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Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis
FEMS Immunology and Medical Microbiology 38 (2003) 29^34
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Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis Fre¤de¤ric Descamps a , Fre¤de¤ric Brouta a , Sandy Vermout a , Michel Monod b , Bertrand Losson a , Bernard Mignon a; a
Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Lie'ge, B-43 Sart-Tilman, 4000 Lie'ge, Belgium b Laboratoire de Mycologie, Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Received 18 December 2002; received in revised form 6 March 2003; accepted 9 March 2003 First published online 1 April 2003
Abstract A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat. 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Keratinase ; Subtilisin-like protease ; ELISA; Lymphoproliferation test; Microsporum canis; Pichia pastoris
1. Introduction Feline dermatophytosis is a super¢cial skin infection caused most of the time by the zoophilic dermatophyte Microsporum canis [1]. This fungus represents a major problem in catteries, where the infection can be endemic [2] and is responsible for a frequently reported zoonosis [3]. Therefore, the set-up of a successful immunoprophylaxis against M. canis in cat is recommended by the WHO (World Health Organization) and ISHAM (International Society of Human and Animal Mycology) [4]. A M. canis 31.5-kDa subtilase (SUB3) appears to be an interesting antigen candidate because it can induce a cell-mediated immune response in experimentally infected guinea pigs [5]. SUB3 is also considered as a potential major fungal
* Corresponding author: Tel.: (32)-4-366 40 99; Fax: (32)-4-366 40 97. E-mail address : [email protected] (B. Mignon).
virulence factor. Indeed, it is the major polypeptide secreted by the fungus cultivated in a minimal keratin-enriched medium [6]. Moreover, its keratinolytic activity was demonstrated [6], and its in vivo expression was shown both in naturally infected cats [7] and experimentally infected guinea pigs [5]. More recently, we have demonstrated that SUB3 belongs to a protease family of three homologous subtilases [8], an additional datum suggesting its importance for the fungal biology. Production and puri¢cation of SUB3 from the native micro-organism being extremely time-consuming [6], the use of recombinant technology is indispensable to further study the role of this keratinase in both fungal pathogenicity and host immune response. The methylotrophic yeast Pichia pastoris appears to be a suitable recombinant expression system because of its phylogenetic relationship with ¢lamentous fungi. It presents many of the advantages of higher eukaryotic systems, including post-translational modi¢cations, while being easy to manipulate [9^11]. Here we have investigated P. pastoris as a means of producing secreted recombinant SUB3 (r-SUB3). The antigenic properties of the r-SUB3 were then assessed in a guinea pig experimental infection model.
0928-8244 / 03 / $22.00 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(03)00101-9
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2. Materials and methods 2.1. Experimental infection of guinea pigs and blood sampling Fourteen speci¢c pathogen-free 3-month-old female guinea pigs of the Hartley strain (BpK Universal, Humberside, UK) were cutaneously infected with M. canis as previously described [8]. They were randomly divided into four subgroups of three to four guinea pigs each. Negative controls consisted of two guinea pigs exposed to the same procedure except that the inoculum did not contain any infective material. From day 0 to day 57 post-infection, blood samples were collected at fortnightly intervals by intracardiac puncture performed under general anesthesia. Animal experiments were approved by the local ethic committee (University of Lie'ge). 2.2. Clinical follow-up Animal infection was monitored weekly using clinical and mycological criteria. Clinical criteria were erythema, alopecia, squamosis and crusts. For each clinical criterion, a subjective score from zero to three was attributed by a single examiner throughout the experiment. The presence of £uorescent hair under Wood’s light and the growth of M. canis on Sabouraud dextrose agar (containing cycloheximide and chloramphenicol) represented mycological criteria. The haircoat toothbrushing method [12] was used to inoculate the culture plates. A score of zero or one was attributed to each mycological criterion. For each animal, a global score was calculated by adding clinical and mycological scores. Results were then expressed as a median global score.
errors. The plasmid containing the isolated cDNA was digested with XhoI and NotI, for which sites were previously designed at the 5P extremity of the RT-PCR primers [8], and cloned into the expression vector pPICZKB, generating plasmid pP-cSUB3. Plasmid DNA was prepared using a Qiagen plasmid Midi kit from one E. coli clone harboring a correct construct and digested with DraI before P. pastoris transformation. 2.5. Expression of the M. canis recombinant SUB3 in P. pastoris P. pastoris was transformed by electroporation with 10 Wg of linearized pP-cSUB3 using the following parameters : 1000 V, 329 6 and 40 WF. As a negative control, 10 Wg of pPicZKB linearized by DraI was used. Transformants able to grow on yeast extract peptone dextrose agar containing 100 Wg ml31 zeocin (Invitrogen) were assumed to harbor the construct at the correct yeast genomic location by integration events in the AOX1 locus. According to the supplier’s instructions, 20 transformants were used to inoculate 40 ml of bu¡ered glycerol complex medium (BMGY ; Invitrogen) [0.1 M potassium phosphate bu¡er, pH 6.0, containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 1% (v/v) glycerol and 4U1035 % (w/v) biotin]. Cultures were grown at 30‡C in a shaking incubator (260 rpm) until they reached an absorbance of 4 at 600 nm. Then, cells were harvested by centrifugation (1500Ug for 5 min), resuspended in 8 ml of bu¡ered methanol complex medium [same medium as BMGY except that glycerol was replaced by methanol 0.5% (v/v)] and incubated under the same conditions for 6 additional days. Expression of r-SUB3 was induced by daily adding a volume of methanol to a ¢nal concentration of 0.5% (v/v).
2.3. Strains and plasmids M. canis strain IHEM 15221 (Brussels, Belgium) was used for guinea pig infection. P. pastoris KM71 H (Invitrogen, Carlsbad, CA, USA), plasmid pCR0 4Blunt-TOPO0 (Invitrogen) and the Escherichia coli^P. pastoris shuttle vector pPicZKB (Invitrogen) were used to express recombinant SUB3 (r-SUB3). 2.4. Construction of the expression plasmid The expression plasmid was constructed by cloning a 1149-bp reverse-transcription polymerase chain reaction (RT-PCR) product encoding SUB3 including its prosequence [8] in the multiple cloning site of the plasmid pPicZKB. The isolated cDNA was inserted into the plasmid vector pCR0 4Blunt-TOPO0 as described in the Manual Version E of the Zero Blunt0 TOPO0 PCR cloning kit for sequencing (Invitrogen). The cloned fragment was sequenced using a Perkin-Elmer sequencer (Norwalk, CT, USA) to verify the absence of possible RT-PCR-induced
2.6. Protein extract analysis, proteolytic assays, puri¢cation procedures and N-terminal sequencing In order to identify clones expressing r-SUB3 and to monitor its time-course production, a sample of each culture supernatant was harvested daily, subjected to trichloroacetic precipitation and to sodium dodecyl sulfate^ polyacrylamide gel electrophoresis (SDS^PAGE) under reducing conditions [13]. Keratinolytic and subtilisin-like activities were measured using keratin azure (Sigma, Munich, Germany) and N-succinyl-Ala-Ala-Pro-Phe-pnitroanilide (AAPF-pNa; Sigma), respectively, as previously described by Mignon et al. [6]. Puri¢cation of r-SUB3 was performed as follows : culture supernatant was separated from cells by centrifugation (1500Ug for 10 min), concentrated by ultra¢ltration on an Amicon YM 10 ¢lter (Amicon, Beverly, MA, USA), dialyzed against bicine bu¡er (50 mM bicine, pH 8.2) and applied onto a carboxymethyl-agarose column previously equilibrated in the same bu¡er. The bound protein was eluted
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with a linear gradient of salt (30 ml-30 ml, 1 M NaCl, pH 8.2). All procedures were carried out at 4‡C. N-terminal amino acid sequencing of the puri¢ed protein was performed by Edman degradation using a Perkin-Elmer sequencer type Procise. Protein concentrations were determined by the method of Bradford [14]. Hexose content was determined with the Gel Code Glycoprotein Staining kit (Pierce). 2.7. Enzyme-linked immunosorbent assay (ELISA) Antigen, reference antisera and donkey anti-guinea pig Igs (Chemicon, Temecula, CA, USA) were appropriately diluted after standard checkerboard titration (data not shown). All washes were repeated four times. Polystyrene microtiter plates (Maxisorp F16, Nunc, Roskilde, Denmark) were coated overnight at room temperature with 100 Wl per well of 20 Wg ml31 r-SUB3 solution in 0.01 M phosphate-bu¡ered saline (PBS), pH 7.4. Odd rows were sensitized with the antigen while even ones remained free of antigen as control wells. After washing with PBS, plates were satured for 1 h at 37‡C with saturation bu¡er (PBS containing 1% (w/v) bovine serum albumin (Sigma)) and then washed with PBS containing 0.05% Tween 20 (PBS-T). Serum samples were diluted 1:100 in dilution bu¡er (saturation bu¡er containing 0.05% Tween 20 (v/ v)). 100 Wl serum samples were added for 1 h at 37‡C to two antigen-coated and two control wells. After washing with PBS-T, 100 Wl of biotin-conjugated donkey anti-guinea pig Igs diluted 1:20 000 in dilution bu¡er was added for 1 h at 37‡C. The plates were then washed with PBS-T and 100 Wl of a streptavidine-peroxidase solution (Amersham Pharmacia Biotech, Uppsala, Sweden) diluted 1:1000 in dilution bu¡er was added for 30 min at room temperature. After washing with PBS-T, peroxidase activity was revealed by the addition of 100 Wl of a solution containing tetramethylbenzidin and hydrogen peroxide (Coris BioConcept, Namur, Belgium). The reaction was stopped after 20 min at room temperature with 50 Wl of 1 M sulfuric acid, and the absorbance at 450 nm was immediately measured with a spectrophotometer (Multiskan RC, Thermolabsystems, Helsinki, Finland). On each plate, positive and negative reference antisera were also included in duplicates. The positive reference antiserum was collected by Mignon et al. [5] from a guinea pig immunized with native SUB3. The negative reference antiserum was a pool of all the sera obtained from the guinea pigs before infection. The test was performed three times. For each serum tested, the optical density (OD) was de¢ned as the mean of the absorbance di¡erences between the antigen-sensitized wells and the control ones. Results were then expressed as OD percentages obtained as follows: OD percentage = (ODx 3ODn )/(ODp 3ODn ), where ODx is the OD obtained with the tested serum, ODp and ODn are the ODs obtained on the same plate with the positive and the negative reference antisera, respec-
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tively. Statistical comparison between pre- and post-infection antibody levels was performed with a Wilcoxon Signed Rank Test. A P-value 9 0.05 was considered signi¢cant. 2.8. Lymphocyte blastogenesis test The recombinant SUB3 was evaluated in vitro for its ability to induce T-cell proliferation with peripheral blood mononuclear cells obtained from control and infected guinea pigs. Brie£y, 2 ml of heparinized blood from each guinea pig was diluted 1:2 in PBS, pipetted over 3 ml of Ficoll^Paque solution (Amersham) in a 15-ml conical tube and centrifuged for 30 min at 400Ug. The peripheral blood mononuclear cell ring was harvested and washed twice with PBS for 10 min at 400Ug. Cells obtained from blood of guinea pigs belonging to the same subgroup were mixed together and diluted at a ¢nal concentration of 1U106 ml31 in RPMI-1640 medium (Bio Media, Boussens, Belgium) containing 2% horse serum (Gibco BRL, Paisley, UK), 1% non-essential amino acids, 1% sodium pyruvate, 10 IU ml31 penicillin, 0.1 mg ml31 streptomycin and 50 WM 2-mercaptoethanol. Aliquots of the cell suspension (250 Wl) were incubated in 96-well sterile tissueculture plates (Corning, NY, USA) containing in quadruplicates either PBS (negative control wells), 1 Wg concanavalin A (positive control wells) or 400 ng heat-inactivated r-SUB3 (test wells). Plates were cultured for 3 days at 37‡C in an atmosphere containing 5% CO2 and pulsed with 1 WCi of [3 H]thymidine (Amersham) for 24 h. Cells were harvested onto glass-¢ber ¢lters (Skatron, Sterling, VA, USA) using a Skatron cell harvester. The dried ¢lter discs were transferred to vials containing 4 ml of scintillation £uid (Ecoscint A, National Diagnostics, Hessle Hull, UK), and incorporation of [3 H]thymidine was quanti¢ed for 3 min with a Beckman LS 5000 CE scintillation counter (Beckman Instruments, Fullerton, CA, USA). The results of the LBTs were expressed as a stimulation index (SI), calculated as the ratio of the mean count per minute (cpm) in the test wells divided by the mean cpm in the negative control wells. Statistical comparison of the postinfection lymphoproliferative response with the pre-infection response was performed with a paired t-test. A P-value 9 0.05 was considered signi¢cant.
3. Results and discussion 3.1. Production of r-SUB3 A cDNA fragment encoding SUB3 with its propeptide was cloned into the pPICZK expression cassette downstream the sequence coding for the K-factor signal peptide of Saccharomyces cerevisiae. The propeptide of many secreted proteinases has been found to be essential and speci¢c for the correct folding as well as the secretion of the
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10 8
9766-
6 Score
4531-
4 2 0
210
Fig. 1. Expression of M. canis IHEM 15221 r-SUB3 by P. pastoris. Puri¢ed fraction eluted after cation-exchange chromatography. Sample was loaded on a 12% SDS polyacrylamide gel, which was stained with Coomassie brilliant blue R-250. Molecular mass standards (in kDa) are shown on the left.
mature domain of the enzyme ([15,16] ; for review, see [17]). SDS^PAGE and enzymatic activity towards AAPF-pNa revealed that all tested zeocin-resistant P. pastoris clones were expressing r-SUB3 as the major protein in the culture supernatant (data not shown). P. pastoris transformed only with the parent vector showed neither AAPF-pNa-hydrolyzing activity nor 31.5 kDa proteinase secretion (data not shown). A yield of approximately 150 Wg protein per ml was obtained with all the expressing clones, suggesting that none of them had integrated several copies of the SUB3 cDNA. One colony was then arbitrarily selected for mid-scale production of the r-SUB3, which was puri¢ed to homogeneity by a single step of cation-exchange chromatography (Fig. 1) thanks to an unusually high isoelectric point [8]. This step allowed a 49% recovery rate of the protease. The N-terminal amino acid sequence of r-SUB3 was determined to be ALTTQ, like that of the native protease [6]. Like SUB3 isolated from M. canis culture supernatant and despite the presence in its nucleotide sequence of three putative N-glycosylation sites [8], r-SUB3 was not glycosylated. No di¡erence in speci¢c activity was found between the two proteins (data not shown). 3.2. Antibody response to r-SUB3 during cutaneous infection Fourteen guinea pigs were experimentally infected. Typical dermatophytic lesions appeared on the scari¢ed area of all the infected guinea pigs around 7 days after infection. Median global scores relative to the extension of the lesions during the experiment are given in Fig. 2. The maximum score was observed around day 21 post-infection. Afterwards lesions disappeared progressively. All the guinea pigs were clinically cured 2 months post-infection. No lesions developed in control animals (data not shown). Using the ELISA, no signi¢cant increase of anti-r-SUB3
20 30 40 50 60 70 Day post infection Fig. 2. Kinetics of median global scores assessing the extension of dermatophytic lesions in M. canis experimentally infected guinea pigs (see Section 2 for details). Vertical bars indicate values ranging from 25th percentile to 75th percentile.
10
Igs was detected in guinea pigs after cutaneous infection (Fig. 3). However, considerable variation was observed between the animals, and individual analysis of the data indicated that one out of 14 guinea pigs signi¢cantly developed r-SUB3 Igs throughout the infection period. No speci¢c antibodies were detected in non-infected control animals. These results are in agreement with those previously observed using native SUB3 as antigen for speci¢c antibody detection in guinea pigs and cats [5,18]. It con¢rms that SUB3 is not responsible for the antibody response raised against the M. canis exo-antigen [5,18], the crude fungal supernatant from which native SUB3 was previously puri¢ed [6], and that other proteins to be identi¢ed induce speci¢c antibody responses during infection. The role of this humoral immune response, as it is observed for antibody responses to other dermatophyte antigens [2,19^21], remains largely misunderstood. Generally, no apparent correlation is observed between the level of circulating antibodies and susceptibility to dermatophyte infection [19] or recovery from disease [20,21].
Optical density (%)
14-
150
100
50
15 29 43 57 Day post infection Fig. 3. Kinetics of the median antibody response (optical density %) to recombinant SUB3 in M. canis experimentally infected guinea pigs tested in ELISA. Vertical bars indicate values ranging from 25th percentile to 75th percentile.
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3.3. Cell-mediated immune response to r-SUB3 A strong and statistically signi¢cant (P = 0.037) lymphoproliferative response to r-SUB3 was recorded on day 43 post-infection, while a moderate, not signi¢cant, increase was noticed from day 0 to day 29 post-infection (Table 1). This is in agreement with the existence of positive delayedtype hypersensitivity (DTH) reactions to native SUB3 previously demonstrated in vivo in guinea pigs infected with M. canis [5]. In the control guinea pigs, the lymphocyte responses remained at a basal level throughout the experiment (data not shown). Other authors working on other dermatophytes and using crude, non-puri¢ed antigens, observed in experimentally infected guinea pigs [22] and mice [23] a suppression of lymphocyte reactivity to T- and Bcell mitogens when cutaneous lesions were maximal. They attributed this suppression to induction of T-suppressor cell activity [23] and/or a host or fungal serum suppressor factor [22]. In this study, there was no evidence of nonspeci¢c suppression of responses to the mitogen ConA (Table 1), although the possibility of a serum-suppressive factor could not be excluded, as horse serum rather than contemporary autologous serum was used. Unlike the humoral response, the cell-mediated immune response is considered to be essential for the resolution of clinical dermatophytosis and development of acquired resistance [24,25]. Indeed, the development of cutaneous DTH reactions toward dermatophyte antigens was associated with resolution of lesions and resistance to reinfection, whereas lack of DTH was associated with susceptibility to infection or chronic infection [26^28]. Moreover, adoptive immunity to experimentally induced dermatophytosis in mice could be achieved by the transfer of Thelper cells from infected to susceptible mice [29]. Sparkes et al. [21] assessed the kinetics of immune responses to soluble crude M. canis antigens in experimentally infected cats and observed a clear temporal relationship between the development of substantially elevated lymphocyte proliferative responses and the onset of disease regression. In our study, the increase of anti-SUB3 cell-mediated imTable 1 Lymphocyte proliferative responses to M. canis IHEM 15221 r-SUB3 and ConA in experimentally infected guinea pigs Days post-infection
0 15 29 43 57 a
Lymphoproliferative response (SI)a r-SUB3
ConA
1.001 X 0.287 2.064 X 1.844 2.064 X 1.845 9.267 X 6.661b 5.060 X 3.829
70.500 X 35.761 64.931 X 32.906 137.080 X 74.170 108.062 X 21.735 107.918 X 58.466
Values are means X standard deviations for SI calculated as the ratio of the cpm in test wells to the negative control wells. Four SI values were obtained for each day, corresponding to the SI means calculated for each infected guinea pig subgroup. b Signi¢cantly di¡erent (P 6 0.05) from value obtained on day 0 (prior to infection) as calculated by paired t-test.
33
mune response on day 15 post-infection preceded the onset of disease regression. However, a maximal response was observed on day 43 post-infection, when lesions were almost totally cleared. In this context, the role of the speci¢c cellular immune response to SUB3 needs to be further investigated. Interestingly, Woodfolk et al. [30] demonstrated that Trichophyton rubrum Tri r 2, a SUB3 homologous protease [8], elicits either immediate hypersensitivity or DTH cutaneous reactions according to the individuals. They suggested that hyporesponsiveness to speci¢c antigenic determinants (particularly those of Tri r 2) could contribute to the persistence of T. rubrum infection in humans [31]. In this context, it would be interesting to evaluate the cellular immune response to SUB3 in dermatophytic cats, especially in chronically infected ones, which constitute a major zoonotic hazard [32]. Moreover, the eventual protective role of the anti-SUB3 cell-mediated immune response should be investigated in vaccination trials. The recombinant expression of SUB3 provides a valuable tool for addressing these issues.
Acknowledgements We thank Jacques Detry and Humbert Gianfreda for excellent technical assistance. This work was supported by Grant 3.4534.01 from Fonds de la Recherche Scienti¢que Me¤dicale (FRSM). F.D. and F.B. are recipients of a studentship of FRIA (Fonds pour la Formation a' la Recherche dans l’Industrie et dans l’Agriculture, rue d’Egmont 5, 1000 Brussels, Belgium).
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[8] Descamps, F., Brouta, F., Monod, M., Zaugg, C., Baar, D., Losson, B. and Mignon, B. (2002) Isolation of a Microsporum canis gene family encoding three subtilisin-like proteases which are expressed in vivo during hair infection. J. Invest. Dermatol. 119, 830^835. [9] Cregg, J.M., Vedvick, T.S. and Raschke, W.C. (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology 11, 905^910. [10] Rosenfeld, S.A. (1999) Use of Pichia pastoris for the expression of recombinant proteins. Methods Enzymol. 306, 154^169. [11] Cereginho, J.L. and Cregg, J.M. (2000) Heterologous expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45^66. [12] Mac Enzie, D.W.R. (1961) The extra-human occurrence of Trichophyton tonsurans var. sulfureum in a residential school. Sabouraudia 1, 58^64. [13] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [14] Bradford, M.M. (1976) A rapid and sensitive method for the quanti¢cation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. [15] Beggah, S., Le¤chenne, B., Reichard, U., Foundling, S. and Monod, M. (2000) Intra- and intermolecular events direct the propeptidemediated maturation of the Candida albicans secreted aspartic proteinase Sap1p. Microbiology 146, 2765^2773. [16] Ikemura, H. and Inouye, M. (1988) In vitro processing of pro-subtilisin produced in Escherichia coli. J. Biol. Chem. 263, 12959^12963. [17] Eder, J. and Fersht, A.R. (1995) Pro-sequence-assisted protein folding. Mol. Microbiol. 16, 609^614. [18] Mignon, B.R., Coignoul, F., Leclipteux, T., Focant, C. and Losson, B.J. (1999) Histopathological pattern and humoral immune response to a crude exo-antigen and puri¢ed keratinase of Microsporum canis in symptomatic and asymptomatic infected cats. Med. Mycol. 37, 1^9. [19] Cox, W.A. and Moore, J.A. (1968) Experimental Trichophyton verrucosum infections in laboratory animals. J. Comp. Pathol. 78, 35^41. [20] Dahl, M.V. (1987) Immunological resistance to dermatophyte infections. Adv. Dermatol. 2, 305^320.
[21] Sparkes, A.H., Stokes, C.R. and Gru¡ydd-Jones, T.J. (1995) Experimental Microsporum canis infection in cats: correlation between immunological and clinical observations. J. Med. Vet. Mycol. 33, 177^ 184. [22] Green, F. and Balish, E. (1979) Suppression of in vitro lymphocyte transformation during an experimental dermatophyte infection. Infect. Immun. 26, 554^562. [23] Calderon, R.A. and Hay, R.J. (1984) Cell-mediated immunity in experimental murine dermatophytosis I. Temporal aspects of T-suppressor activity caused by Trichophyton quinckeanum. Immunology 53, 457^464. [24] Sohnle, P.G. (1989) Dermatophytosis. In: Immunology of the Fungal Diseases (Cox, R.A., Ed.), pp. 1^27. CRC Press, Boca Raton, FL. [25] Svejgaard, E. (1985) Immunologic investigations of dermatophytes and dermatophytosis. Semin. Dermatol. 4, 201^221. [26] Jones, H.E., Reinhardt, J.H. and Rinaldi, M.G. (1973) A clinical, mycological, and immunological survey for dermatophytosis. Arch. Dermatol. 108, 61^65. [27] Jones, H.E., Reinhardt, J.H. and Rinaldi, M.G. (1974) Acquired immunity to dermatophytes. Arch. Dermatol. 109, 840^848. [28] Jones, H.E., Reinhardt, J.H. and Rinaldi, M.G. (1974) Model dermatophytosis in naturally infected subjects. Arch. Dermatol. 110, 369^374. [29] Calderon, R.A. and Hay, R.J. (1984) Cell-mediated immunity in experimental murine dermatophytosis II. Adoptive transfer of immunity to dermatophyte infection by lymphoid cells from donors with acute or chronic infections. Immunology 53, 465^472. [30] Woodfolk, J.A., Wheatley, L.M., Piyasena, R.V., Benjamin, D.C. and Platts-Mills, T.A.E. (1998) Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. J. Biol. Chem. 273, 29489^29496. [31] Woodfolk, J.A., Sung, S.S.J., Benjamin, D.C., Lee, J.K. and PlattsMills, T.A.E. (2000) Distinct human T cell repertoires mediate immediate and delayed-type hypersensitivity to the Trichophyton antigen, Tri r 2. J. Immunol. 165, 4379^4387. [32] Mignon, B. and Losson, B. (1997) Prevalence and characterization of Microsporum canis carriage in cats. J. Med. Vet. Mycol. 35, 249^256.
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Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic subtilisin-like serine protease from Microsporum canis Fre¤de¤ric Descamps a , Fre¤de¤ric Brouta a , Sandy Vermout a , Michel Monod b , Bertrand Losson a , Bernard Mignon a; a
Department of Parasitology and Parasitic Diseases, Faculty of Veterinary Medicine, University of Lie'ge, B-43 Sart-Tilman, 4000 Lie'ge, Belgium b Laboratoire de Mycologie, Service de Dermatologie, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland Received 18 December 2002; received in revised form 6 March 2003; accepted 9 March 2003 First published online 1 April 2003
Abstract A secreted 31.5-kDa keratinolytic subtilase (SUB3; AJ431180) is thought to be a Microsporum canis virulence factor and represents a candidate for vaccination trials. In this study, the recombinant keratinase (r-SUB3) was produced by the Pichia pastoris expression system and purified to homogeneity. Recombinant SUB3 displayed identical biochemical properties with the native protease. Experimentally cutaneously infected guinea pigs showed specific lymphoproliferative response towards r-SUB3, while no specific humoral immune response was induced except for one animal. The heterologous expression of SUB3 provides a valuable tool for addressing further investigations on the role of this keratinase in the specific cellular immune response and on its use in vaccination trials in the cat. 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Keratinase ; Subtilisin-like protease ; ELISA; Lymphoproliferation test; Microsporum canis; Pichia pastoris
1. Introduction Feline dermatophytosis is a super¢cial skin infection caused most of the time by the zoophilic dermatophyte Microsporum canis [1]. This fungus represents a major problem in catteries, where the infection can be endemic [2] and is responsible for a frequently reported zoonosis [3]. Therefore, the set-up of a successful immunoprophylaxis against M. canis in cat is recommended by the WHO (World Health Organization) and ISHAM (International Society of Human and Animal Mycology) [4]. A M. canis 31.5-kDa subtilase (SUB3) appears to be an interesting antigen candidate because it can induce a cell-mediated immune response in experimentally infected guinea pigs [5]. SUB3 is also considered as a potential major fungal
* Corresponding author: Tel.: (32)-4-366 40 99; Fax: (32)-4-366 40 97. E-mail address : [email protected] (B. Mignon).
virulence factor. Indeed, it is the major polypeptide secreted by the fungus cultivated in a minimal keratin-enriched medium [6]. Moreover, its keratinolytic activity was demonstrated [6], and its in vivo expression was shown both in naturally infected cats [7] and experimentally infected guinea pigs [5]. More recently, we have demonstrated that SUB3 belongs to a protease family of three homologous subtilases [8], an additional datum suggesting its importance for the fungal biology. Production and puri¢cation of SUB3 from the native micro-organism being extremely time-consuming [6], the use of recombinant technology is indispensable to further study the role of this keratinase in both fungal pathogenicity and host immune response. The methylotrophic yeast Pichia pastoris appears to be a suitable recombinant expression system because of its phylogenetic relationship with ¢lamentous fungi. It presents many of the advantages of higher eukaryotic systems, including post-translational modi¢cations, while being easy to manipulate [9^11]. Here we have investigated P. pastoris as a means of producing secreted recombinant SUB3 (r-SUB3). The antigenic properties of the r-SUB3 were then assessed in a guinea pig experimental infection model.
0928-8244 / 03 / $22.00 6 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0928-8244(03)00101-9
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2. Materials and methods 2.1. Experimental infection of guinea pigs and blood sampling Fourteen speci¢c pathogen-free 3-month-old female guinea pigs of the Hartley strain (BpK Universal, Humberside, UK) were cutaneously infected with M. canis as previously described [8]. They were randomly divided into four subgroups of three to four guinea pigs each. Negative controls consisted of two guinea pigs exposed to the same procedure except that the inoculum did not contain any infective material. From day 0 to day 57 post-infection, blood samples were collected at fortnightly intervals by intracardiac puncture performed under general anesthesia. Animal experiments were approved by the local ethic committee (University of Lie'ge). 2.2. Clinical follow-up Animal infection was monitored weekly using clinical and mycological criteria. Clinical criteria were erythema, alopecia, squamosis and crusts. For each clinical criterion, a subjective score from zero to three was attributed by a single examiner throughout the experiment. The presence of £uorescent hair under Wood’s light and the growth of M. canis on Sabouraud dextrose agar (containing cycloheximide and chloramphenicol) represented mycological criteria. The haircoat toothbrushing method [12] was used to inoculate the culture plates. A score of zero or one was attributed to each mycological criterion. For each animal, a global score was calculated by adding clinical and mycological scores. Results were then expressed as a median global score.
errors. The plasmid containing the isolated cDNA was digested with XhoI and NotI, for which sites were previously designed at the 5P extremity of the RT-PCR primers [8], and cloned into the expression vector pPICZKB, generating plasmid pP-cSUB3. Plasmid DNA was prepared using a Qiagen plasmid Midi kit from one E. coli clone harboring a correct construct and digested with DraI before P. pastoris transformation. 2.5. Expression of the M. canis recombinant SUB3 in P. pastoris P. pastoris was transformed by electroporation with 10 Wg of linearized pP-cSUB3 using the following parameters : 1000 V, 329 6 and 40 WF. As a negative control, 10 Wg of pPicZKB linearized by DraI was used. Transformants able to grow on yeast extract peptone dextrose agar containing 100 Wg ml31 zeocin (Invitrogen) were assumed to harbor the construct at the correct yeast genomic location by integration events in the AOX1 locus. According to the supplier’s instructions, 20 transformants were used to inoculate 40 ml of bu¡ered glycerol complex medium (BMGY ; Invitrogen) [0.1 M potassium phosphate bu¡er, pH 6.0, containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 1% (v/v) glycerol and 4U1035 % (w/v) biotin]. Cultures were grown at 30‡C in a shaking incubator (260 rpm) until they reached an absorbance of 4 at 600 nm. Then, cells were harvested by centrifugation (1500Ug for 5 min), resuspended in 8 ml of bu¡ered methanol complex medium [same medium as BMGY except that glycerol was replaced by methanol 0.5% (v/v)] and incubated under the same conditions for 6 additional days. Expression of r-SUB3 was induced by daily adding a volume of methanol to a ¢nal concentration of 0.5% (v/v).
2.3. Strains and plasmids M. canis strain IHEM 15221 (Brussels, Belgium) was used for guinea pig infection. P. pastoris KM71 H (Invitrogen, Carlsbad, CA, USA), plasmid pCR0 4Blunt-TOPO0 (Invitrogen) and the Escherichia coli^P. pastoris shuttle vector pPicZKB (Invitrogen) were used to express recombinant SUB3 (r-SUB3). 2.4. Construction of the expression plasmid The expression plasmid was constructed by cloning a 1149-bp reverse-transcription polymerase chain reaction (RT-PCR) product encoding SUB3 including its prosequence [8] in the multiple cloning site of the plasmid pPicZKB. The isolated cDNA was inserted into the plasmid vector pCR0 4Blunt-TOPO0 as described in the Manual Version E of the Zero Blunt0 TOPO0 PCR cloning kit for sequencing (Invitrogen). The cloned fragment was sequenced using a Perkin-Elmer sequencer (Norwalk, CT, USA) to verify the absence of possible RT-PCR-induced
2.6. Protein extract analysis, proteolytic assays, puri¢cation procedures and N-terminal sequencing In order to identify clones expressing r-SUB3 and to monitor its time-course production, a sample of each culture supernatant was harvested daily, subjected to trichloroacetic precipitation and to sodium dodecyl sulfate^ polyacrylamide gel electrophoresis (SDS^PAGE) under reducing conditions [13]. Keratinolytic and subtilisin-like activities were measured using keratin azure (Sigma, Munich, Germany) and N-succinyl-Ala-Ala-Pro-Phe-pnitroanilide (AAPF-pNa; Sigma), respectively, as previously described by Mignon et al. [6]. Puri¢cation of r-SUB3 was performed as follows : culture supernatant was separated from cells by centrifugation (1500Ug for 10 min), concentrated by ultra¢ltration on an Amicon YM 10 ¢lter (Amicon, Beverly, MA, USA), dialyzed against bicine bu¡er (50 mM bicine, pH 8.2) and applied onto a carboxymethyl-agarose column previously equilibrated in the same bu¡er. The bound protein was eluted
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with a linear gradient of salt (30 ml-30 ml, 1 M NaCl, pH 8.2). All procedures were carried out at 4‡C. N-terminal amino acid sequencing of the puri¢ed protein was performed by Edman degradation using a Perkin-Elmer sequencer type Procise. Protein concentrations were determined by the method of Bradford [14]. Hexose content was determined with the Gel Code Glycoprotein Staining kit (Pierce). 2.7. Enzyme-linked immunosorbent assay (ELISA) Antigen, reference antisera and donkey anti-guinea pig Igs (Chemicon, Temecula, CA, USA) were appropriately diluted after standard checkerboard titration (data not shown). All washes were repeated four times. Polystyrene microtiter plates (Maxisorp F16, Nunc, Roskilde, Denmark) were coated overnight at room temperature with 100 Wl per well of 20 Wg ml31 r-SUB3 solution in 0.01 M phosphate-bu¡ered saline (PBS), pH 7.4. Odd rows were sensitized with the antigen while even ones remained free of antigen as control wells. After washing with PBS, plates were satured for 1 h at 37‡C with saturation bu¡er (PBS containing 1% (w/v) bovine serum albumin (Sigma)) and then washed with PBS containing 0.05% Tween 20 (PBS-T). Serum samples were diluted 1:100 in dilution bu¡er (saturation bu¡er containing 0.05% Tween 20 (v/ v)). 100 Wl serum samples were added for 1 h at 37‡C to two antigen-coated and two control wells. After washing with PBS-T, 100 Wl of biotin-conjugated donkey anti-guinea pig Igs diluted 1:20 000 in dilution bu¡er was added for 1 h at 37‡C. The plates were then washed with PBS-T and 100 Wl of a streptavidine-peroxidase solution (Amersham Pharmacia Biotech, Uppsala, Sweden) diluted 1:1000 in dilution bu¡er was added for 30 min at room temperature. After washing with PBS-T, peroxidase activity was revealed by the addition of 100 Wl of a solution containing tetramethylbenzidin and hydrogen peroxide (Coris BioConcept, Namur, Belgium). The reaction was stopped after 20 min at room temperature with 50 Wl of 1 M sulfuric acid, and the absorbance at 450 nm was immediately measured with a spectrophotometer (Multiskan RC, Thermolabsystems, Helsinki, Finland). On each plate, positive and negative reference antisera were also included in duplicates. The positive reference antiserum was collected by Mignon et al. [5] from a guinea pig immunized with native SUB3. The negative reference antiserum was a pool of all the sera obtained from the guinea pigs before infection. The test was performed three times. For each serum tested, the optical density (OD) was de¢ned as the mean of the absorbance di¡erences between the antigen-sensitized wells and the control ones. Results were then expressed as OD percentages obtained as follows: OD percentage = (ODx 3ODn )/(ODp 3ODn ), where ODx is the OD obtained with the tested serum, ODp and ODn are the ODs obtained on the same plate with the positive and the negative reference antisera, respec-
31
tively. Statistical comparison between pre- and post-infection antibody levels was performed with a Wilcoxon Signed Rank Test. A P-value 9 0.05 was considered signi¢cant. 2.8. Lymphocyte blastogenesis test The recombinant SUB3 was evaluated in vitro for its ability to induce T-cell proliferation with peripheral blood mononuclear cells obtained from control and infected guinea pigs. Brie£y, 2 ml of heparinized blood from each guinea pig was diluted 1:2 in PBS, pipetted over 3 ml of Ficoll^Paque solution (Amersham) in a 15-ml conical tube and centrifuged for 30 min at 400Ug. The peripheral blood mononuclear cell ring was harvested and washed twice with PBS for 10 min at 400Ug. Cells obtained from blood of guinea pigs belonging to the same subgroup were mixed together and diluted at a ¢nal concentration of 1U106 ml31 in RPMI-1640 medium (Bio Media, Boussens, Belgium) containing 2% horse serum (Gibco BRL, Paisley, UK), 1% non-essential amino acids, 1% sodium pyruvate, 10 IU ml31 penicillin, 0.1 mg ml31 streptomycin and 50 WM 2-mercaptoethanol. Aliquots of the cell suspension (250 Wl) were incubated in 96-well sterile tissueculture plates (Corning, NY, USA) containing in quadruplicates either PBS (negative control wells), 1 Wg concanavalin A (positive control wells) or 400 ng heat-inactivated r-SUB3 (test wells). Plates were cultured for 3 days at 37‡C in an atmosphere containing 5% CO2 and pulsed with 1 WCi of [3 H]thymidine (Amersham) for 24 h. Cells were harvested onto glass-¢ber ¢lters (Skatron, Sterling, VA, USA) using a Skatron cell harvester. The dried ¢lter discs were transferred to vials containing 4 ml of scintillation £uid (Ecoscint A, National Diagnostics, Hessle Hull, UK), and incorporation of [3 H]thymidine was quanti¢ed for 3 min with a Beckman LS 5000 CE scintillation counter (Beckman Instruments, Fullerton, CA, USA). The results of the LBTs were expressed as a stimulation index (SI), calculated as the ratio of the mean count per minute (cpm) in the test wells divided by the mean cpm in the negative control wells. Statistical comparison of the postinfection lymphoproliferative response with the pre-infection response was performed with a paired t-test. A P-value 9 0.05 was considered signi¢cant.
3. Results and discussion 3.1. Production of r-SUB3 A cDNA fragment encoding SUB3 with its propeptide was cloned into the pPICZK expression cassette downstream the sequence coding for the K-factor signal peptide of Saccharomyces cerevisiae. The propeptide of many secreted proteinases has been found to be essential and speci¢c for the correct folding as well as the secretion of the
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10 8
9766-
6 Score
4531-
4 2 0
210
Fig. 1. Expression of M. canis IHEM 15221 r-SUB3 by P. pastoris. Puri¢ed fraction eluted after cation-exchange chromatography. Sample was loaded on a 12% SDS polyacrylamide gel, which was stained with Coomassie brilliant blue R-250. Molecular mass standards (in kDa) are shown on the left.
mature domain of the enzyme ([15,16] ; for review, see [17]). SDS^PAGE and enzymatic activity towards AAPF-pNa revealed that all tested zeocin-resistant P. pastoris clones were expressing r-SUB3 as the major protein in the culture supernatant (data not shown). P. pastoris transformed only with the parent vector showed neither AAPF-pNa-hydrolyzing activity nor 31.5 kDa proteinase secretion (data not shown). A yield of approximately 150 Wg protein per ml was obtained with all the expressing clones, suggesting that none of them had integrated several copies of the SUB3 cDNA. One colony was then arbitrarily selected for mid-scale production of the r-SUB3, which was puri¢ed to homogeneity by a single step of cation-exchange chromatography (Fig. 1) thanks to an unusually high isoelectric point [8]. This step allowed a 49% recovery rate of the protease. The N-terminal amino acid sequence of r-SUB3 was determined to be ALTTQ, like that of the native protease [6]. Like SUB3 isolated from M. canis culture supernatant and despite the presence in its nucleotide sequence of three putative N-glycosylation sites [8], r-SUB3 was not glycosylated. No di¡erence in speci¢c activity was found between the two proteins (data not shown). 3.2. Antibody response to r-SUB3 during cutaneous infection Fourteen guinea pigs were experimentally infected. Typical dermatophytic lesions appeared on the scari¢ed area of all the infected guinea pigs around 7 days after infection. Median global scores relative to the extension of the lesions during the experiment are given in Fig. 2. The maximum score was observed around day 21 post-infection. Afterwards lesions disappeared progressively. All the guinea pigs were clinically cured 2 months post-infection. No lesions developed in control animals (data not shown). Using the ELISA, no signi¢cant increase of anti-r-SUB3
20 30 40 50 60 70 Day post infection Fig. 2. Kinetics of median global scores assessing the extension of dermatophytic lesions in M. canis experimentally infected guinea pigs (see Section 2 for details). Vertical bars indicate values ranging from 25th percentile to 75th percentile.
10
Igs was detected in guinea pigs after cutaneous infection (Fig. 3). However, considerable variation was observed between the animals, and individual analysis of the data indicated that one out of 14 guinea pigs signi¢cantly developed r-SUB3 Igs throughout the infection period. No speci¢c antibodies were detected in non-infected control animals. These results are in agreement with those previously observed using native SUB3 as antigen for speci¢c antibody detection in guinea pigs and cats [5,18]. It con¢rms that SUB3 is not responsible for the antibody response raised against the M. canis exo-antigen [5,18], the crude fungal supernatant from which native SUB3 was previously puri¢ed [6], and that other proteins to be identi¢ed induce speci¢c antibody responses during infection. The role of this humoral immune response, as it is observed for antibody responses to other dermatophyte antigens [2,19^21], remains largely misunderstood. Generally, no apparent correlation is observed between the level of circulating antibodies and susceptibility to dermatophyte infection [19] or recovery from disease [20,21].
Optical density (%)
14-
150
100
50
15 29 43 57 Day post infection Fig. 3. Kinetics of the median antibody response (optical density %) to recombinant SUB3 in M. canis experimentally infected guinea pigs tested in ELISA. Vertical bars indicate values ranging from 25th percentile to 75th percentile.
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3.3. Cell-mediated immune response to r-SUB3 A strong and statistically signi¢cant (P = 0.037) lymphoproliferative response to r-SUB3 was recorded on day 43 post-infection, while a moderate, not signi¢cant, increase was noticed from day 0 to day 29 post-infection (Table 1). This is in agreement with the existence of positive delayedtype hypersensitivity (DTH) reactions to native SUB3 previously demonstrated in vivo in guinea pigs infected with M. canis [5]. In the control guinea pigs, the lymphocyte responses remained at a basal level throughout the experiment (data not shown). Other authors working on other dermatophytes and using crude, non-puri¢ed antigens, observed in experimentally infected guinea pigs [22] and mice [23] a suppression of lymphocyte reactivity to T- and Bcell mitogens when cutaneous lesions were maximal. They attributed this suppression to induction of T-suppressor cell activity [23] and/or a host or fungal serum suppressor factor [22]. In this study, there was no evidence of nonspeci¢c suppression of responses to the mitogen ConA (Table 1), although the possibility of a serum-suppressive factor could not be excluded, as horse serum rather than contemporary autologous serum was used. Unlike the humoral response, the cell-mediated immune response is considered to be essential for the resolution of clinical dermatophytosis and development of acquired resistance [24,25]. Indeed, the development of cutaneous DTH reactions toward dermatophyte antigens was associated with resolution of lesions and resistance to reinfection, whereas lack of DTH was associated with susceptibility to infection or chronic infection [26^28]. Moreover, adoptive immunity to experimentally induced dermatophytosis in mice could be achieved by the transfer of Thelper cells from infected to susceptible mice [29]. Sparkes et al. [21] assessed the kinetics of immune responses to soluble crude M. canis antigens in experimentally infected cats and observed a clear temporal relationship between the development of substantially elevated lymphocyte proliferative responses and the onset of disease regression. In our study, the increase of anti-SUB3 cell-mediated imTable 1 Lymphocyte proliferative responses to M. canis IHEM 15221 r-SUB3 and ConA in experimentally infected guinea pigs Days post-infection
0 15 29 43 57 a
Lymphoproliferative response (SI)a r-SUB3
ConA
1.001 X 0.287 2.064 X 1.844 2.064 X 1.845 9.267 X 6.661b 5.060 X 3.829
70.500 X 35.761 64.931 X 32.906 137.080 X 74.170 108.062 X 21.735 107.918 X 58.466
Values are means X standard deviations for SI calculated as the ratio of the cpm in test wells to the negative control wells. Four SI values were obtained for each day, corresponding to the SI means calculated for each infected guinea pig subgroup. b Signi¢cantly di¡erent (P 6 0.05) from value obtained on day 0 (prior to infection) as calculated by paired t-test.
33
mune response on day 15 post-infection preceded the onset of disease regression. However, a maximal response was observed on day 43 post-infection, when lesions were almost totally cleared. In this context, the role of the speci¢c cellular immune response to SUB3 needs to be further investigated. Interestingly, Woodfolk et al. [30] demonstrated that Trichophyton rubrum Tri r 2, a SUB3 homologous protease [8], elicits either immediate hypersensitivity or DTH cutaneous reactions according to the individuals. They suggested that hyporesponsiveness to speci¢c antigenic determinants (particularly those of Tri r 2) could contribute to the persistence of T. rubrum infection in humans [31]. In this context, it would be interesting to evaluate the cellular immune response to SUB3 in dermatophytic cats, especially in chronically infected ones, which constitute a major zoonotic hazard [32]. Moreover, the eventual protective role of the anti-SUB3 cell-mediated immune response should be investigated in vaccination trials. The recombinant expression of SUB3 provides a valuable tool for addressing these issues.
Acknowledgements We thank Jacques Detry and Humbert Gianfreda for excellent technical assistance. This work was supported by Grant 3.4534.01 from Fonds de la Recherche Scienti¢que Me¤dicale (FRSM). F.D. and F.B. are recipients of a studentship of FRIA (Fonds pour la Formation a' la Recherche dans l’Industrie et dans l’Agriculture, rue d’Egmont 5, 1000 Brussels, Belgium).
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