1 Department of Biology, Technion Israel Institute of Technology, Haifa 32000, Israel
2 Department of Molecular Microbiology, Faculty of Medicine, Technion Israel Institute of Technology, Haifa 32000, Israel
3 Department of Plastic Surgery, Rambam Medical Center, Haifa, Israel
Correspondence
Israela Berdicevsky
Israelab{at}tx.technion.ac.il
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ABSTRACT |
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INTRODUCTION |
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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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METHODS |
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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 [
-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
-D-glucanase ml1 (InterSpex), 5 mg Driselase ml1 (InterSpex) and 0·05 mg chitinase ml1 (Sigma), and incubated for 4 h with shaking at 70 r.p.m. and 30 °C. Then 108109 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 35004000, Sigma), 10 mM Tris pH 7·5, 50 mM CaCl2. The tubes were incubated with PEG solution for 56 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 ml1 in RSD. Transformants appeared after 14 days' growth at 30 °C, and were transferred to SDA with 150 µg hygromycin ml1. 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.
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RESULTS AND DISCUSSION |
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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
).
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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.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 February 2004;
revised 4 May 2004;
accepted 13 May 2004.
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