking. Total RNA was extracted (Collart & Oliviero, 1993
) and further purified using RNeasy (Qiagen). RNA was treated with DNase as described previously (Green et al., 2004
) and purified again with RNeasy. cDNA was synthesized using the SuperScript First Strand Synthesis System (Invitrogen) with 1 µg RNA as the template. Following synthesis, cDNA was diluted 1 : 5 with sterile MilliQ water. PCR reactions contained 100 nM of each primer, 1x Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen), 1x ROX reference dye (Invitrogen) and 5 µl diluted cDNA in a final volume of 25 µl. PCR reactions were run on the ABI Prism 7000 Sequence Detection System (SDS) with a 2 min 50 °C UDG incubation step, and 95 °C incubation for 2 min, followed by 40 cycles of 95 °C (15 s) and 60 °C (1 min). All primer pairs produced a single amplicon with a uniform melting curve as determined by the dissociation profile of the product. A standard curve was constructed for each primer set with 1 : 10, 1 : 25, 1 : 50, 1 : 100, 1 : 250 and 1 : 500 dilutions of cDNA. The slopes of the standard curves were within 10 % of 100 % efficiency. CT values were determined using the AUTOANALYSE features of the SDS software.
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Preparation of PALS-GFP strains for flow cytometry analysis.
Frozen stocks of C. albicans strains were streaked onto YPD agar plates and incubated at 37 °C for 24 h. A single colony of each strain was resuspended in 1 ml sterile YPD liquid and vortexed vigorously to resuspend the cells. Twenty microlitres of this suspension were used to inoculate 10 ml fresh YPD liquid or YNB+Glc medium. Cultures were incubated for 16 h at 37 °C with 200 r.p.m. shaking. An aliquot of each starter culture was observed microscopically to ensure that only yeast forms were present. Cells were collected by centrifugation, washed twice in sterile PBS (per litre: 10 g NaCl; 0·25 g KCl; 1·43 g Na2HPO4; pH 7·2) and counted in duplicate. One hundred millilitres of fresh growth medium was inoculated at a density of 1x106 cells ml1 and incubated at 37 °C with 200 r.p.m. shaking. Every hour, an aliquot of each culture was removed and the cells were collected by centrifugation, washed twice in PBS and analysed by flow cytometry. An additional aliquot of culture was fixed with glutaraldehyde (final concentration 1 %) and used for triplicate optical density readings. OD620 readings were taken in a microplate format using the iEMS Reader MF (Thermo Labsystems).
PALS-GFP reporter strains were also grown in RPMI medium and YPD with 10 % serum for flow cytometry analysis. Starter cultures were prepared in YPD medium as described above. C. albicans cells were harvested by centrifugation, washed three times in PBS and counted in triplicate. Cells were resuspended in 20 ml RPMI or YPD with 10 % serum at a concentration of 5x106 cells ml1 and incubated in a 37 °C water bath with 200 r.p.m. shaking. An aliquot of each culture was collected and analysed by flow cytometry every 15 min for 1 h. The percentage germ tube formation was determined for each replicate at 1 h. The reporter strains and CAI12 exhibited 9099 % germ tubes with length greater than or equal to the diameter of the mother yeast, suggesting that disruption of the small allele of each ALS gene did not result in altered filamentation in RPMI (data not shown).
Flow cytometry.
Flow cytometry was performed using a Beckman Coulter EPICS XL machine. This instrument is equipped with a 15 mW air-cooled argon laser with an excitation wavelength of 488 nm. For fluorescence analysis, a region was set on a histogram, which represented side-angle light scatter versus forward-angle light scatter for a population of CAI12 yeast-form cells. The fluorescence was then gated on this region. Ten thousand events were collected at the medium flow rate. Fluorescence was measured on the FL1 fluorescence channel equipped with a 525 nm emission bandpass filter. Geometric mean fluorescence values for each time point were calculated using WINLIST software (Verity).
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RESULTS |
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PALS-GFP reporter constructs were also verified by RT-PCR analysis. Previous work showed that expression of all eight ALS genes could be detected by RT-PCR using ALS-specific primers and RNA extracted from cells grown overnight at 37 °C in YPD medium (Green et al., 2004). RT-PCR analysis was conducted using GFP-specific primers GFPRTF and GFPRTR (Table 1
) and RNA extracted from a YPD culture of each PALS-GFP reporter strain grown overnight at 37 °C. Strain CAI12, which does not encode GFP sequences, was included as a negative control. Amplification of RNA without reverse transcription was used as a further negative control to ensure that the signals obtained resulted from the cDNA rather than from contaminating genomic DNA. All DNase-treated RNA preparations produced negative results (data not shown). RT-PCR products of the predicted size for the GFP primers indicated that GFP was transcribed from each ALS locus in a manner similar to that observed in the wild-type parent strain (Fig. 1
; Green et al., 2004
). Real-time RT-PCR was also used to verify that GFP production in the reporter strains matched ALS gene expression in CAI12; these results are presented with the flow cytometry data below.
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The PALS-GFP reporter strains grown in YNB+Glc (Table 5) showed generally similar GMF readings to those from YPD-grown cells. However, some notable exceptions were observed. For example, in YNB, ALS1 transcription did not reach the levels observed in YPD. The PALS7-GFP strain did not show the transient increase in GMF values around 3 h, but instead showed a slight steady increase as the culture aged. The PALS9-GFP strain had GMF readings that were more elevated relative to the control compared to the values observed when the strain was grown in YPD. Overall, the control and reporter strains grew more slowly and to a lower density in YNB than in YPD.
Flow cytometry analysis of C. albicans PALS-GFP germ tubes
The PALS-GFP reporter strains were assayed for fluorescence changes during germ tube formation in RPMI at 37 °C (Table 7) and in YPD with 10 % serum (Table 8
). Germ tube growth was faster in the serum-containing medium, so instead of measuring fluorescence every 15 min for 1 h as for RPMI-grown cells (Table 7
), measurements were taken every 10 min for 0·5 h (Table 8
). In both media, PALS1-GFP was the earliest and most strongly up-regulated gene, followed both temporally and in intensity by PALS3-GFP. PALS3-GFP expression increased dramatically when germ tubes became visible by microscopic observation. GMF readings for the PALS5-GFP, PALS6-GFP, PALS7-GFP and PALS9-GFP strains were similar to those for the CAI12 negative control, suggesting at most, low levels of promoter activity under the growth conditions tested. Real-time RT-PCR measurements of transcript copy number supported the flow cytometry results. Transcript copy numbers were 500 000±36 000 for ALS1, 490 000±180 000 for ALS3, 110±32 for ALS5, 5·6±0·8 for ALS6, 6·0±2·8 for ALS7 and 490±120 for ALS9, compared to 3 600 000±530 000 for the TEF1 housekeeping gene. Transcript copy numbers were also measured for ALS2 (180 000±9900) and ALS4 (1500±590). Kinetics of germ tube formation differed between the RPMI-grown PALS-GFP cells in the flow cytometry experiment and those of strain CAI12 used for real-time RT-PCR analysis. Microscopic evaluation of germ tube length for both culture conditions showed that germ tubes on the cells from the real-time analysis were longer than those from the flow cytometry analysis that had been grown for the same amount of time. The PALS-GFP reporter strains were matched to CAI12 for growth rate and did not show differences in germ tube formation when incubated in the same experiment. The only difference between the experiments was the size of the culture flask used (50 ml for reporter strains in flow cytometry vs 500 ml for CAI12 for real-time analysis), and this may have contributed to the quicker germ tube formation for the wild-type strain. These growth rate differences may have also accounted for the different patterns of relative abundance of ALS1 and ALS3 transcripts from the flow cytometry data (where the values appeared different) and the real-time analysis (where the values were the same). Regardless of these differences between the flow cytometry and real-time RT-PCR analyses, the main conclusions from the two methods supported each other and indicated that some ALS genes were transcribed at high levels while others were relatively quiet.
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DISCUSSION |
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The PALS-GFP reporter strains were used to follow ALS gene expression over time in two cultures where yeast forms were grown and two cultures that yielded germ tubes. The overall conclusion from these analyses was that the ALS family has genes that are regulated by large increases and decreases in transcriptional activity and also those that show a more consistent low-level activity. Although ALS1 expression has been discussed in previous publications (Hoyer et al., 1995; Fu et al., 2002
; Zhao et al., 2004
), this report is believed to be the first to follow ALS1 expression over a growth curve. Results for all media tested showed that ALS1 expression increases dramatically when cells are placed into fresh medium, whether the conditions promote growth as yeast or hyphal forms. These observations suggest that Als1p functions in response to new environments or perhaps in the release of cells from stationary phase.
Studies here also corroborated the hypha-specific regulation of ALS3. This relationship between the morphological form and ALS3 transcription was demonstrated originally by Northern blotting (Hoyer et al., 1998a). ALS3 transcription increases when germ tubes are visible microscopically, in contrast to transcription of ALS1, which occurs almost immediately after C. albicans cells are placed into the hyphal-induction media tested (Zhao et al., 2004
). This pattern of regulation suggests that Als1p is localized at the initial point where the germ tube emerges from the mother yeast, a localization that has been demonstrated by immunostaining methods (Fu et al., 2002
). Localization of Als3p awaits production of a specific antiserum or utilization of an alternative method, but from the data here, it is likely that Als3p will be localized farther down the germ tube length than Als1p. Direct comparisons between als1
/als1
and als3
/als3
strains in adhesion to vascular endothelial and buccal epithelial cells showed the dramatic contribution to C. albicans adhesion by Als3p (Zhao et al., 2004
). Hypha-specific production of Als3p helps to explain the increased adhesive capacity of germ tubes relative to yeast forms (Calderone & Braun, 1991
). The GFP strains described here could be used to study ALS gene expression during prolonged filamentation, although an alternative technique such as fluorescence microscopy would have to be used since longer filamentous forms are not amenable to flow cytometry analysis.
Construction of the PALS2-GFP strain was not completed, consistent with previous observations that integration of constructs at the ALS2 locus is problematic (Zhao et al., 2005). However, the real-time RT-PCR data showed that ALS2 transcription is stronger than transcription of the other ALS genes at 16 h in YPD-grown cells and that ALS2 transcription is strongly up-regulated during germ tube formation in both RPMI and serum-containing media. The YPD result from this analysis matches previous observations that the ALS2 message is one of the last to disappear from RT-PCR analysis of a dilution series of cDNA made from an overnight culture of YPD-grown C. albicans cells (Green et al., 2004
). In that previous work, the ALS1 transcript appeared equally as strong as that from ALS2. The disparity between ALS1 results from the two studies can be explained by noting that ALS1 transcription increases when YPD-grown cells are washed in PBS (C. B. Green & L. L. Hoyer, unpublished observation). The PBS washing step was included in the previous analysis (Green et al., 2004
), but not in the analysis described in this paper. Transcript copy number also increased for ALS4 during germ tube formation in RPMI, although not to the same magnitude as for ALS2 transcription.
In comparison with the strong transcriptional responses described above, those from other genes were relatively mild, with ALS6 and ALS7 showing the lowest transcriptional activity within the family. This result matches previous studies where ALS6 and ALS7 expression most frequently fell below the detection limit for our standard RT-PCR assay (Green et al., 2004). The low level of transcription for ALS6 and ALS7, and also for ALS5, limits the value of the PALS-GFP reporter strains and flow cytometry for assessment of transcriptional activity. In some cases, the transcript copy numbers are so low that it is questionable whether these genes are really active at all under the conditions tested. Work by Zhang et al. (2003)
showed ALS7 message on a Northern blot of 20 µg poly(A) RNA, consistent with the conclusion of low-level transcriptional activity in cultures similar to our YPD growth conditions. In data presented here, GFP transcript could be amplified by RT-PCR from all of the PALS-GFP reporter strains, consistent with the conclusion of activity from each of the ALS promoters. Despite the low level of transcript, the signal from the ALS7 promoter increased in a statistically significant manner following 2 h growth in fresh YPD medium. These growth conditions can be exploited to learn more about Als7p function by microarray analysis of als7/als7 mutant strains. Expression data for the other ALS genes will be used similarly.
Despite the technical differences between the various approaches to studying ALS gene expression, the results from the methods are very similar. If the level of transcription is positively associated with protein production, the data would suggest that much less Als5p, Als6p and Als7p will be present on the C. albicans cell surface than Als1p, Als2p or Als3p. Reagents and methods that can discriminate between the various Als proteins are required to test this hypothesis. Insight into the levels of the various Als proteins that are likely to be present on the C. albicans cell surface also provides guidance for the selection of methods to study Als protein function. For example, using overexpression methods to study an Als protein that is present in small quantities on the C. albicans cell might lead to experimental artefacts.
Comparison between data for the various ALS genes also highlights the tight control of ALS expression in C. albicans. For example, in the SC5314 background, three ALS genes occupy an approximately 20 kb contiguous region of chromosome 6 where ALS1 is localized between ALS5 and ALS9 (Zhao et al., 2003). Considering the transcriptional abundance from the ALS1 locus, minimal expression of ALS5 and ALS9 illustrate the fine regulation of ALS gene expression. Functional comparisons can also be inferred from gene expression data. One example is to consider ALS1, ALS3 and ALS5, which are more than 85 % identical within the 5' domain of each coding region. By comparison with data from studies of Saccharomyces cerevisiae
-agglutinin (Wojciechowicz et al., 1993
), this region of the ALS gene has been proposed to encode the Als adhesive functional domain (Hoyer, 2001
). Gene expression data suggest that high levels of Als1p are present on newly budding yeast forms and newly forming germ tubes, high levels of Als3p are deposited on the surface of elongating germ tubes, and scant amounts of Als5p potentially are present on either growth form. These data suggest that there is a need for large amounts of Als1p as cells begin to divide, large amounts of Als3p as germ tubes lengthen, and only small amounts of Als5p throughout growth under the conditions tested. These functional clues will be combined with information from other approaches to further define the roles of each Als protein and functional interrelationships within the Als family.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 October 2004;
revised 8 December 2004;
accepted 26 December 2004.
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