Received 8 August 2012
Purification, crystallization and preliminary X-ray diffraction analysis of the Staphylococcus epidermidis extracellular serine protease Esp
Krishnan Vengadesan,a Kevin Macon,b Shinya Sugumoto,c Yoshimitsu Mizunoe,c Tadayuki Iwasec and Sthanam V. L. Narayanab*
aUNESCO Regional Centre for Biotechnology (RCB), Gurgaon, Haryana 122 016, India,bCenter for Biophysical Sciences and Engineering, School of Optometry, University of Alabama at Birmingham, Birmingham, AL 35294, USA, and cDepartment of Bacteriology, School of Medicine, Jikei University, Minato-Ku, Tokyo 105-8461, Japan
Esp, an extracellular serine protease from Staphylococcus epidermidis, has been shown to inhibit S. aureus biofilm formation and nasal colonization. The full-length 27 kDa pro-Esp was purified and digested with thermolysin to obtain mature Esp. The mature Esp containing 216 residues crystallized in space group P21, with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, = 98.2° and one molecule in the asymmetric unit, with an estimated solvent content of 42%. A diffraction data set has been collected to 1.8 Å resolution on a rotating-anode home-source facility.
Bacterial biofilms are increasingly being recognized as a major cause of persistent infections, particularly in patients receiving medical implants such as ventilation tubes, in-dwelling catheters, artificial heart valves etc. After initial adhesion and invasion, biofilm formation is the key step involved in Staphylococcus epidermidis virulence during device-associated infections (Rohde et al., 2007, 2010; Mack et al., 2006). Biofilms are composed of extracellular polysaccharides, proteins and other nutrients essential for microbial survival. Pathogens embedded in such biofilms are less susceptible than their planktonic counterparts to antibiotics and biocides (Gilbert et al., 2002; Foster, 2005), and are also impervious to destructive inflammatory processes of the host (Vuong et al., 2004). Generally, Gram-positive bacteria are less susceptible to human complement, probably because of their thick peptidoglycan layer. However, human polymorphonuclear neutrophils (PMNs) can eliminate microbes when brought into close contact (Vuong et al., 2004). Interestingly, a typical biofilm of S. epidermidis can activate the complement better than its planktonic colleagues, as shown by measurement of the released anaphylotoxin C3a (Kristian et al., 2008). However, deposition of C3b on the target cells was diminished in biofilm-embedded S. epidermidis, which would contribute to its escape from PMN killing, in addition to complement evasion (Kristian et al., 2008).
Bacteria, which were once thought of as organisms that rarely interact, have recently been found to lead highly social lives in biofilms (West et al., 2006). Central to such cooperative living is their ability to detect the local cell density and coordinate their behaviors (Bassler & Losick, 2006). This detection ability, termed quorum sensing, involves the secretion and detection of auto-inducer molecules that accumulate in the biofilm in proportion to the respective cell density. Based on the auto-inducer density, microbes modulate behaviors such as surface attachment (Dunne, 2002), the production of `sticky' extracellular polymeric substances that are conducive to the transfer of genetic material (Sakuragi & Kolter, 2007), sporulation (Grossman, 1995) and secretion of virulence factors (Miller et al., 2002; Zhu et al., 2002). In contrast to such cooperative abilities of microbes, Iwase et al. (2010) have recently identified a staphylococcal species that exhibits an ability to regulate the survival of other classes of pathogens in biofilms (Iwase et al., 2010).
S. epidermidis, a Gram-positive commensal bacterium, is part of the normal human flora and is a dominant colonizer of the upper respiratory tract, especially the nostrils (Iwase et al., 2010). The nostrils are also primary reservoirs for another human bacterial pathogen, S. aureus, which is responsible for illnesses ranging from minor skin infections (e.g. pimples, impetigo and boils) to life-threatening diseases such as pneumonia, endocarditis and meningitis. The recent emergence of methicillin-resistant and vancomycin-resistant S. aureus has been identified as being responsible for a large number of infections and deaths all over the world (Klein et al., 2007; Lowy, 1998; Hiramatsu et al., 1997), necessitating an urgent need for identifying new therapies that do not elicit resistance. Understanding the mode of S. aureus biofilm inhibition by S. epidermidis could open an avenue for an alternative therapeutic strategy.
Epidemiological studies on staphylococcal nasal colonization in the nasal cavities of volunteers have revealed the presence of both S. epidermidis and S. aureus in some populations, and the presence of S. epidermidis along with the absence of S. aureus in many others (Iwase et al., 2010). Further co-culturing in the nasal cavities helped Iwase et al. (2010) to identify two types of S. epidermidis: one type can cohabit with S. aureus, while the other inhibits the growth of the latter in a dose-dependent manner (Iwase et al., 2010). More interestingly, culture supernatants of inhibitory S. epidermidis destroyed previously formed S. aureus biofilm, while the non-inhibitory-type cell culture had no effect.
Iwase et al. (2010) isolated a 27 kDa protein from the inhibitory-type S. epidermidis cell culture and identified it as a serine protease, previously suggested to be pro-Esp from oral strains of S. epidermidis (Moon et al., 2001; Dubin et al., 2001). The pro-Esp, also previously known as SspA or GluSE (Ohara-Nemoto et al., 2002; Moon et al., 2001), contains a 66-residue pro-peptide at the N-terminal end and a mature 216-residue polypeptide in the C-terminal region (Fig. 1). Mature Esp processed from pro-Esp by an unknown protease exhibits significant sequence similarity (59.1%) to S. aureus V8 protease (Ohara-Nemoto et al., 2002). There is limited similarity in the pro-peptide segments (<25%) of the Esp and V8 serine proteases, and the mature Esp lacks 55 residues that comprise the multiple PNN/PDN tripeptides observed in the C-terminus of active V8 (Ohara-Nemoto et al., 2002). In this paper, we report the cloning, purification, activation, crystallization and preliminary crystallographic characterization of mature Esp. The mature Esp crystals diffracted to 1.8 Å resolution using a rotating-anode home-source facility and the diffraction data collected were of good quality and were suitable for structure determination.
| || Figure 1 |
A schematic diagram of pro-Esp. Pro-Esp is composed of signal peptide (SP; Met1-Ala27), pro-peptide (PP; Lys28-Ser66) and the following mature Esp (Val67-Gln282). The arrow at the top indicates the thermolysin cleavage site.
S. epidermidis JK16 strain was obtained from the American Type Culture Collection (ATCC). The primers Esp-pET-NdeII (5'-CATATGAAAAAGAGATTTTTATCTATATG-3') and Esp-pET-BamHI (5'-GGATCCTTACTGAATATTTATATCAG-3') harboring NdeI and BamHI restriction sites, respectively, were used to PCR-amplify the sequence of esp (coding Met1-Gln282) from chromosomal DNA of S. epidermidis strain ATCC 12228 (gene accession No. AAO05142.1), which was cloned into the pET28b plasmid (EMD Biosciences/Novagen). The resulting pET28-His-proEsp plasmid, which includes a His6 tag at the N-terminus, was transformed into Escherichia coli BL21 (DE3) cells.
Overnight cultures (10 ml) of stationary-phase E. coli were used to inoculate 1.0 l Luria-Bertani (LB) broth supplemented with 100 µg ml-1 ampicillin. The cells were allowed to grow at 310 K until the culture reached an OD600 nm of 0.6. Protein expression was induced by the addition of isopropyl -D-1-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM and the culture was incubated overnight at 303 K. The cells were harvested by centrifugation at 2568g (Beckman Allegra 6R Centrifuge) for 20 min at 277 K and were resuspended in lysis buffer [50 mM sodium phosphate pH 7.5, 300 mM NaCl, EDTA-free protease-inhibitor cocktail tablet (Roche), 10%(v/v) glycerol, 5 mM imidazole]. The cells were lysed using a French press (76 MPa) and the soluble supernatant was collected by centrifugation at 18 384g for 20 min at 277 K. The supernatant was loaded onto a column containing 8 ml Ni-NTA Superflow Agarose resin pre-equilibrated with lysis buffer. The column was washed with ten bed volumes of buffer A (50 mM sodium phosphate pH 7.4, 300 mM NaCl) and the bound protein was eluted with a linear gradient of 0-100% buffer B (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 300 mM imidazole). Fractions containing the pro-Esp, as determined by SDS-PAGE, were pooled.
In light of the high sequence similarity and similar enzymatic properties of S. epidermidis Esp and S. aureus V8, the metalloprotease thermolysin employed for the activation of pro-V8 was used for cleavage of the (Ser66-Val67) pro-Esp N-terminal pro-peptide (Yoshikawa et al., 1992; Ohara-Nemoto et al., 2002). The N-terminal His6-tag-fused pro-Esp was digested by thermolysin at 310 K for 4 h and the proteolysis was stopped by the addition of 5 mM EDTA; the cleavage was confirmed by SDS-PAGE. The proteolytic activity of mature Esp was tested using an asocasein assay (Sugimoto et al., 2011) as follows: the product of thermolysin-treated recombinant protein was diluted into a reaction mixture consisting of 1% asocasein, 100 mM Tris-HCl pH 8.0, 5 mM EDTA. After incubation at 310 K for 5 h, an equal volume of 5% trichloroacetic acid (TCA) was added to stop the reaction. The soluble material was recovered by centrifugation at 2123g for 10 min and the absorbance was measured at 400 nm.
The mature Esp was further purified by gel-filtration chromatography on a Superdex 75 10/30 column (GE Healthcare) in buffer consisting of 20 mM Tris-HCl pH 7.2, 150 mM NaCl. For crystallization, the protein was concentrated to 22 mg ml-1 using an Amicon ultrafiltration system.
Initial crystallization conditions were screened at 293 K by the sitting-drop vapor-diffusion method with the commercial crystallization screens Crystal Screen HT, Index HT (Hampton Research) and Wizard Screens I and II (Emerald BioSystems) using a Crystal Phoenix robot (Art Robbins Instruments) and 96-well Intelli-Plates (Art Robbins). Each crystallization drop was prepared by mixing 0.2 µl reservoir solution and 0.2 µl protein solution. Crystals were observed in several conditions after a week and the conditions were optimized manually with the help of laboratory-made solutions and using the hanging-drop vapor-diffusion method. Crystals used for diffraction data collection were obtained from drops consisting of equal volumes (1 µl) of protein solution (14 mg ml-1 in 20 mM Tris-HCl buffer pH 7.2, 150 mM NaCl) and well solution (1 µl) that were equilibrated against a 1 ml reservoir. Thin rod-shaped crystals (Fig. 2) obtained from a condition with 0.25 M potassium acetate, 22%(w/v) PEG 3350 in the well were used for data collection.
| || Figure 2 |
Crystals of mature Esp obtained by the hanging-drop vapor-diffusion method using 0.25 M potassium acetate and 22% PEG 3350 as precipitants at room temperature. The dimensions of the largest crystal in the drop are 0.5 × 0.04 × 0.02 mm.
Native diffraction data were collected to 1.8 Å resolution (Fig. 3) using an R-AXIS IV imaging-plate detector mounted on an in-house Rigaku rotating-anode X-ray generator operating at 100 mA and 50 kV. Diffraction data were collected from a single crystal over a range of 360° in 1° steps at a 130 mm crystal-to-detector distance and with 5 min exposure per frame. The cryoprotectant was prepared by mixing 20 µl 100%(v/v) ethylene glycol and 80 µl well solution consisting of 0.25 M potassium acetate, 22%(w/v) PEG 3350. The selected crystal was picked up using a small nylon loop and swiped through the prepared cryoprotectant solution before being positioned in the liquid-nitrogen stream and X-ray beam. The diffraction data were processed with d*TREK (Pflugrath, 1999) and the crystallographic data statistics are presented in Table 1.
| || Figure 3 |
X-ray diffraction image of a native mature Esp crystal collected using an R-AXIS IV imaging-plate detector mounted on an in-house Rigaku rotating-anode X-ray generator operating at 100 mA and 50 kV.
The native mature Esp crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 39.5, b = 61.2, c = 42.5 Å, = 98.2°. Matthews coefficient calculations (Matthews, 1968) suggested the presence of one molecule in the asymmetric unit (calculated VM of 2.14 Å3 Da-1, estimated solvent content of 42%).
The pro-Esp of S. epidermidis was purified and activated by thermolysin to obtain mature Esp. Single crystals of mature Esp suitable for diffraction studies were obtained in a week from a condition containing a mixture of 0.25 M potassium acetate and 22%(w/v) PEG 3350 as a precipitant. The Esp crystals diffracted to 1.8 Å resolution and belonged to the monoclinic space group P21. The VM calculation suggested that the asymmetric unit is likely to contain one molecule, with a solvent content of 42%. Attempts to solve the crystal structure of mature Esp by molecular replacement using the coordinates of S. aureus V8 (PDB entry 2o8l ; Prasad et al., 2004) as the starting model have not been successful. Attempts to use a selenomethionine derivative for MAD/SAD phasing are in progress; there are two suitable methionine residues for replacement with selenomethionine in mature Esp.
This work was supported by a grant from the NIH to SVLN (grant No. AI073521); TI is funded by the Uehara Memorial Foundation and the Science Research Promotion Fund of `The Promotion and Mutual Aid Corporation for Private Schools of Japan'.
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