A new procedure for the cloning, expression and purification of the β-carbonic anhydrase from the pathogenic yeast Malassezia globosa, an anti-dandruff drug target

Abstract

Malassezia yeasts are almost exclusively the single eukaryotic members of the fungal flora of the skin. Malassezia globosa and Malassezia restricta are found on the skin of practically all humans. Malassezia globosa is highly implicated in the pathogenesis of dandruff and its genome encodes for only one carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the β-class (MgCA). It has been indeed demonstrated that in many pathogenic microorganisms, CAs are essential for their life cycle and their inhibition can lead to growth impairment and defects. In the previous work, the recombinant MgCA was investigated for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target. MgCA was cloned as GST-fusion protein, but the yield was rather low and the protein was often found in inclusion bodies. Here, we propose an alternative procedure consisting in cloning the recombinant MgCA as His-Tag fusion protein. This procedure resulted in a good method to express and purify the active recombinant MgCA, and the protein recovery was better with respect to that used for preparing MG-CA (β-CA cloned as GST-fusion protein).

• Carbonic anhydrases
• enzyme kinetics
• fusion protein
• Malassezia
• protonography

Introduction

Carbon dioxide (CO₂) and its hydration product bicarbonate () are substrates and products of various metabolic reactions in the cells of different living organisms1–7. CO₂ is the final product of the respiration in all domains of life, from microorganisms to mammals8–10; bicarbonate () is a substrate for several carboxylating enzymes involved in biosynthetic pathways such as biosynthesis of fatty acids, amino acids and nucleotides11–14. The interconversion of CO₂ and is spontaneously balanced to maintain the equilibrium between dissolved inorganic CO₂, carbonic acid (H₂CO₃), bicarbonate () and carbonate (). Almost, all life on earth depend on the production of sugars from sunlight and CO₂ (photosynthesis) and the metabolic breakdown (respiration) of those sugars to produce the energy needed for movement, growth and reproduction8,9,14,15. The uncatalyzed hydration–dehydration reaction CO₂ + H₂O ⇄  + H⁺ is slow at physiological pH and thus, in biological systems, the reaction is accelerated by enzymatic catalysts, called carbonic anhydrases (CAs, EC 4.2.1.1). The conversion of CO₂ to bicarbonate and protons is a physiologically relevant reaction in all life kingdoms8,10,14,16–18. Virtually, CA isozymes have been found in all mammalian tissues and cell types, acting as CO₂ transporter or involving in other physiological processes. CAs are also found in plants, fungi, algae, protozoa, cyanobacteria and bacteria8,19–25. Six different, genetically distinct CA families are known to date: α-, β-, γ-, δ-, ζ- and η-CAs8,20,22. Many representatives of all these enzyme classes have been crystallized and characterized in detail. α-CAs are normally monomers and rarely dimers; β-CAs are dimers, tetramers or octamers; γ-CAs are trimers. Only the ζ-CA crystallized has three slightly different active sites on the same polypeptide chain26, whereas no X-ray crystal structures of δ- and η-CAs are available up to now. CAs contain Zn²⁺ in their active site, coordinated by three histidine residues and a water molecule/hydroxide ion (in the α- and γ-CAs) or two Cys and one His residues (in the β class), with the fourth ligand being a water molecule/hydroxide ion22,23,27. In the η-CAs, the coordination pattern of the Zn(II) ion is totally different from the other classes, with one Gln and two His acting as ligands21.

It has been demonstrated that in many pathogenic microorganisms (bacteria, fungi and protozoa), CAs are essential for their life cycle2 and their inhibition can lead to growth impairment and defects22. CAs are abundant in fungi22, but these enzymes have only recently been characterized in detail. Moreover, the physiological concentrations of CO₂/ induce prominent virulence in Candida albicans (filamentation) and Candida glabrata or Cryptococcus neoformans (capsule biosynthesis) through direct activation of the fungal adenylyl cyclase, which is involved in the fungal differentiation13,28–34. These findings suggest a link between cAMP signaling and CO₂/ sensing capable of influencing the fungal metabolism and pathogenesis33–36. Nonpathogenic and pathogenic fungi, such as Saccharomyces cerevisiae, C. albicans and C. glabrata, present only a single copy of CA belonging to a β-CA class; while in other fungi it is possible to find multiple copies of β-CA- and α-CA-encoding genes29,37–39. Candida albicans CA, CaNce103, and the analogous CA, CgNce103, encoded by the genome of C. glabrata, are essential for pathogenesis of these fungi in niches where the available CO₂ is limited (e.g. the skin)29,32,33. Again, the CA, named Can2, identified in the genome of C. neoformans is essential for its growth29,31–33,40.

Malassezia yeasts are almost exclusively the single eukaryotic members of the microbial flora of the skin. Malassezia have been recognized for more than 150 years41,42 as members of the human cutaneous flora and etiologic agents of certain skin diseases. At the begining of 1800, it was noted that yeast cells and filaments were present in the skin scales of patients with pityriasis versicolor41,42, whereas yeast cells, but no filaments, were observed in scales from healthy scalp, seborrheic dermatitis scalp, and dandruff. Dandruff is a frequent pathological skin condition confined to the scalp and is characterized by flaking with minimal to absent inflammation. Malassezia genus includes seven species, the three former taxa (M. furfur, M. pachydermatis, and M. sympodialis) and four new taxa (M. globosa, M. obtusa, M. restricta, and M. slooffiae)43. With the exception of M. pachydermatis, all species afore-mentioned require lipids for their growth44. Moreover, M. globosa and M. restricta are found on the skin of practically all humans44. It has been demonstrated that oleic acid responsible of the sebum of the scalp is produced from the hydrolysis of triglycerides by M. globosa lipases. These data highly implicate M. globosa in the pathogenesis of dandruff. Similar to the above-mentioned fungi/yeasts, M. globosa genome contains only one β-CA, denominated MgCA22,45,46. This enzyme has been cloned and investigated several years ago for its inhibition profile with sulfonamides, which in models of dandruff infection were able to protect animals from the fungal infection, allowing us to propose this enzyme as a new antidandruff target46. In the previous work, a chimeric MgCA was produced having at the N-terminal the GST amino acid sequence, but the MgCA yield was rather low and the protein was often found in inclusion bodies46.

Here, we propose an alternative procedure for the preparation of recombinant MgCA encoded in the genome of M. globosa, the yeast responsible for dandruff, following a different cloning strategy compared with that described by Hewitson et al.46. We propose to use a synthetic gene encoding MgCA to which a tag containing six His residues has been attached at the amino terminal part for allowing the purification with Ni(II)-based affinity columns. The yield and purity of MgCA obtained in this way was better compared with that of the earlier paper44.

Materials and methods

Gene identification

The identification of the gene encoding for M. globosa β-CA (MgCA) was performed at the link http://www.ncbi.nlm.nih.gov/genome/selecting the genome of “M. globosa”. The β-CA gene of M. globosa (accession number: XP_001730815.1) was identified running the “BLAST” program and using the nucleotide sequences of β-CAs as query sequence47,48.

Construct preparation, protein expression and purification

The GeneArt Company, specialized in gene synthesis, designed the synthetic M. globosa gene encoding for the β-CA, and containing 4 base pair sequences (CACC) necessary for directional cloning at the 5′ end of the MgCA gene. The fragment was subsequently cloned into the expression vector pET100/D-TOPO (Invitrogen, Carlsbad, CA), creating the plasmid pET100D-Topo/MgCA. In order to confirm the integrity of the M. globosa gene and the fact that no errors occurred at the ligation sites, the vector containing the fragment was sequenced. Competent Escherichia coli BL21 (DE3) Codon Plus cells were transformed with pET100/D-Topo/MgCA, grown at 37 °C, induced with 1 mM IPTG. Zn(SO₄) was added after 30 min and after additional growth for 3 h, cells were harvested and disrupted by sonication at 4 °C in 20 mM buffer phosphate, pH 8.0. Following sonication, the sample was centrifuged at 1200×g at 4 °C for 30 min. The supernatant was dialyzed against 0.02M phosphate buffer (pH 8.0) containing 0.01 M imidazole and 0.5M NaCl at 4 °C and loaded onto a His-select HF Nickel affinity column (1.0 by 1.0 cm, GE Healthcare, Little Chalfont, Buckinghamshire, UK). The column was equilibrated with 0.02M phosphate buffer (pH 8.0) containing 0.01 M imidazole and 0.5M NaCl at a flow rate of 1.0 ml/min. The VchCAβ was eluted with 0.02M phosphate buffer (pH 8.0) containing 0.5 M NaCl and 0.25 mM imidazole at a flow rate of 1.0 ml/min. Active fractions (1 ml) were collected and combined for a total volume of 5 ml. Subsequently, they were dialyzed, concentrated and analyzed by SDS-PAGE. At this stage of purification, the enzyme was at least 95% pure and the obtained recovery was of 0.03 mg of the recombinant yeast MgCA.

Sequence analysis

Multialignment of amino acid sequences was performed using the program MUSCLE (MUltiple Sequence Comparison by Log-Expectation), a new computer program for creating multiple alignments of protein sequence49.

SDS-PAGE

Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) was performed as described by Laemmli using 12% gels50.

Protonography

Wells of 12% SDS-gel were loaded with bCA and MgCA mixed with loading buffer without 2-mercaptoethanol and without boiling the samples, in order to avoid protein denaturation. The gel was run at 180V until the dye front ran off the gel. Following the electrophoresis, the 12% SDS-gel was subject to protonography to detect the bCA and MgCA hydratase activity on the gel as described by Capasso and coworkers3,19,20.

Enzyme kinetics

An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO₂ hydration activity51. Bromothymol blue (at a concentration of 0.2 mM) has been used as an indicator, working at the absorbance maximum of 557 nm, with 10–20 mM TRIS (pH 8.3) as buffer, and 20 mM Na₂SO₄ for maintaining constant ionic strength (this anion is not inhibitory and has a K_I> 200 mM against this enzyme), following the initial rates of the CA-catalyzed CO₂ hydration reaction for a period of 10–100 s. The CO₂ concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each measurement at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (1–10 mM) were prepared in distilled-deionized water and dilutions up to 0.01 μM were done thereafter with distilled-deionized water. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using the Cheng–Prusoff equation whereas the kinetic parameters for the uninhibited enzymes from the Lineweaver–Burk plots, as reported earlier, and represent the mean from at least three different determinations.

Results and discussion

The full nucleotide sequence encoding for MgCA showed an open-reading frame of 241 residues polypeptide chain which contained all the typical features of a β-CA, including the three residues that are involved in the catalytic mechanism of the enzyme (two cysteines and one histidine, see Figure 1). The predicted molecular mass of the enzyme from its amino acid sequence is of 26.98 kDa. We performed a BLAST search to compare MgCA amino acid sequence to other protein sequences present in the data bank. The result of BLAST analysis showed that the first 50 top-ranking library sequences were represented by CAs from fungi and bacteria (Figure 2). Intriguingly, MgCA showed 77% identity with the β-CA from M. sympodialis, the only malasseziacea present in the top raking list generated by BLAST program (Figure 2). Malassezia sympodialis has been reported as the most frequent skin-colonizing yeast in both patients with atopic eczema, as well as in healthy individuals43,44. It has also been associated with folliculitis and catheter-related infections43,44. In animals, it has caused otitis externa (in cats), whereas it is a known commensal of swine52,53.

Figure 1. Nucleotide and translated amino acid (bold letters) sequences of MgCA. The open reading frame is indicated by capital letters. Zinc ligands are indicated in red, while the catalytic dyad involved in the activation of the zinc-coordinated water in blue. .

Figure 2. Blast output reporting the first 33 sequences. By going down the list, it is possible to see less than perfect matches, slowly degrading as the corresponding score decreases and the E value increases. The E value is an assessment of the statistical significance of the score. E value close to 1 is a warning that the alignment is not reliable.

The recombinant MgCA was isolated and purified to homogeneity at room temperature from E. coli (DE3) codon plus cell extract. Carbonic anhydrase activity was recovered in the soluble fraction of the cell extract obtained after sonication and centrifugation. Using the affinity column (His-select HF Nickel affinity gel), MgCA was purified to apparent homogeneity, as indicated by SDS–PAGE (Figure 3, lane B). The analysis by SDS–PAGE of MgCA showed a band of about 29 kDa (monomeric form) under reducing condition (Figure 3). The recovery of purified protein concentration was 1 mg of purified protein using the cloning, expression and purification aforementioned and starting from 2 L of bacterial culture.

Figure 3. SDS-PAGE of the recombinant MgCA purified from E. coli codon plus cells. Lane A, molecular markers, M.W. starting from the top: 250, 150, 100, 75, 50, 37, 25 and 20; Lane B, purified SspCA from His-tag affinity column.

Using the stopped-flow technique, the kinetic parameters were determined for the newly purified recombinant MgCA using CO₂ as a substrate. MgCA showed a significant catalytic activity, with a k_cat of 9.2 × 10⁵ s⁻¹ and a k_cat/K_m of 8.3 × 10⁷ M^–1 × s ⁻ 1. Table 1 shows a comparison of the kinetic parameters of MgCA with those of MG-CA (β-CA cloned as GST-fusion protein)46, hCA I and II (α-CA from Homo sapiens, isoform I and II, respectively) and Can2 (β-CA from C. neoformans). It may be observed that MgCA and MG-CA showed kinetic parameters about 2 times higher than the other β-CA reported in Table 1 (Can2), and four times higher than the human α-CA isoform I. Moreover, data from Table 1 show that the MgCA cloned as His-fusion protein has kinetic data similar to those of the GST-fusion protein reported earlier, but the activity of the His-tagged MgCA is better. It is interesting to note that both proteins obtained with two different cloning strategies are not affected by acetazolamide inhibition. Intriguingly, this lack of effectiveness of AAZ for inhibiting a β-class enzyme has been observed only for Cab, an enzyme isolated and characterized from the Archaeon Methanobacterium thermoautotrophicum.

Table 1. Kinetic parameters for the CO₂ hydration reaction catalyzed by MG-CA (GST-fusion protein), MgCA (His-fusion protein), hCA I and II (α-CA from H. sapiens, isoform I and II, respectively) and Can2 (β-CA from C. neoformans).

Cryptonym	Activity level	Class	Kcat (s^−1)	kcat/km M^−1 × s^−1	KI (Acetazolamide) μM
hCA I	Moderate	α	2.0 × 10^5	5.0 × 10^7	0.25
hCA II	Very high	α	1.4 × 10^6	1.5 × 10^8	0.012
Can2	Moderate	β	3.9 × 10^5	4.3 × 10^7	0.010
MgCA	Moderate	β	9.2 × 10^5	8.3 × 10^7	74
MG-CA	Moderate	β	8.6 × 10^5	6.9 × 10^7	76

Inhibition data with the clinically used sulfonamide acetazolamide (5-acetamido-1,3,4- thiadiazole-2-sulfonamide) are also provided.

The hydratase activity of MgCA on the SDS–PAGE gel was investigated and compared with that obtained for bovine bCA (α-CA). The gels were run under denaturing and non-reducing conditions. The protonogram, shown in Figure 4, was obtained loading on the SDS–PAGE samples of MgCA and bCA at 10 μg/well. As described in the experimental section, the protonography is based on monitoring the pH variation in the gel due to the CA-catalyzed conversion of CO₂ to bicarbonate and protons. The protonogram was thereafter stained with bromothymol blue, which is a widely used pH indicator. This dye appears blue in its deprotonated form, while its color changes to yellow in the protonated form. Thus, the production of H⁺ ions during the CO₂ hydration reaction, due to the CA hydratase activity, lowers the pH of the solution until the color transition point of the dye is reached, that is, at pH 6.8. The developed protonogram showed a yellow band at a molecular weight of 29 kDa corresponding to the β-hydrates activity of MgCA (Figure 4). The protonogram showed only one band corresponding to the monomer of MgCA (29 kDa). As described in the literature, β-CA fold resembles neither that of α-CA nor that of γ-CA. In the β-CAs, pair of monomers are joined to form an active enzyme54. By using the protonogram, we observed a yellow band corresponding to the β-monomer position. This is possible because following the electrophoresis, SDS was removed from the gel. This procedure potentially led to the rearrangement of β-CA monomers in the gel, reconstituting the active dimeric form of the enzyme. β-CA activity corresponding to the dimeric band is absent in the protonogram because the concentration of SDS used in the loading buffer determines the dissociation of the MgCA dimer.

Figure 4. MgCA protonogram. The yellow bands correspond to the hydratase activity on the gel responsible for the drop of pH from 8.2 to the transition point of the dye. Incubation time was of 60 s. β-CA is present in the monomeric (29 kDa) form (see text for details). Lane A, molecular markers, M.W. starting from the top: 250, 150, 100, 75, 50, 37, 25 and 20 kDa; Lane B: MgCA; Lane C: commercial bovine CA (bCA).

Conclusion

The results of this study are significant, because they establish that the MgCA fusion protein cloned as His-Tag fusion protein is a good method to express and purify the active recombinant protein; the yield and purification efficiency of MgCA fusion protein by affinity column (His-select HF Nickel affinity gel) is better compared with that used for preparing MG-CA (β-CA cloned as GST-fusion protein)46; the CO₂ hydratase activity by using protonography allows us to speculate that this technique may also be extended to enzymes belonging to other CA classes such as the β-CA (MgCA) from M. globosa.

Declaration of interest

We thank the Distinguished Scientist Fellowship Program (DSFP) at KSU for funding this project. This work was also financed in part by an FP7 EU project (Dynano). The authors report no conflict of interest.