Microbiology (2004), 150, 2785–2790
DOI 10.1099/mic.0.27094-0
Green fluorescent protein (GFP) as a vital marker
for pathogenic development of the dermatophyte
Trichophyton mentagrophytes
Gil Kaufman,1,2 Benjamin A. Horwitz,1 Ruthi Hadar,1 Yehuda Ullmann3
and Israela Berdicevsky2
1,2
Department of Biology1 and Department of Molecular Microbiology, Faculty of Medicine2,
Technion – Israel Institute of Technology, Haifa 32000, Israel
Correspondence
Israela Berdicevsky
[email protected]
3
Department of Plastic Surgery, Rambam Medical Center, Haifa, Israel
Received 12 February 2004
Revised 4 May 2004
Accepted 13 May 2004
Skin infections by dermatophytes of the genus Trichophyton are widespread, but methods
to investigate the molecular basis of pathogenicity are only starting to be developed. The initial
stages of growth on the host can only be studied by electron microscopy, which requires fixing
the tissue. This paper shows that restriction-enzyme-mediated integration (REMI) provides
stable expression of the green fluorescent protein (GFP) in a clinical isolate of Trichophyton
mentagrophytes. Under control of a constitutively active fungal promoter, GFP renders the
hyphae fluorescent both in culture and in a recently developed model using human skin explants.
Stages of infection and penetration into the skin layers were visualized by confocal microscopy.
The stages of infection can thus be followed using GFP as a vital marker, and this method will
also provide, for the first time, a means to follow gene expression during infection of skin by
dermatophyte fungi.
INTRODUCTION
Dermatophytes (Weitzman & Summerbell, 1995) are pathogenic fungi that invade the outermost skin layers (stratum
corneum) and keratinized structures derived from the
epidermis, causing infections of the skin, hair and nails.
Relatively little is known about the stages of infection,
which include contact and adherence of fungal conidia
(Zurita & Hay, 1987), germination (Aljabre et al., 1992),
invasive growth and development of an active lesion
(Richardson & Aljabre, 1993). Animal models (Hanel
et al., 1990; Treiber et al., 2001) are available but these
may not accurately reflect the properties and composition
of human skin. Alternatives are sheets of stratum corneum
(Aljabre et al., 1992) or corneocyte cells (Aljabre et al., 1993).
We have recently developed a model system using skin
explants that are a by-product of plastic surgery (Duek
et al., 2004). In order to characterize initial adherence and
invasion steps, these ex vivo skin sections, which are of
full human epidermis thickness, are infected and examined
by scanning and transmission electron microscopy. The
green fluorescent protein (GFP) was introduced as a marker
to follow the development of fungal pathogens on and in
their hosts, in both basidiomycete (Spellig et al., 1996) and
ascomycete (Maor et al., 1998) pathogens of plants. Since
Abbreviations: GFP, green fluorescent protein; REMI, restrictionenzyme-mediated integration.
0002-7094 G 2004 SGM
then, GFP under the control of constitutive and inducible
fungal promoters has been widely used to follow gene
expression and development in phytopathogens (Lev, 2003;
Poggeler et al., 2003; Chen et al., 2003; Viterbo et al., 2002;
Wasylnka & Moore, 2002; Kahmann & Basse, 2001) and in
Candida, Histoplasma (Kugler et al., 2000) and Aspergillus
fumigatus (Langfelder et al., 2001). The GFP reporter provides a unique way to visualize gene expression as a function
of time and location, within the living host tissue.
Nevertheless, no such experiments have been done on
dermatophytes, partly because it has been difficult to
construct transgenic strains. Following an initial report of
transformation to hygromycin resistance using a promoter
from Cochliobolus heterostrophus (Gonzalez et al., 1989),
there appears to have been no further work in this direction. Several methods are available to introduce transgenes
into fungi: protoplast fusion (Gonzalez et al., 1989; Lev &
Horwitz, 2003) electroporation (Robinson & Sharon, 1999),
biolistic bombardment and Agrobacterium (de Groot et al.,
1998). In this study, we employed protoplast fusion and
standard fungal expression signals to express GFP in a
dermatophyte. We tested restriction-enzyme-mediated
integration (REMI) (Lu et al., 1994) as a means to stabilize
the integrated DNA. The infection process was followed by
visualizing fluorescence in the human skin explant model.
The results indicate that GFP is useful in following the
pathogenicity of Trichophyton mentagrophytes in human skin.
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G. Kaufman and others
METHODS
Fungal strains and culture. A clinical wild-type of T. mentagro-
phytes var. mentagrophytes was obtained from Rambam Hospital,
Haifa. Wild-type and transgenic strains were grown axenically
on Sabouraud dextrose [glucose] agar (SDA, Difco) (containing
0?05 mg chloramphenicol and 0?5 mg cycloheximide ml21) at 30 uC
for 14–21 days. For hygromycin B selection, SDA was prepared
with a final concentration of 150 mg hygromycin B ml21
(Calbiochem). Mycelium pieces, approximately 0?5 cm2, were used
to inoculate human skin explants obtained as a by-product from
plastic surgery and immersed in sterilized skin graft fluid (SGF),
as described previously (Duek et al., 2004). Plates with mineral
medium (MgSO4.7H2O, 1 g l21; KH2PO4, 0?1 g l21; FeSO4.7H2O,
10 mg l21; ZnSO4.7H2O, 5 mg l21) and 0?25 % bovine elastin
(Sigma) were used to test the potential virulence of the wild-type
and transgenic isolates.
5–6 min at room temperature. At the end of the incubation, 1 ml
STC was added to each tube, and the contents were gently mixed
again. Regeneration medium was held at 55 uC prior to use. The
content of each tube was divided into two to four portions. Each
portion was poured into a plate containing 20 ml liquid regeneration medium and immediately mixed (gently but quickly, with a
pipette). Twenty micrograms of plasmid gGFP (Maor et al., 1998)
were used to transform 108 protoplasts. After the PEG treatment the
protoplast suspension was mixed into 10 ml regeneration Saboraud
dextrose medium (RSD: SDA with 2 % sorbitol) at 55 uC, then
incubated for 36 h at 30 uC, and overlaid with 200 mg hygromycin B
ml21 in RSD. Transformants appeared after 14 days’ growth at
30 uC, and were transferred to SDA with 150 mg hygromycin ml21.
For REMI, 100 units of BglII were added to the transformation reaction (Lu et al., 1994), and 20 mg of plasmid gGFP digested with
BglII was used for the transformation.
Confocal microscopy. Fungal mycelium grown axenically on skin
DNA manipulations. gGFP, a vector used for the expression of the
green fluorescent protein, contained hph and sgfp genes fused to the
Aspergillus nidulans trpC promoter and terminator (Maor et al.,
1998). Plasmid DNA was purified with a midiprep kit according to
the supplier’s directions (Qiagen). Genomic DNA was isolated from
mycelium ground in liquid nitrogen as described for plant tissue
(Edwards et al., 1991), and 1?25 mg used as template for PCR and
genomic Southern analysis. About 5 mg genomic DNA was digested
for each lane, with the indicated restriction enzymes. The gel was
blotted to Hybond-N+ (Amersham), cross-linked with UV. A
probe corresponding to the coding region of GFP was labelled with
[a-32P]dCTP (Amersham) by random priming, and the blot hybridized according to the manufacturer’s recommendations, by the
method of Church & Gilbert (1984). The signal was detected by
overnight exposure with a phosphorimager (Fuji). The primers
3074#GFP (59-GAGCTGAAGGGCATCGACTT-39) and 3075#GFP
(59-CTTGTGCCCCAGGATGTTG-39) were used in PCR amplification to detect gfp, ment-hph1 (59-GAGGGCGAAGAATCTCGTGC39) and ment-hph2 (59-CACTGACGGTGTCGTCCATC-39) for the
hph gene, and ment-act1 (59-CGCCCCAGCCTTCTACGTC-39)
and ment-act2 (59-CGGTCGGAGATACCTGGGTAC-39) for the
T. mentagrophytes act (actin) gene. The amplified PCR fragments
were purified from gels with the Qiaex II kit according to the
supplier’s directions (Qiagen) and DNA sequencing was carried out
on an automated fluorescence sequencer (Applied Biosystems
PRISM 3100 at the sequencing lab, Faculty of Medicine, Technion).
The sequences were edited using the manufacturer’s software.
BLASTN and FASTA sequence homology analyses were performed
through the National Center for Biotechnology Information (NCBI)
website.
DNA-mediated transformation. Transformation was essentially as
described by Turgeon et al. (1987) and Gonzalez et al. (1989) with
modifications. Conidia were germinated by shaking in Sabouraud
dextrose broth (SDB) medium at 200 r.p.m. and 30 uC for 19 h;
the germlings were transferred to osmoticum containing 5 mg b-Dglucanase ml21 (InterSpex), 5 mg Driselase ml21 (InterSpex) and
0?05 mg chitinase ml21 (Sigma), and incubated for 4 h with shaking
at 70 r.p.m. and 30 uC. Then 108–109 protoplasts were separated
from the mycelium and cell debris by filtering through two layers of
sterile gauze. The filtrate was centrifuged for 5 min at 4500 r.p.m.
and washed twice with STC (sorbitol/Tris/calcium: 1?2 M sorbitol,
10 mM Tris pH 7?5, 50 mM CaCl2), after which 108 protoplasts
were resuspended in 200 ml STC. Three aliquots of 60 % PEG solution, 200, 200 and 800 ml, were added to each tube, with gentle
mixing after each addition. PEG solution consisted of 60 % PEG
(polyethylene glycol, MW 3500–4000, Sigma), 10 mM Tris pH 7?5,
50 mM CaCl2. The tubes were incubated with PEG solution for
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explants was visualized by a Nikon E600 fluorescent microscope and
Radiance 2000 confocal laser scanning microscope. Images were
processed by Image Pro Plus software. Samples of infected and
non-infected skin were scanned under the same conditions and at a
depth of 12 layers with intervals of 0?82 mm.
Stability and virulence. The genetic stability of the transgenic
isolates was assessed by monitoring GFP expression and hygromycin
resistance. Purified single colonies of each transgenic isolate were
grown and subcultured a total of three times on a plate of nonselective medium (SDA without hygromycin) or selective medium (with
hygromycin) and also were transferred from plates without hygromycin to plates with hygromycin. The virulence was assessed by the
ability of the transgenic isolates to infect and expand into skin
explants and to degrade keratin and elastin.
RESULTS AND DISCUSSION
REMI as a method for transformation in
T. mentagrophytes
Transformation of genes into dermatophytes is difficult.
Protoplast transformation, biolistic bombardment and
Agrobacterium-mediated
integration
were
initially
tested. Candidate transformants were obtained only from
protoplast-fusion-mediated transformation (Gonzalez et al.,
1989), and the hygromycin resistance marker detected by
PCR (data not shown). The hygromycin resistance of these
colonies generally proved to be unstable upon subsequent
transfer. We reasoned that REMI might provide stable
integration, since linear DNA integrated at the restriction site would not be eliminated by a reversal of the
single-crossover integration event. REMI indeed improves
the transformation frequency in some filamentous fungi
(Lu et al., 1994). As indicated in Table 1, more stable
hygromycin-resistant and fluorescent transformants were
obtained with the REMI method than by transformation
with circular DNA. The transformants were able to grow
on high concentrations of hygromycin B (250 mg ml21).
We believe this to be the first report of REMI, along
with protoplast transformation, as a successful method for
insertion and expression of transgenes in the dermatophyte
T. mentagrophytes.
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Microbiology 150
GFP expression in Trichophyton on skin
expected gfp sequence (data not shown). The bacterial hph
gene conferring hygromycin resistance was also detected
by PCR amplification of genomic DNA of the transformants, but not of the wild-type. The T. mentagrophytes
actin gene was detected in the transformants and the wildtype (Fig. 1a). Southern analysis indicated integration at
the recognition site of the enzyme used (BglII) to linearize
the transforming DNA; a single band migrating at the size
of the integrated vector was detected in both transformants.
The integration sites in the two transformants were different, as shown by digestion with SphI, which cuts the
transgene 742 bp upstream of the start codon of GFP.
Furthermore, the pattern obtained with the two enzymes is
consistent with single-copy integration (Fig. 1b).
Table 1. Circular plasmid transformation vs BglII-restricted
plasmid transformation
‘Stable’ denotes transformants which remained hygromycin resistant following transfer on non-selective medium. ‘Total’ indicates
the number of resistant colonies initially isolated.
Plasmid digested with BglII
Fluorescent
1
2
0
4
6
1
Total Fluorescent Stable Total
12
14
8
0
0
0
1
1
0
3
5
2
Insertion and expression of GFP in
T. mentagrophytes
The level of fluorescence varied among transformants. A
strongly fluorescent isolate (REMI6) was used for confocal
microscopy. REMI is expected to create mutations by
random insertion. In one mutant, the GFP fluorescence
was not uniform, but rather localized to intracellular regions
that appear to correspond to vacuoles (data not shown).
This distribution might be the result of a REMI-mediated
integration or rearrangement at the integration site or
elsewhere in the genome. Hyphae of transformant REMI6
(Fig. 2b) and REMI5 (Fig. 2c) grown in medium were
Two fluorescent transformants were chosen for molecular
and microscopic analysis. The transgenes were detected
by PCR amplification and Southern analysis (Fig. 1). The
‘humanized’ version of the Aquorea victoria gene encoding
the green fluorescent protein (gfp) was detected by PCR
amplification of genomic DNA of the transformants, but
not of the wild-type isolate. One product amplified with
gfp-specific primers was sequenced and was indeed the
(a)
(b)
Fig. 1. Detection of the transgenes. (a)
PCR amplification with primers for gfp, hph
(hygromycin phosphotransferase) and act
(actin control). The bands are consistent
with the predicted sizes indicated on the
left. The plasmid control indicates product
amplified from plasmid gGFP, and the
following two lanes show products amplified
from genomic DNA from the wild-type (WT)
and a fluorescent transformant (REMI6).
(b) Southern analysis: genomic DNA of wildtype and transformants was digested with
the indicated enzymes and the blot probed
with the GFP coding region.
23
9.4
6.5
4.3
426 bp
hph
2.3
626 bp
act
http://mic.sgmjournals.org
WT
gfp
97 bp
(a)
SphI
REMI6
WT
kbp
REMI5
REMI6
WT
Plasmid
control
BglII
REMI6
1
2
3
Stable
Circular plasmid
REMI5
Expt
(b)
(c)
Fig. 2. Confocal images of wild-type (a)
and transformants REMI6 (b) and REMI5 (c),
grown for 14 days on SDA with 150 mg
hygromycin B ml”1.
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G. Kaufman and others
(a)
Frame 1
Frame 2
Frame 3
Frame 4
Frame 5
Frame 6
Frame 1
Frame 2
Frame 3
Frame 4
Frame 5
Frame 6
(b)
Fig. 3. Confocal images of human skin explants: (a) infected with transformant REMI6; (b) non-infected. Each successive
frame was taken at an interval of 0?82 mm depth from the previous frame. Bars, 20 mm. In (a), the large arrow indicates fungal
hyphae. The small arrows follow invasion of a hypha into the skin: in frame 1, the arrow points to the first appearance of the
fluorescent cell; with increasing depth in successive optical sections 2–6, the arrows show the extension of this invading
hypha. Keratinocytes are visible by their weak autofluorescence in both (a) and (b). In non-infected skin (b) no fluorescent
hyphae are visible.
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Microbiology 150
GFP expression in Trichophyton on skin
clearly fluorescent, as compared with a wild-type control
under the same conditions (Fig. 2a).
(a)
GFP as a vital marker to follow infection of
T. mentagrophytes in human skin explants
The pathogen was visualized during development on skin
explants (Fig. 3a). The fluorescent hyphae could be clearly
seen at a distance from the infection point on skin and at
a depth of 9?8 mm from the skin surface. The skin cells
are visible due to their weak autofluorescence, which
decreases with depth (Fig. 3a, b). Infection of skin layers
by T. mentagrophytes was detected previously (Duek et al.,
2004) by scanning and transmission electron microscopy.
The pathogenesis of T. mentagrophytes in human skin
explants was followed by the directions of the spreading
and the branching of the elongated hyphae in two dimensions, vertical and horizontal, on the skin (data not shown)
and invading into the skin layers. Other fungal elements,
microconidia and arthroconidia, were not detected on
the skin explants or inside them. The ability to follow
T. mentagrophytes pathogenicity stages in skin explants
without modifying or damaging the sample, as in fixation,
makes the analysis more accurate and reliable. This is apparently the second report of the construction of a transgenic
dermatophyte, following the first report of transformation
to hygromycin resistance (Gonzalez et al., 1989). GFP has
been used to follow development and gene expression in
plant–fungal interactions, but despite the phylogenetic
closeness of dermatophytes to other ascomycetes, very
little work of this kind has been done in dermatophytes.
It will now be possible to employ these methods to study
the dermatophyte–skin interaction and to quantitate the
fungal biomass in the infected skin at different stages of
infection. Furthermore, our application of REMI opens the
way to using this technique to generate tagged mutations
in dermatophytes.
T. mentagrophytes transformants are
genetically stable and virulent
(b)
Fig. 4. Growth on selective medium and elastin degradation.
(a) Ability of the different strains to grow on selective medium:
SDA plus 150 mg hygromycin B ml”1. The wild-type (panel 1)
is unable to grow on hygromycin plates, while transformants
REMI6 (panel 2) and REMI5 (panel 3) are able to grow. (b) The
ability of the wild-type (panel 1), and the transformants REMI6
(panel 2) and REMI5 (panel 3) to digest elastin (0?25 % in
mineral medium), is visible as a clear halo around the colonies.
the different transformants. REMI6 showed identical ability
to digest elastin as the wild-type, in contrast to REMI5,
which showed a lesser ability. The locus of integration into
the genome might affect enzyme production or secretion.
ACKNOWLEDGEMENTS
We thank Edith Vissuss-Toby and Ofer Shenkar for expert assistance
with confocal microscopy at the Faculty of Medicine. This work was
supported in part by the Vice-President’s Research fund, Technion
Research and Development Foundation.
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