Abstract
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 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.
METHODS
Fungal strains and culture.
A clinical wild-type of T. mentagrophytes 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 ml−1) at 30 °C for 14–21 days. For hygromycin B selection, SDA was prepared with a final concentration of 150 μg hygromycin B ml−1 (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 l−1; KH2PO4, 0·1 g l−1; FeSO4.7H2O, 10 mg l−1; ZnSO4.7H2O, 5 mg l−1) and 0·25 % bovine elastin (Sigma) were used to test the potential virulence of the wild-type and transgenic isolates.
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 μg used as template for PCR and genomic Southern analysis. About 5 μg 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 [α-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 (5′-GAGCTGAAGGGCATCGACTT-3′) and 3075#GFP (5′-CTTGTGCCCCAGGATGTTG-3′) were used in PCR amplification to detect gfp, ment-hph1 (5′-GAGGGCGAAGAATCTCGTGC-3′) and ment-hph2 (5′-CACTGACGGTGTCGTCCATC-3′) for the hph gene, and ment-act1 (5′-CGCCCCAGCCTTCTACGTC-3′) and ment-act2 (5′-CGGTCGGAGATACCTGGGTAC-3′) 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 °C for 19 h; the germlings were transferred to osmoticum containing 5 mg β-d-glucanase ml−1 (InterSpex), 5 mg Driselase ml−1 (InterSpex) and 0·05 mg chitinase ml−1 (Sigma), and incubated for 4 h with shaking at 70 r.p.m. and 30 °C. 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 μl STC. Three aliquots of 60 % PEG solution, 200, 200 and 800 μl, 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 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 °C 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 °C, then incubated for 36 h at 30 °C, and overlaid with 200 μg hygromycin B ml−1 in RSD. Transformants appeared after 14 days' growth at 30 °C, and were transferred to SDA with 150 μg hygromycin ml−1. For REMI, 100 units of BglII were added to the transformation reaction (Lu et al., 1994), and 20 μg of plasmid gGFP digested with BglII was used for the transformation.
Confocal microscopy.
Fungal mycelium grown axenically on skin 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 μm.
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 μg ml−1). 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.
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.
Insertion and expression of GFP in T. mentagrophytes
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 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 wild-type (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⇓).
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 wild-type and transformants was digested with the indicated enzymes and the blot probed with the GFP coding region.
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. 2⇓b) and REMI5 (Fig. 2c⇓) grown in medium were clearly fluorescent, as compared with a wild-type control under the same conditions (Fig. 2a⇓).
Confocal images of wild-type (a) and transformants REMI6 (b) and REMI5 (c), grown for 14 days on SDA with 150 μg hygromycin B ml−1.
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. 3⇓a). The fluorescent hyphae could be clearly seen at a distance from the infection point on skin and at a depth of 9·8 μm 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.
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 μm depth from the previous frame. Bars, 20 μm. 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.
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
The transgenic isolates were repeatedly transferred and then stored on non-selective media, and in contrast to the wild-type they were resistant to hygromycin B and continued to express GFP. No major differences were noticed in the growth rate or the morphology of the colony in comparison to the wild-type on non-selective medium (not shown). The transformants grew well on selective medium (Fig. 4a⇓). The virulence of the transformants was measured by the ability to infect and invade the human skin explants by confocal microscopy (Fig. 3⇑) and to digest keratin (data not shown) and elastin (Fig. 4b⇓).
Growth on selective medium and elastin degradation. (a) Ability of the different strains to grow on selective medium: SDA plus 150 μg 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 genetic stability of the transformants shows that REMI is a useful method to insert genes and to maintain stable transformants and expression of the transgene.
The virulence of the transformants was maintained, although their ability to digest elastin was variable between 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.
Acknowledgments
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.