Previous Article | Next Article 
Journal of Bacteriology, June 2001, p. 3761-3769, Vol. 183, No. 12
Zentrum für Infektionsforschung,
Universität Würzburg, D-97070 Würzburg, Germany
Received 22 January 2001/Accepted 26 March 2001
The opportunistic fungal pathogen Candida albicans
can switch spontaneously and reversibly between different cell forms, a capacity that may enhance adaptation to different host niches and
evasion of host defense mechanisms. Phenotypic switching has been
studied intensively for the white-opaque switching system of strain
WO-1. To facilitate the molecular analysis of phenotypic switching, we
have constructed homozygous ura3 mutants from strain WO-1 by targeted gene deletion. The two URA3 alleles
were sequentially inactivated using the
MPAR-flipping strategy, which is based
on the selection of integrative transformants carrying a mycophenolic
acid (MPA) resistance marker that is subsequently deleted again by
site-specific, FLP-mediated recombination. To investigate a
possible cell type-independent switching in the expression of
individual phase-specific genes, two different reporter genes that
allowed the analysis of gene expression at the single-cell level were
integrated into the genome, using URA3 as a selection
marker. Fluorescence microscopic analysis of cells in which a
GFP reporter gene was placed under the control of
phase-specific promoters demonstrated that the opaque-phase-specific SAP1 gene was detectably expressed only in opaque cells
and that the white-phase-specific WH11 gene was
detectably expressed only in white cells. When
MPAR was used as a reporter gene, it
conferred an MPA-resistant phenotype on opaque but not white cells in
strains expressing it from the SAP1 promoter, which was
monitored at the level of single cells by a significantly enlarged size
of the corresponding colonies on MPA-containing indicator plates.
Similarly, white but not opaque cells became MPA resistant when
MPAR was placed under the control of
the WH11 promoter. The analysis of these reporter
strains showed that cell type-independent phase variation in the
expression of the SAP1 and WH11 genes did
not occur at a detectable frequency. The expression of these
phase-specific genes of C. albicans in vitro, therefore,
is tightly linked to the cell type.
Pathogenic microorganisms
must be able to adapt to changing environmental conditions during the
course of an infection to ensure survival and growth in different host
niches. This adaptation is achieved by the regulated expression of
appropriate sets of genes whose products are needed by the pathogen at
a particular stage of an infection, in response to corresponding
environmental signals (23). In addition, many prokaryotic
and eukaryotic pathogens have the capacity to generate variants with
altered properties in a reversible and often random fashion, a
phenomenon known as phase or antigenic variation (10).
Such variability not only provides an opportunity to produce progeny
that are better adapted to a special host niche, but it also enables
evasion of the host's immune response to surface-exposed or secreted
antigens produced by the initially infecting organisms. Certain traits
can be both advantageous and disadvantageous for the microorganism,
depending on the host niche or infection stage. The capacity to alter
their expression state independently of environmental signals may
enhance pathogens' ability to cause disease (6, 12, 21, 31,
45).
Antigenic variation can be achieved by different molecular mechanisms,
including inversion of promoter elements or structural genes by
site-specific recombination (1, 20), differential methylation of regulatory sequences (7), insertion and
deletion of single nucleotides, oligonucleotide repeats, or mobile
genetic elements into either promoter or coding regions (12, 44,
46, 50), or movement of silent genes into expression loci by
homologous recombination (11, 13, 22). These specific
mechanisms ensure that variants are generated at a much higher
frequency than would be possible by random mutation alone but still
result in a semistable phenotype to allow the expansion of new variants
that eventually take over the population, especially after passing
through infection bottlenecks (29).
The opportunistic fungal pathogen Candida albicans displays
a remarkable capacity to alter its cellular characteristics. Apart from
a transition between growth in yeast and hyphal forms, generally referred to as dimorphism, C. albicans can reversibly switch
between different cell types that are distinguishable by the appearance of the resulting colonies on agar plates (35). In contrast
to the above-mentioned bacterial and protozoal examples of antigenic variation, where usually a single trait is affected, phenotypic switching in C. albicans alters many different cellular
properties, including morphology, adhesion, antigenicity, and
pathogenicity, in a programmed, coordinated fashion. Most C. albicans strains are capable of phenotypic switching, but the
process has been most intensely studied using the white-opaque
switching of strain WO-1 as a model system. This strain predominantly
switches between the typical budding yeast form, which produces
hemispherical, white colonies, and an elongated cell type, which forms
flat, opaque colonies (34). A growing list of
phase-specifically expressed genes has been discovered, including the
white-phase-specific genes WH11 and EFG1 and the
opaque-phase-specific genes SAP1 (PEP1), SAP3, OP4, and CDR3 (2, 25, 26,
37, 38, 47). The transition between the white and opaque phases
involves the coordinated activation of opaque-phase-specific genes and
deactivation of white-phase-specific genes and vice versa, which has
been postulated to be governed by a master regulatory switch, the
nature of which is still unknown. Switching among a set of preselected
cell types may allow a more efficient adaptation to new host niches
than randomly altering individual cellular properties
(36).
The specific role of many phase-specific genes has not yet been
elucidated, but some of them may have a function in the maintenance of
the distinct cellular phenotype. The WH11 gene encodes a
protein with homology to the heat shock protein Hsp12 and is localized in the cytoplasm of white cells (33). The SAP
genes encode secreted aspartic proteinases that have been shown to
contribute to the virulence of WO-1 and other C. albicans
strains (14, 16). However, Sap antigens are also
recognized by the host's immune system (19). Sap antigens
have been detected on the surfaces of C. albicans organisms
(4, 5, 32) and may thus allow the host to recognize and
destroy the invading cells. On the other hand, forced expression of the
white-phase-specific gene WH11 in opaque cells resulted in
enhanced virulence in a mouse model of systemic candidiasis
(17), whereas forced expression of the opaque-phase-specific gene SAP1 in white cells increased the
virulence of these cells in a model of cutaneous infection
(16). It is therefore conceivable that phase variation of
single genes might also be advantageous under conditions that otherwise
favor the maintenance of a specific cell type and could thus occur
independently from switching between the white and opaque phases by a
different molecular mechanism. Such an additional level of variability
would further enhance the flexibility of an infecting C. albicans strain.
To date, the expression of phase-specific genes has been analyzed only
at the population level, either by Northern hybridization or by using a
luciferase reporter enzyme (25, 26, 38, 40). A rare
switching in individual cells from the expressed to a nonexpressed state of an otherwise phase-specific gene would therefore not have been
detected. Similarly, activation of such a gene in the "wrong" cell
type also would probably not be detected, since this could be explained
by the presence of a minor proportion of the other cell type in
populations of white or opaque cells.
Recently, the GFP gene, encoding the green fluorescent
protein (GFP) from Aequorea victoria, has been adapted to
the noncanonical codon usage of C. albicans, allowing
researchers to monitor the expression of target genes at the level of
single cells (8, 27). In addition, we have developed a
method for the genetic manipulation of C. albicans wild-type
strains (48). Sequential gene disruption can now also be
performed in uridine-prototrophic strains like WO-1 by using a marker
conferring resistance against mycophenolic acid (MPA) for the selection
of transformants and its subsequent deletion by FLP-mediated,
site-specific recombination (MPAR
flipping). In the present study, we have used these new molecular tools
to analyze the expression of phase-specific genes in strain WO-1 at the
single-cell level.
Strains and growth media.
The C. albicans strains
used in this study are listed in Table 1.
Strains were subcultured separately in the white and opaque phases at
room temperature on agar plates containing Lee's medium, pH 6.8 (3), and 5 µg of phloxine B ml
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3761-3769.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Analysis of Phase-Specific Gene Expression at the
Single-Cell Level in the White-Opaque Switching System of
Candida albicans
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1,
which selectively stains opaque colonies pink (35). For
routine growth of the strains, YPD liquid medium (10 g of yeast
extract, 20 g of peptone, and 20 g of glucose per liter) was
used. Cells were grown overnight in YCB-BSA (23.4 g of yeast carbon
base, 4 g of bovine serum albumin per liter [pH 4.0]) to induce
the SAP2 promoter for excision of the
MPAR flipper from MPA-resistant
transformants. Screening for MPA-sensitive derivatives was performed
after growth on minimal agar plates (6.7 g of yeast nitrogen base
without amino acids [Bio 101, Vista, Calif.], 20 g of glucose,
0.77 g of complete supplement medium [Bio 101], and 15 g of
agar per liter) containing 1 µg of MPA ml1
(48). Uridine (100 µg ml1) was
added to the media to support growth of ura3 mutant strains.
TABLE 1.
C. albicans strains used in this study
Plasmid constructions.
For inactivation of the
URA3 gene, two different plasmid constructions were made.
First, a DNA fragment comprising the URA3 coding region plus
420 bp of the upstream and 126 bp of the downstream region that was
originally cloned as a SalI-PstI fragment into pBluescript (27) was removed by
SalI-BamHI digestion and cloned into pUC18 to
yield pURA7. An internal fragment from positions 22 to 228 of the
URA3 coding region was subsequently deleted by inverse PCR
with the primers URA9
(5'-GCTCTCTCACCGCGGTCTTAGTGTTGAC-3') and URA8
(5'-CCTATGAATCCACTCGAGAACCATTATTAG-3'), thereby
introducing SacII and XhoI sites (underlined)
into which a SacII-XhoI fragment containing the
MPAR flipper from pSFI1 (48)
was inserted instead of the deleted URA3 sequences,
resulting in pSFIU2 (Fig. 1A). To delete
the whole URA3 gene, larger flanking regions were obtained
by screening a C. albicans fosmid library (kindly provided
by Stewart Scherer, Acacia Biosciences, Richmond, Calif.) using the
insert from pURA7 as a probe. A 4.7-kb XhoI-SacI
fragment containing the URA3 gene from a positive clone
(16H6) was ligated into pBluescript. Sequence analysis of the resulting
plasmid, pURA300, allowed us to design primers for the amplification of
URA3-flanking regions. The upstream region from positions
2462 to 394 was obtained by PCR with the primers URA15
(5'-TTTGTGCATGCTGTATTTCCAAAACG-3') and URA16
(5'-TGTTTCCGCGGATACCATCCAAATCAATTCC-3') containing introduced SphI and SacII sites
(underlined), respectively. The downstream region ranging from
positions 114 to 1102 behind the stop codon was amplified with the
primers URA17
(5'-CTATTTACAATCTCGAGGTGGTCCTTC-3') and URA18
(5'-CCATTAATTGCGAGCTCTGCTACTGGA-3') containing
introduced XhoI and SacI sites (underlined),
respectively. The SacII-XhoI MPAR flipper fragment from pSFI1 was then
ligated together with the SphI-SacII
URA3 upstream and the XhoI-SacI
URA3 downstream fragments into the
SphI/SacI-digested pUC18, resulting in pSFIU4
(Fig. 1A). The SphI-SacI URA3
deletion/disruption fragments from pSFIU4 and pSFIU2 were then
used to replace the wild-type URA3 alleles in C. albicans WO-1.
|
|
|
C. albicans transformation.
C.
albicans strains were transformed by electroporation
(15) with gel-purified linear DNA fragments from the
plasmids described above. MPA-resistant transformants were selected on
minimal agar plates containing 10 µg of MPA
ml1. Single colonies were picked after 5 to 7 days of growth at 30°C and restreaked on the same medium.
Uridine-prototrophic transformants of ura3 mutants were
selected on minimal agar plates without uridine. After confirmation of
the correct allelic replacements, white and opaque colonies of the
strains were identified by plating on agar plates containing Lee's
medium and 5 µg of phloxine B ml1 and then
maintained separately on the same plates at room temperature.
Isolation of chromosomal DNA and Southern hybridization. Genomic DNA from C. albicans strains was isolated as described by Millon et al. (24). DNA (10 µg) was digested with appropriate restriction enzymes, separated on a 1% (wt/vol) agarose gel, and, after ethidium bromide staining, transferred by vacuum blotting onto a nylon membrane and fixed by UV cross-linking. Southern hybridization with enhanced chemiluminescence (ECL)-labeled probes was performed with the ECL labeling and detection kit from Amersham (Braunschweig, Germany) according to the instructions of the manufacturer.
Fluorescence microscopy. White and opaque cells of the strains were grown separately or as mixed populations overnight at 25°C in liquid Lee's medium, pH 6.8, and aliquots of the cultures were spotted on microscope slides. Fluorescence was detected using a Zeiss Axiolab microscope equipped for epifluorescence microscopy with a 50-W mercury high-pressure bulb and with the Zeiss fluorescein-specific filter set at 09.
Screening for MPA-resistant and MPA-sensitive white and opaque
colonies.
The MPA resistance conferred by the expression of the
PSAP1-MPAR and
PWH11-MPAR reporter
gene fusions was initially assessed by suspending cells from white or
opaque colonies of strain WO-1 and the strains carrying these fusions
in water and plating appropriate dilutions on agar plates (7.5-cm
diameter) containing Lee's medium supplemented with 5 µg of phloxine
B ml1 and 7.5 µg of MPA
ml1 at a density of about 50 cells per plate.
The plates were incubated at 25°C for 5 days or at room temperature
for 7 days, after which the colony size was scored. To screen large
numbers of cells of the reporter strains, suspended cells from
different colonies were treated in the same way but using larger plates
(14-cm diameter) and a density of approximately 300 cells per plate.
All colonies whose diameters differed by at least 20% from the average
were isolated, and the cells were reexamined for
MPAR expression in the same way.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Construction of ura3 mutants of strain WO-1 by targeted gene deletion. To facilitate the genetic manipulation of strain WO-1, we constructed a ura3 mutant of this strain by targeted gene deletion using the MPAR-flipping strategy (48). A fragment comprising about 0.4 kb of upstream and 0.1 kb of downstream sequences in addition to the coding region of the URA3 gene is sufficient for selection of prototrophic transformants of C. albicans ura3 mutants. In plasmid pSFIU4 (Fig. 1A) almost all URA3 sequences present in the selection marker were replaced by the MPAR flipper cassette. Strain WO-1 was transformed with the insert from this plasmid, and MPA-resistant transformants were analyzed by Southern hybridization. The two URA3 alleles in WO-1 can be distinguished by an EcoRV restriction site polymorphism downstream of the coding region (Fig. 1A) and are located on 4.9- and 2.4-kb EcoRV fragments (Fig. 1B, lane 1). Two independent transformants in which either URA3-1 (WUM1A) (Fig. 1B, lane 2) or URA3-2 (WUM1B) (lane 3) had been replaced by the deletion cassette were selected, and the MPAR flipper was subsequently excised by induced, FLP-mediated recombination as described previously (48). All 10 tested MPA-sensitive derivatives of the two parent strains exhibited the hybridization pattern expected after specific deletion of the MPAR flipper cassette. The presence of a new 3.7-kb EcoRV fragment in both WUM2A (Fig. 1B, lane 4) and WUM2B (lane 5) demonstrates that, during integration into the URA3-2 allele, the polymorphic EcoRV site had been eliminated by the homologous cloned URA3 downstream sequence that did not contain this site. The insert from pSFIU4 was then used again to delete the remaining URA3 wild-type allele in strains WUM2A and WUM2B. MPA-resistant transformants were selected and screened for uridine auxotrophy. Southern hybridization analysis demonstrated the correct replacement of the second URA3 allele in the resulting strains, WUM3A (Fig. 1B, lane 6) and WUM3B (lane 7). Excision of the MPAR flipper from these strains resulted in strains WUM5A (Fig. 1B, lane 8) and WUM5B (lane 9). The presence of a 1.2-kb hybridizing EcoRV fragment in strain WUM5A demonstrates that, in this case, the polymorphic EcoRV site had been maintained after integration of the cassette into the URA3-2 allele.
In addition to the desired intrachromosomal recombination between FRT sites flanking the MPAR flipper, deletion of the cassette after the second round of excision could also have occurred by mitotic interchromosomal recombination between the centromere-proximal FRT site of the MPAR flipper and the FRT site present on the homologous chromosome. Such an event is not distinguishable from intrachromosomal recombination when the same replacement construct is used for deletion of both alleles of a target gene. Although interchromosomal recombination was never observed in previous experiments in which we used the flipper strategy for sequential disruption of different genes in various C. albicans strains (28, 48), the possibility remained that this might occur more frequently at the URA3 locus and, in that case, would lead to mutants that had become homozygous for all chromosomal regions located centromere distal to the site of crossover. To test for this possibility, we generated a second disruption construct in which only a part of the URA3 coding region was replaced by the MPAR flipper, such that different flanking regions were present on both sides of the cassette compared with the first deletion cassette (Fig. 1A). The insert from plasmid pSFIU2 was then used for a new transformation of the heterozygous strain WUM2A. A resulting transformant, strain WUM4A (Fig. 1B, lane 10), in which the remaining URA3 wild-type allele had been correctly replaced by the disruption cassette was used to excise the MPAR flipper after induction of the FLP gene. All seven tested MPA-sensitive derivatives exhibited the 2.3-kb fragment expected after specific excision of the MPAR flipper, demonstrating that interchromosomal recombination between FRT sites located on homologous chromosomes did not occur at an appreciable frequency also at the URA3 locus. One of these derivatives in which the two URA3 alleles had been inactivated with different deletion/disruption constructs (strain WUM6A) (Fig. 1B, lane 11) was kept for further analysis. For clarity, the different inactivated URA3 alleles were designated ura3
::FRT (obtained with pSFIU4) and
ura3::FRT (obtained with pSFIU2). All integration
and excision events were also confirmed with a URA3
downstream fragment (Fig. 1A) as an additional probe for Southern
analysis of the strains, yielding the expected hybridization patterns
(data not shown).
Phenotypic analysis of the ura3 mutants. The three independently constructed ura3 mutants of strain WO-1, strains WUM5A, WUM5B, and WUM6A, were then tested for possible differences in their growth characteristics. All three ura3 mutants exhibited identical growth in uridine-supplemented YPD or minimal medium. In addition, reintroduction of the URA3 gene into strain WUM5A (see below) restored growth in media without uridine to the same level as that of the parent strain WO-1.
All three ura3 mutants displayed the white-opaque switching typical of strain WO-1, as monitored at the colony level on agar plates containing Lee's medium supplemented with phloxine B and by the morphology of individual cells observed under the microscope (see also below).Integration specificity in WO-1 ura3 mutants using the URA3 gene as a selection marker. The homozygous ura3 mutants WUM5A and WUM6A were both derived from the heterozygous mutant WUM2A but differ from each other in that virtually all URA3 sequences contained in the URA3 selection marker were removed from the genome of WUM5A whereas a considerable portion of these sequences was still present in one of the disrupted URA3 alleles of WUM6A. The presence of these sequences might interfere with specific integration at other genomic loci when the URA3 marker is used for selection of transformants. To test for the specificity of integration, the two strains were transformed with a DNA fragment containing a fusion of the SAP1 promoter and a C. albicans-adapted GFP gene. The cassette contained the URA3 selection marker and was flanked on both sides by SAP1 sequences, such that specific integration into one of the SAP1 alleles should occur by a double-crossover event, resulting in allelic replacement (Fig. 2A).
The two SAP1 alleles in strain WO-1 and its ura3 derivatives can be distinguished by a BglII restriction site polymorphism in the SAP1 upstream region (Fig. 2A) and are located on 7.0- and 6.6-kb BglII fragments (Fig. 2B, lane 1). Of seven prototrophic transformants of strain WUM5A, three had integrated the PSAP1-GFP fusion into the SAP1-1 allele, as exemplified by strain WUGS1A (Fig. 2B, lane 2), and two had integrated the reporter gene fusion in the SAP1-2 allele, as in strain WUGS1B (lane 3). The two other transformants had undergone additional recombination events, but the disrupted URA3 loci remained unchanged in all seven transformants (not shown). Of 12 tested prototrophic transformants of strain WUM6A, four contained the fusion in the SAP1-1 allele and six had it in the SAP1-2 allele. In the two remaining transformants, the URA3 allele disrupted in the second mutagenesis round was reconstituted. These results demonstrate that the presence of sequences homologous to the URA3 selection marker can indeed promote integration of the marker into its original locus, but overall, the specificity of targeted integration at a heterologous site was high and comparable between the two strains.Analysis of phase-specific gene expression at the single-cell level. In white and opaque cells of strain WO-1, the expression of certain genes is coupled to the cell type, the WH11 gene being white phase specific and other genes, like SAP1, being detectably expressed only in opaque cell cultures. However, phase variation of single genes might be advantageous also under conditions that otherwise favor the maintenance of a specific cell type and could thus occur independently from switching between the white and opaque phases. If such a gene-specific phase variation happened at relatively low frequency, as observed in other pathogenic microorganisms, it might not be detected with the methods used up to now to analyze phenotypic switching in C. albicans. GFP can be used as a reporter of gene expression at the level of single cells. We therefore analyzed the expression of the opaque-phase-specific SAP1 gene in single cells using strains carrying a transcriptional fusion of the SAP1 promoter with the GFP gene integrated into either of the two SAP1 alleles (see above). In addition, the GFP gene was also placed under the control of the white-phase-specific gene WH11 and introduced into the genome of strain WUM5A (Fig. 3A). From six uridine-prototrophic transformants, five had correctly integrated the reporter gene fusion into one of the WH11 alleles, as demonstrated by the appearance of an expected 4.0-kb EcoRI fragment in addition to the 3.7-kb fragment representing the wild-type WH11 gene after Southern hybridization with a WH11-specific probe. Two independent transformants (strains WUGW11A and WUGW11B) (Fig. 2B, lanes 2 and 3) were selected for further study.
Analysis of the reporter strains by epifluorescence microscopy demonstrated that GFP was a useful reporter for qualitative analysis of phase-specific gene expression at the single-cell level. White and opaque cells of strain WO-1 can be distinguished by their morphologies, white cells being round to oval (length <1.5 times the width) and opaque cells being significantly elongated (length >2 times the width). Opaque but not white cells of strains WUGS1A and WUGS1B expressing GFP under the control of the SAP1 promoter fluoresced, and vice versa: fluorescence was observed in white but not opaque cells of strains WUGW11A and WUGW11B expressing GFP under the control of the WH11 promoter (Fig. 4). Activation of the WH11 gene in an opaque cell should therefore be detectable as a fluorescent elongated cell in opaque cell populations of strains WUGW11A and -B, whereas activation of the SAP1 gene in a white cell would be detected as a fluorescent round cell in white cell populations of strains WUGS1A and -B. Upon screening such populations of the reporter strains by epifluorescence microscopy (>104 cells of each strain), we could not detect expression of SAP1 or WH11 in the wrong cell type. It should be noted that the elongate morphology of opaque cells may sometimes not be distinct when the cells are viewed from one of the poles. However, the few fluorescent cells detected in opaque cell populations of strains WUGWH11A and -B had the appearance of white cells, with none of them being elongated, and very likely represented cells that had undergone switching. Similarly, all fluorescent cells detected in white cell populations of strains WUMS1A and -B matched the opaque phenotype. In no case could a fluorescent cell unambiguously be allocated to the wrong phase. These results demonstrated, at the single-cell level, that expression of the phase-specific SAP1 and WH11 genes is linked to the cell type.
|
Phase variation of SAP1 and WH11 expression does not occur independently of white-opaque switching in vitro. The results obtained with the strains carrying the GFP reporter gene fusions showed that a switch from the "OFF" to the "ON" state of the phase-specific SAP1 and WH11 genes does not occur in the wrong cell type, since single fluorescent cells of the corresponding phase would have been easily detected in the background of a nonfluorescent cell population. This indicates that activation of phase-specific genes is under the tight control of the postulated master switching regulator. Nevertheless, a switch from the ON to the OFF state for individual phase-specific genes might occur even in the normally appropriate cell type by an independent mechanism. We also searched for white cells that do not detectably express WH11 and for opaque cells that do not detectably express SAP1 by looking at several hundred individual cells of white cell populations of strains WUGW11A and -B and of opaque cell populations of strains WUGS1A and -B first by phase-contrast and then by epifluorescence microscopy. In this way we were unable to detect such an ON-OFF switch. However, at an expected low frequency, a corresponding nonfluorescent cell might be missed in a population of fluorescent cells. For this reason, we devised a second reporter system in which a drug resistance gene was placed under the control of phase-specific promoters. Since the MPAR marker confers resistance against MPA upon cells in which it is expressed, a promoterless MPAR gene was fused to the SAP1 and WH11 promoters and integrated into the corresponding loci in strain WUM5A with the help of the URA3 selection marker (Fig. 2A and 3A). Two transformants carrying the PSAP1-MPAR fusion inserted in either of the two SAP1 alleles (strains WUMS1A and WUMS1B) (Fig. 2B, lanes 4 and 5) and two transformants in which the PWH11-MPAR fusion was integrated in one of the WH11 alleles (strains WUMW11A and WUMW11B) (Fig. 3B, lanes 4 and 5) were selected for analysis.
The strains carrying these reporter gene fusions should express an MPA-resistant phenotype only in the appropriate cell type in which the promoter is activated, i.e., only opaque cells of the strains with the PSAP1-MPAR fusion should exhibit enhanced MPA resistance and only white cells of the strains with the PWH11-MPAR fusion should become MPA resistant. Figure 5 shows that growth of the parent strain WO-1 was sensitive to the presence of 7.5 µg of MPA ml1 in the medium. Opaque cells displayed a
higher sensitivity than white cells, since they formed smaller colonies
than white cells, although in the absence of MPA opaque colonies are
larger than white colonies due to their flat morphology. The expression
of the MPAR gene resulted in the expected
phenotype. The strains carrying the MPAR
gene under the control of the SAP1 promoter exhibited
enhanced MPA resistance in the opaque phase, whereas the sensitivity of white cells remained unchanged. In contrast, the strains carrying the
MPAR gene under the control of the
WH11 promoter showed the reverse phenotype, although,
for unknown reasons, opaque cells exhibited slightly enhanced
growth on plates with or without MPA compared with the parent strain
WO-1. Nevertheless, these results demonstrate that the reporter system
worked sufficiently well in both white and opaque cells to allow
screening for colonies that did not express the
MPAR gene.
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The white-opaque switching of strain WO-1 has served for many years as a model system to study phenotypic switching in C. albicans and has led to the discovery of phase-specific genes, expression or down-regulation of which might provide selective advantages in specific host niches encountered by the fungus at particular stages of an infection (36). An ade2 mutant generated by chemical mutagenesis of strain WO-1 has been used in the past as a host for genetic analyses of phenotypic switching, for example, to introduce luciferase reporter gene fusions and to allow misexpression of phase-specific genes (16, 17, 18, 39, 40, 41). However, until recently no ura3 mutant of strain WO-1 was available that could be used for genetic manipulations requiring a recyclable marker, like sequential gene disruption. While our work was in progress, the generation of a ura3-auxotrophic derivative from the WO-1 ade2 mutant was reported, and this, for the first time, enabled the construction of a specific mutant (efg1/efg1) in this strain background by targeted gene deletion using the URA3 blaster strategy (42). The ura3 mutants described in our present study have the advantage that they were generated directly from strain WO-1 by two independent rounds of specific allelic replacement of both URA3 copies and, therefore, have not suffered from unspecific mutagenesis procedures or selection of mitotic recombinants. Although the MPAR marker in principle can be used for all genetic manipulations in any C. albicans wild-type strain, the availability of a specifically generated ura3 mutant is desirable for model strains like WO-1. First, using URA3 as a selection marker, prototrophic transformants can be recovered after only 2 days of selection on uridine-deficient medium, whereas the primary isolation of MPA-resistant transformants usually requires about 1 week, with additional time needed for clone purification. Therefore, the generation of mutants by sequential gene disruptions is significantly accelerated when the URA3 gene can be used as selection marker. Second, many already available URA3-based genetic constructions (e.g., gene disruption cassettes) that have been used for the molecular analysis of the C. albicans model strain CAI4 can probably be used directly also in the ura3 mutants of WO-1. Finally, the availability of a second selection marker like URA3 allows the alternative use of MPAR as a reporter gene, as demonstrated in the present study for the analysis of phase-specific gene expression at the level of individual colonies to detect switch events in the cells from which they were derived.
We have used two different reporter systems, GFP and
MPAR, that allowed the analysis of
phase-specific gene expression at the single-cell level, with the aim
of detecting a possible cell type-independent switching in the
expression status of individual genes. Our results demonstrate that the
expression of the phase-specific WH11 and SAP1
genes is tightly coupled to the white or opaque cell morphology, since
all white cells but no opaque cells examined expressed the WH11 gene, and all opaque cells but no white cells expressed
the SAP1 gene. A significant activation of a phase-specific
gene in the wrong cell type should have been easily detected with both reporter systems, since forced misexpression of the WH11 and
SAP1 genes does not affect the cell morphology (16,
17). It should be noted that both reporter systems are useful
only for qualitative rather than quantitative analyses, and we probably
would have been unable to detect a low level of gene expression.
However, a true morphology-independent phase variation in the
expression of a gene would be expected to result in expression levels
in the wrong cell type that are comparable to those in the correct phase and, vice versa, in down-regulation of the gene in the normally appropriate cell type to the level of the wrong phase. According to the
fluorescence signals obtained with the GFP gene and the difference in MPA resistance between cells expressing and not expressing the MPAR gene, such a phase
variation should have been detected with our reporter systems.
Therefore, we conclude that cell type-independent switching of
individual phase-specific genes does not occur at an appreciable
frequency (<104) under the in vitro conditions
tested, at least not at a frequency comparable to that of the
white-opaque switching itself (34). However, this does not
exclude such a possibility during infection. In fact, extreme
differences in the frequency of antigenic variation in vitro and in
vivo have been observed in other pathogens (49). Therefore, we intend to analyze switching of phase-specific genes in
different animal models of candidiasis. The
MPAR-based reporter system will be
especially useful for this purpose, since clones of possible variants
would be detected after simple plating of recovered cells and then be
directly accessible for an analysis of the underlying molecular changes.
Phenotypic switching in C. albicans has been proposed to be under the control of a master regulatory switch, since many different cellular properties are altered in a programmed, coordinated fashion (36). However, the nature of this master regulator is unknown, as is the pathway leading to activation and deactivation of phase-specific genes. Inactivation of the SIR2 gene results in deregulated phenotypic switching in the C. albicans strain CAI4 (30), but switching in this strain is by far less well characterized than the white-opaque switching of strain WO-1. Such specific mutations can now also be introduced into strain WO-1 to assess their effect on white-opaque switching. The reporter gene fusions described in this study will be useful for addressing the question of whether in such regulatory mutants the expression of phase-specific genes is still linked to cell morphology or decoupled from the cell type. This approach should allow the dissection of the signal transduction pathway from the master regulatory switch to the different genes whose expression is controlled by phenotypic switching.
| |
ACKNOWLEDGMENTS |
|---|
This study was supported by the Bundesministerium für Bildung und Forschung (BMBF grant O1 K1 8906-0).
We thank Klaus Schröppel for his advice concerning growth of strain WO-1.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Zentrum für Infektionsforschung, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany. Phone: 49-931-31 21 52. Fax: 49-931-31 25 78. E-mail: joachim.morschhaeuser{at}mail.uni-wuerzburg.de.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Abraham, J. M.,
C. S. Freitag,
J. R. Clements, and B. I. Eisenstein.
1985.
An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli.
Proc. Natl. Acad. Sci. USA
82:5724-5727 |
| 2. |
Balan, I.,
A.-M. Alarco, and M. Raymond.
1997.
The Candida albicans CDR3 gene codes for an opaque-phase ABC transporter.
J. Bacteriol.
179:7210-7218 |
| 3. |
Bedell, G. W., and D. R. Soll.
1979.
Effects of low concentrations of zinc on the growth and dimorphism of Candida albicans: evidence for zinc-resistant and -sensitive pathways for mycelium formation.
Infect. Immun.
26:348-354 |
| 4. |
Borg, M., and R. Rüchel.
1988.
Expression of extracellular acid proteinase by proteolytic Candida species during experimental infection of oral mucosa.
Infect. Immun.
56:626-631 |
| 5. | Borg-von-Zepelin, M., S. Beggah, K. Boggian, D. Sanglard, and M. Monod. 1998. The expression of the secreted aspartyl proteinases Sap4 to Sap6 from Candida albicans in murine macrophages. Mol. Microbiol. 28:543-554[CrossRef][Medline]. |
| 6. |
Borst, P., and G. Rudenko.
1994.
Antigenic variation in African trypanosomes.
Science
264:1872-1873 |
| 7. | Braaten, B. A., X. Nou, L. S. Kaltenbach, and D. A. Low. 1994. Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli. Cell 76:577-588[CrossRef][Medline]. |
| 8. |
Cormack, B. P.,
G. Bertram,
M. Egerton,
N. A. R. Gow,
S. Falkow, and A. J. P. Brown.
1997.
Yeast-enhanced green fluorescent protein (yGFP): a reporter of gene expression in Candida albicans.
Microbiology
143:303-311 |
| 9. | De Backer, M. D., P. T. Magee, and J. Pla. 2000. Recent developments in molecular genetics of Candida albicans. Annu. Rev. Microbiol. 54:463-498[CrossRef][Medline]. |
| 10. | Deitsch, K. W., E. R. Moxon, and T. E. Wellems. 1997. Shared themes of antigenic variation and virulence in bacterial, protozoal, and fungal infections. Microbiol. Mol. Biol. Rev. 61:281-293[Abstract]. |
| 11. | Haas, R., and T. F. Meyer. 1986. The repertoire of silent pilus genes in Neisseria gonorrhoeae: evidence for gene conversion. Cell 44:107-115[CrossRef][Medline]. |
| 12. | Hammerschmidt, S., R. Hilse, J. P. van Putten, R. Gerardy-Schahn, A. Unkmeir, and M. Frosch. 1996. Modulation of cell surface sialic acid expression in Neisseria gonorrhoeae via a transposable genetic element. EMBO J. 15:192-198[Medline]. |
| 13. | Hoeijmakers, J. H. J., A. C. C. Frasch, A. Bernards, P. Borst, and G. A. M. Cross. 1980. Novel expression-linked copies of the genes for variant surface antigens in trypanosomes. Nature 284:78-80[CrossRef][Medline]. |
| 14. | Hube, B., D. Sanglard, F. Odds, D. Hess, M. Monod, W. Schäfer, A. J. P. Brown, and N. A. R. Gow. 1997. Disruption of each of the secreted aspartyl proteinase genes SAP1, SAP2, and SAP3 of Candida albicans attenuates virulence. Infect. Immun. 65:3529-3538[Abstract]. |
| 15. |
Köhler, G. A.,
T. C. White, and N. Agabian.
1997.
Overexpression of a cloned IMP dehydrogenase gene of Candida albicans confers resistance to the specific inhibitor mycophenolic acid.
J. Bacteriol.
179:2331-2338 |
| 16. |
Kvaal, C.,
S. A. Lachke,
T. Srikantha,
K. Daniels,
J. McCoy, and D. R. Soll.
1999.
Misexpression of the opaque-phase-specific gene PEP1 (SAP1) in the white phase of Candida albicans confers increased virulence in a mouse model of cutaneous infection.
Infect. Immun.
67:6652-6662 |
| 17. | Kvaal, C., T. Srikantha, and D. R. Soll. 1997. Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence. Infect. Immun. 65:4468-4475[Abstract]. |
| 18. |
Lockhart, S. R.,
M. Nguyen,
T. Srikantha, and D. R. Soll.
1998.
A MADS box protein consensus binding site is necessary and sufficient for activation of the opaque-phase-specific gene OP4 of Candida albicans.
J. Bacteriol.
180:6607-6616 |
| 19. |
Macdonald, F., and F. C. Odds.
1980.
Inducible proteinase of Candida albicans in diagnostic serology and the pathogenesis of systemic candidosis.
J. Med. Microbiol.
13:423-435 |
| 20. |
Marrs, C. F.,
W. W. Ruehl,
G. K. Schoolnik, and S. Falkow.
1988.
Pilin gene phase variation of Moraxella bovis is caused by an inversion of the pilin genes.
J. Bacteriol.
170:3032-3039 |
| 21. | Martinez, J. J., M. A. Mulvey, J. D. Schilling, J. S. Pinkner, and S. J. Hultgren. 2000. Type 1 pilus-mediated bacterial invasion of bladder epithelial cells. EMBO J. 19:2803-2812[CrossRef][Medline]. |
| 22. | Meier, J. T., M. I. Simon, and A. G. Barbour. 1985. Antigenic variation is associated with DNA rearrangements in a relapsing fever Borrelia. Cell 41:403-409[CrossRef][Medline]. |
| 23. |
Mekalanos, J. J.
1992.
Environmental signals controlling expression of virulence genes in bacteria.
J. Bacteriol.
174:1-7 |
| 24. |
Millon, L.,
A. Manteaux,
G. Reboux,
C. Drobacheff,
M. Monod,
T. Barale, and Y. Michel-Briand.
1994.
Fluconazole-resistant recurrent oral candidiasis in human immunodeficiency virus-positive patients: persistence of Candida albicans strains with the same genotype.
J. Clin. Microbiol.
32:1115-1118 |
| 25. |
Morrow, B.,
T. Srikantha, and D. R. Soll.
1992.
Transcription of the gene for a pepsinogen, PEP1, is regulated by the white-opaque switching in Candida albicans.
Mol. Cell. Biol.
12:2997-3005 |
| 26. |
Morrow, B.,
T. Srikantha,
J. Anderson, and D. R. Soll.
1993.
Coordinate regulation of two opaque-phase-specific genes during white-opaque switching in Candida albicans.
Infect. Immun.
61:1823-1828 |
| 27. | Morschhäuser, J., S. Michel, and J. Hacker. 1998. Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation. Mol. Gen. Genet. 257:412-420[CrossRef][Medline]. |
| 28. | Morschhäuser, J., S. Michel, and P. Staib. 1999. Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination. Mol. Microbiol. 32:547-556[CrossRef][Medline]. |
| 29. | Moxon, E. R., P. B. Rainey, M. A. Novak, and R. E. Lenski. 1994. Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr. Biol. 4:24-33[CrossRef][Medline]. |
| 30. | Pérez-Martín, J., J. A. Uría, and A. D. Johnson. 1999. Phenotypic switching in Candida albicans is controlled by a SIR2 gene. EMBO J. 18:2580-2592[CrossRef][Medline]. |
| 31. | Roberts, D. J., A. G. Craig, A. R. Berendt, R. Pinches, G. Nash, K. Marsh, and C. I. Newbold. 1992. Rapid switching to multiple antigenic and adhesive phenotypes in malaria. Nature 357:689-692[CrossRef][Medline]. |
| 32. | Schaller, M., H. C. Korting, W. Schäfer, J. Bastert, W. Chen, and B. Hube. 1999. Secreted aspartic proteinase (Sap) activity contributes to tissue damage in a model of human oral candidosis. Mol. Microbiol. 34:169-180[CrossRef][Medline]. |
| 33. |
Schröppel, K.,
T. Srikantha,
D. Wessels,
M. DeCock,
S. R. Lockhart, and D. R. Soll.
1996.
Cytoplasmic localization of the white phase-specific WH11 gene product of Candida albicans.
Microbiology
142:2245-2254 |
| 34. |
Slutsky, B.,
M. Staebell,
J. Anderson,
L. Risen,
M. Pfaller, and D. R. Soll.
1987.
"White-opaque transition": a second high-frequency switching system in Candida albicans.
J. Bacteriol.
169:189-197 |
| 35. | Soll, D. R., B. Morrow, and T. Srikantha. 1993. High-frequency phenotypic switching in Candida albicans. Trends Genet. 9:61-65[CrossRef][Medline]. |
| 36. |
Soll, D. R.
1997.
Gene regulation during high-frequency switching in Candida albicans.
Microbiology
143:279-288 |
| 37. |
Sonneborn, A.,
B. Tebarth, and J. F. Ernst.
1999.
Control of white-opaque phenotypic switching in Candida albicans by the Efg1p morphogenetic regulator.
Infect. Immun.
67:4655-4660 |
| 38. | Srikantha, T., and D. R. Soll. 1993. A white-specific gene in the white-opaque switching system in Candida albicans. Gene 131:53-60[CrossRef][Medline]. |
| 39. | Srikantha, T., B. Morrow, K. Schröppel, and D. R. Soll. 1995. The frequency of integrative transformation at phase-specific genes of Candida albicans correlates with their transcriptional state. Mol. Gen. Genet. 246:342-352[CrossRef][Medline]. |
| 40. |
Srikantha, T.,
A. Klapach,
W. W. Lorenz,
L. K. Tsai,
L. A. Laughlin,
J. A. Gorman, and D. R. Soll.
1996.
The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.
J. Bacteriol.
178:121-129 |
| 41. |
Srikantha, T.,
L. K. Tsai, and D. R. Soll.
1997.
The WH11 gene of Candida albicans is regulated in two distinct developmental programs through the same transcriptional activation sequences.
J. Bacteriol.
179:3837-3844 |
| 42. |
Srikantha, T.,
L. K. Tsai,
K. Daniels, and D. R. Soll.
2000.
EFG1 null mutants of Candida albicans switch but cannot express the complete phenotype of white-phase budding cells.
J. Bacteriol.
182:1580-1591 |
| 43. | Staib, P., M. Kretschmar, T. Nichterlein, G. Köhler, S. Michel, H. Hof, J. Hacker, and J. Morschhäuser. 1999. Host-induced, stage-specific virulence gene activation in Candida albicans during infection. Mol. Microbiol. 32:533-546[CrossRef][Medline]. |
| 44. | Stern, A., M. Brown, P. Nickel, and T. F. Meyer. 1986. Opacity genes in Neisseria gonorrhoeae: control of phase and antigenic variation. Cell 47:61-71[CrossRef][Medline]. |
| 45. |
Stoenner, H. G.,
T. Dodd, and C. Larsen.
1982.
Antigenic variation of Borrelia hermsii.
J. Exp. Med.
156:1297-1311 |
| 46. | Weiser, J. N., J. M. Love, and E. R. Moxon. 1989. The molecular mechanism of phase-variation of Haemophilus influenzae lipopolysaccharide. Cell 59:657-665[CrossRef][Medline]. |
| 47. |
White, T. C.,
S. H. Miyasaki, and N. Agabian.
1993.
Three distinct secreted aspartyl proteinases in Candida albicans.
J. Bacteriol.
175:6126-6133 |
| 48. | Wirsching, S., S. Michel, and J. Morschhäuser. 2000. Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates. Mol. Microbiol. 36:856-865[CrossRef][Medline]. |
| 49. |
Zhang, J. R., and S. J. Norris.
1998.
Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.
Infect. Immun.
66:3689-3697 |
| 50. | Ziebuhr, W., V. Krimmer, S. Rachid, I. Lößner, F. Götz, and J. Hacker. 1999. A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256. Mol. Microbiol. 32:345-356[CrossRef][Medline]. |
Journal of Bacteriology, June 2001, p. 3761-3769, Vol. 183, No. 12
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.12.3761-3769.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Lermann, U., Morschhauser, J.
(2008). Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans. Microbiology
154: 3281-3295
[Abstract] [Full Text] -
Hiller, D., Stahl, S., Morschhauser, J.
(2006). Multiple cis-Acting Sequences Mediate Upregulation of the MDR1 Efflux Pump in a Fluconazole-Resistant Clinical Candida albicans Isolate.. Antimicrob. Agents Chemother.
50: 2300-2308
[Abstract] [Full Text] -
Miller, N. S., Dick, J. D., Merz, W. G.
(2006). Phenotypic Switching in Candida lusitaniae on Copper Sulfate Indicator Agar: Association with Amphotericin B Resistance and Filamentation. J. Clin. Microbiol.
44: 1536-1539
[Abstract] [Full Text] -
Coste, A., Turner, V., Ischer, F., Morschhauser, J., Forche, A., Selmecki, A., Berman, J., Bille, J., Sanglard, D.
(2006). A Mutation in Tac1p, a Transcription Factor Regulating CDR1 and CDR2, Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate Antifungal Resistance in Candida albicans. Genetics
172: 2139-2156
[Abstract] [Full Text] -
Park, Y.-N., Morschhauser, J.
(2005). Tetracycline-Inducible Gene Expression and Gene Deletion in Candida albicans. Eukaryot Cell
4: 1328-1342
[Abstract] [Full Text] -
Brehm-Stecher, B. F., Johnson, E. A.
(2004). Single-Cell Microbiology: Tools, Technologies, and Applications. Microbiol. Mol. Biol. Rev.
68: 538-559
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»