Biochemical and Biophysical Research Communications 354 (2007) 403–408 www.elsevier.com/locate/ybbrc
A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli Linru Wang a
a,b
, Lisa Walrond c, Terry D. Cyr c, Min Lin
a,b,*
Canadian Food Inspection Agency, Animal Diseases Research Institute, 3851 Fallowfield Road, Ottawa, Ont., Canada K2H 8P9 b Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ont., Canada K1H 8M5 c Centre for Biologics Research, Health Canada, Ottawa, Ont., Canada K1A 0L2 Received 7 December 2006 Available online 9 January 2007
Abstract The 86-kDa protein IspC of 774 amino acids in Listeria monocytogenes serotype 4b has been recently identified as the target of humoral immune response to listerial infection and as a novel surface autolysin. A signal peptide is predicted at the N-terminal end of IspC, but no biochemical data has been shown to confirm the presence of the cleavage site of a signal peptidase. To address this and prepare sufficient amount of the protein for biochemical and structural characterization, we present a strategy for efficient expression and purification of IspC and analyze the purified protein by N-terminal sequencing and mass spectrometry. Expression of IspC in Escherichia coli using a pET30a-based expression construct was efficiently improved by incubating the culture at 37 C for 2 h followed by 4 C for 16– 18 h. The recombinant product rIspC remained as a soluble form in the cellular extract and was purified to electrophorectic homogeneity by the combination of metal chelate affinity chromatography with cation-exchange chromatography. The IspC was shown to contain a 23-residue N-terminal signal peptide being processed between Thr 23 and Thr 24 in E. coli, resulting in an 84-kDa mature protein. The highly purified form of rIspC from this study, exhibiting both peptidoglycan hydrolase activity and immunogenicity as previously reported, would facilitate further biochemical, structural, and functional studies of this autolysin. Crown copyright 2007 Published by Elsevier Inc. All rights reserved. Keywords: Listeria monocytogenes; Autolysin; Signal peptide; Peptidoglycan hydrolase
Bacterial autolysins are enzymes that catalyze the specific cleavage of covalent bonds in the cell wall peptidoglycan (murein) from the producing bacterial strains [32] potentially leading to bacteriolysis. They are ubiquitously found in both Gram-positive and Gram-negative bacteria [2,11]. Based on the specific bonds that they cleave, autolysins are classified into several types: N-acetylmuramidases, N-acetylglucosaminidases, N-acetylmuramyl-L-alanine amidases, endopeptidases, and transglycosylases [34]. Bacterial autolysins are involved or implicated in a variety of cellular functions including cell wall turnover, cell division, cell separation, *
Corresponding author. Fax: +1 613 228 6667. E-mail address:
[email protected] (M. Lin).
chemotaxis, biofilm formation, genetic competence, protein secretion, antibiotic-induced lysis, sporulation, and formation of flagella [31,33], and in pathogenesis [3,7,13,14, 25,35]. Multiple autolytic enzymes have been demonstrated and studied in a number of bacterial species [16,26,33], and the purpose of this functional redundancy (if there is any) is not clear. The data accumulated to date in the literature, however, appears to indicate that an individual autolysin in a bacterium performs its unique biological function(s) that is not necessarily overlapped by other autolysins of the same bacterial strains. Thus, for a bacterium of interest, it is necessary to identify and characterize all the autolysins in order to gain a comprehensive understanding of their biological functions and their roles in virulence.
0006-291X/$ - see front matter Crown copyright 2007 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.12.218
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Listeria monocytogenes is a Gram-positive, facultatively anaerobic, intracellular bacterium that causes a severe food-borne disease (listeriosis) with clinical symptoms including septicemia, meningitis, and abortion, mainly affecting immunocompromised individuals, neonates, the elderly, and pregnant women [36]. The disease has a relatively high mortality rate of 20–30% [29,36]. Although 13 serotypes of Listeria are recognized, serotypes 4b, 1/2a, and 1/2b of L. monocytogenes are responsible for almost all human cases of listeriosis [9,23,37] with serotype 4b strains accounting for almost all major outbreaks and a large portion of sporadic cases, suggesting this serotype possesses a virulence potential highly specific to humans [10,18]. Several autolysins have been identified and characterized in L. monocytogenes, including P45 [30], P60 [39], NamA [20] or MurA [8], Ami [5,24], and Auto [6]. The murein-hydrolyzing activity of a flagellin FlaA from L. monocytogenes has been demonstrated [27], but its autolytic activity remains unknown. Recently we have identified and biochemically characterized a novel surface-localized autolysin from L. monocytogenes serotype 4b [38], an 86kDa (deduced molecular mass) protein consisting of 774 amino acids known as the target (designated IspC) of humoral immune response to listerial infection [40]. A signal peptide containing the first 29 N-terminal residues was predicted by SignalP 3.0 using neural networks (http:// www.cbs.dtu.dk/services/SignalP/); removal of it in vivo by a signal peptidase that cleavages between Leu 29 and Gln 30 would yield a mature protein of 82.598 kDa. Analysis of various truncated forms of IspC for cell wall hydrolyzing or binding activity has defined two separate functional domains: the N-terminal catalytic domain (aa 1–197) responsible for the hydrolytic activity and the C-terminal domain (aa 198–774) made up of seven GW (glycine–tryptophan dipeptide) modules responsible for anchoring the protein to the cell wall [38]. In this communication, we report efficient expression of the full-length IspC in E. coli, its purification by chromatographic methods combining metal affinity chromatography with cation-exchange chromatography, and analysis of the purified protein by N-terminal sequencing and mass spectrometry. Biochemical, structural, and functional analysis of the immunogenic autolysin IspC is now possible, since we have developed efficient preparation and purification methods for a sufficient quantity of the recombinant protein.
Materials and methods Chemicals and reagents. Isopropyl-b-D-thiogalactoside (IPTG), phenylmethysulphonyl fluoride (PMSF), and kanamycin were obtained from Sigma (St. Louis, MO, USA). Ni–NTA superflow agarose and anti-histidine tag monoclonal antibody (anti-His mAb) were purchased from Qiagen (Santa Clarita, CA, USA); Horseradish peroxidase (HRP)-conjugated goat–anti-mouse IgG from Jackson ImmunoResearch Laboratories (West Grove, PA, USA); SP Sepharose Fast Flow from Amersham
Biosciences (Baie d’Urfe, Quebec, Canada). All other chemicals and solvents were of commercially available analytical, HPLC or MS grade. Induction of recombinant protein expression in E. coli. The construct pIspC previously created by inserting the ispC ORF into the NdeI and XhoI sites of pET30a [38] was used to produce the recombinant IspC (rIspC) in E. coli Rosetta (DE3)/pLysS (Novagen, Madison, WI, USA). The overnight culture was diluted 1:100 into 10 l fresh LB broth (250 ml per 1 l flask) supplemented with kanamycin (30 lg/ml), and subcultured at 37 C with vigorous shaking until the cell growth reached an OD590 of 0.45 ± 0.05. IPTG (1 mM) was added to induce the expression of rIspC at 37 C for 2 h and then maintained at 4 C overnight. The cells were harvested by centrifugation at 16,900g at 4 C for 10 min and frozen at 80 C until use. SDS–PAGE and Western blotting. SDS–polyacrylamide gel electrophoresis (SDS–PAGE) was performed as described by Laemmli [19], using a 4% stacking gel and a 12% resolving gel in a Bio-Rad minigel apparatus (Bio-Rad, Mississauga, Ontario, Canada). Following electrophoresis, the separated proteins were stained with either Coomassie brilliant blue or electrotransferred onto a nitrocellulose membrane using a Trans-Blot SD semi-dry transfer cell (Bio-Rad, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. The Western blot procedure for analysis of the target protein with anti-His mAb followed by horseradish peroxidase (HRP)-conjugated goat–anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Penn.) was performed essentially as described [22]. Chromatographic procedures. The frozen cell pellets were resuspended in a minimum volume of phosphate-buffered saline (PBS, pH 7.2) containing 1 mM PMSF and lysed by passing through a French Press at 1500 lb/in.2 The homogenates were spun at 27,000g at 4 C for 20 min. The supernatant, mixed with an equal volume of buffer A (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole), was applied at 1 ml/ min to a column (1 · 1.5 cm) of Ni–NTA superflow (Qiagen) that had been pre-equilibrated with buffer A. The column was washed with 30 ml of buffer A, and the proteins were eluted in 1 ml per fraction with 30 ml of 250 mM imidazole in buffer B (25 mM NaH2PO4–NaOH, pH 8.0). The A280 peak fractions containing the target protein, judged by SDS–PAGE and Western blot analysis, were pooled and loaded at 1 ml/min into a column (1 · 2 cm) of SP Sepharose Fast Flow which had been pre-equilibrated with buffer C (10 mM phosphate buffer, pH 8.0, 5% glycerol). After washing with at least 10 times bed volume of buffer C, the rIspC was eluted in a sharp A280 peak following a linear 0–500 mM NaCl gradient in buffer C (30 ml; fraction size, 1 ml). The fractions containing rIspC were pooled, assessed for its purity by SDS–PAGE, and quantified by using the Bradford method [4] with bovine serum albumin (BSA) as a standard. Estimation of protein extinction coefficient. The molar extinction coefficient (e) of IspC at 280 nm was calculated from its amino acid sequence (i.e. the number of tryptophan, tyrosine, and cysteine residues within the protein) [38,40] according to the method of Gill and von Hippel [12]. N-terminal sequencing. The N-terminal sequence of rIspC was determined using Edman degradation chemistry. Following separation of the purified protein by SDS–PAGE and electrotransfer onto a PVDF membrane as described [21], the protein band was visualized by staining with 0.1% (w/v) Ponsceau S and excised for N-terminal sequencing performed at the Biotechnology Research Institute, National Research Council Canada (Montreal, Quebec, Canada). Mass spectrometry. Electrospray ionization (ESI) mass spectrum was acquired on Waters Micromass Global Q-TOF mass spectrometer coupled to a Waters capillary HPLC (Milford, Massachusetts, USA). The mass spectrometer was calibrated with a mixture of proteins: [glu1]-fibrinopeptide B human, horse heart myoglobin, and chicken lysozyme(Sigma). The calibration for intact protein analysis was checked by running cytochrome c, which resulted in a mass accuracy of ±1 amu. The purified IspC at a concentration of 10 pmol/ll in water was loaded onto an Atlantis dC18 trap with a 50-lm, ID silica tubing in place of a capillary HPLC column. The autosampler parameters included sample loading flow rate of 15 ll/min for 3 min, which was then decreased to 5 ll/min for the remainder part of the run. The gradient was delivered at a flow rate of 7 ll/min which was split to a flow rate of through the column 300 nl/min
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through the trap. The linear binary gradient was formed from A, 2% acetonitrile + 0.2% formic acid (FA) and B, 90% methanol + 0.5% acetic acid. The gradient profile was 2% B for 3 min, increased to 100% over the time interval 3.0–12.0 min. The system was maintained at 100% B for 1.5 min, then returned to 2% B in 0.5 min and allowed to re-equilibrate for 15.4 min. The concentrated sample (10 pmol/ll) was also infused directly after dilution 1:4 with the injection buffer (0.2% FA + 0.5% trifluoroacetic acid) and run on the mass spectrometer operated in the positive ion electrospray mode over the mass range 500–2000 amu in the continuum mode, with a scan time of 5.0 s and an interscan time 0.1 s. Other instrument parameters include a capillary voltage of 3.5 kV and a cone voltage of 100 V. Data acquisition and analysis were accomplished using the MassLynx version 4.1 software. Deconvolution of the positive ESI mass spectra was accomplished using MaxEnt1.
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Results and discussion Improvement of IspC expression in E. coli Part of this study aimed to develop a protocol for the generation of sufficient amount of homogeneous rIspC from an E. coli expression host, which is needed for biochemical (i.e. cleavage site on peptidoglycan substrate) and structural analysis (i.e. CD, FITR, Raman, and Xray crystallography). The conditions for induction of protein expression and purification strategy were examined here to achieve this objective. In a previous study [38], we have demonstrated the product of ispC gene coding for an 86-kDa protein of 774 amino acids being expressed in E. coli BL21(DE3)/pLysS cells harboring pIspC under the control of T7 promoter. However, induction with 1 mM IPTG added to the culture (OD590, 0.6–1.0) at 37 C for 3 h led to an apparent cell lysis as indicated by an initial increase followed by a decrease in the OD590 of induced culture. The cell lysis was also observed as formation of fibrous materials in the culture. This, most likely due to the peptidoglycan hydrolytic nature of IspC [38], has resulted in the production of limited amount of rIspC. To overcome this problem, we expressed the protein in E. coli Rosetta (DE3)/pLysS and allowed the induction earlier (OD590, 0.4–0.5) with IPTG at 37 C for a shorter period (2 h only) followed by incubation of the cell culture at 4 C overnight (16–18 h). Under these conditions, the level of IspC expression in E. coli was significantly increased by at least 4-fold, as judged by comparison to the yield of purified protein under the previous expression conditions [38], and the recombinant protein remained in a soluble form. Most likely during expression at a low temperature, the hydrolytic activity of IspC was minimized to prevent the cell lysis and thus improve the protein yield over a longer period of induction. Purification of rIspC The rIspC protein is fused with a 6-histidine tag at its Cterminus which is specifically designed for the ease of protein purification by metal-chelate affinity chromatography [15,28]. The purity of rIspC following chromatography on a column of Ni–NTA superflow was at least 80%, as
Fig. 1. SP Sepharose Fast Flow chromatography of the partially purified rIspC preparation. The rIspC protein, after partial purification by Ni– NTA Superflow affinity chromatography, was applied onto a column of SP Sepharose Fast Flow (1 · 2 cm) and eluted with a 0–500 mM NaCl linear gradient in 30 ml of 10 mM phosphate buffer (pH 8.0) containing 5% glycerol.
estimated by SDS–PAGE (see Fig. 2, lane 5) with one major contaminated protein of 33 kDa. IspC has an alkaline pI of 9.4 calculated from its amino acid sequence. Based on this property, cation-exchange chromatography was explored to further purify the protein using a column of SP Sepharose Fast Flow. The protein was eluted in a sharp peak at 300 mM NaCl following a 0–500 mM NaCl linear gradient (Fig. 1) and showed electrophoretic homogeneity on SDS–PAGE (Fig. 2, lane 6). The yield of the homogeneous IspC was estimated to be approximately 6 mg from 10 l culture. As was the case in our previous studies [38,40], the final preparation of IspC displayed 1
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20.0 Fig. 2. SDS–PAGE analysis of the rIspC protein preparations by chromatographic procedures. Lane 1, protein standards with their molecular mass in kDa (left); lane 2, total proteins from non-induced E. coli/pIspC equivalent to 1 ml of culture with an A590 of 0.2; lane 3, total proteins from IPTG-induced E. coli/pIspC equivalent to 1 ml of culture with an A590 of 0.2; lane 4, the crude soluble protein extract from 1 ml of culture with an A590 of 0.3; lane 5, the protein preparation (6 lg) from Ni– NTA Superflow; lane 6, protein preparation (3 lg) from SP Sepharose Fast Flow.
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(e) of IspC. Gill and von Hippel [12] described a method of calculating e from amino acid sequence with very acceptable limits of errors (to ±5% in most cases) by which the molar extinction coefficient of IspC was calculated to be 1.7851 · 105/M/cm at 280 nm. N-terminal sequencing showed that the sequence of the first 12 N-terminal residues of purified rIspC was NH2TVGGQLQDSLTG. This sequence matched exactly with that of residues 24–35 of the deduced amino acid sequence of IspC [38,40] (Fig. 3). This result further confirmed the identity of purified protein and indicted that the L. monocytogenes IspC contained an N-terminal signal peptide of 23 residues which had been removed by an E. coli signal peptidase-mediated cleavage between Thr 23 and Thr 24. Removal of the 23 amino acid N-terminal signal peptide resulted in a mature protein with a calculated molecular weight of 83.143 kDa. This result is in contrast to the predicted cleavage site for the N-terminal signal peptide between Leu 29 and Gln 30 (Fig. 3) and illustrates that knowledge of a cleavage site requires experimental determination. We have previously demonstrated that IspC was anchored on the cell surface through its C-terminal cell wall binding domain (CWBD) made up of seven GW modules [38]. This, together with the present findings, leads to proposal that the newly synthesized IspC in the cytoplasm is anchored on the cell surface with the aid of the N-terminal signal peptide which directs the targeting of the IspC precursor to the cytoplasmic membrane and then the translocation across this membrane. Following the removal of the signal peptide by a signal peptidase and the release of
peptidoglycan hydrolase activity and immunogenicity only to the anti-serum RaL fromrabbits infected with live L. monocytogenes but not with RaK from rabbits immunized with heat-killed bacteria. This suggests that the structural integrity and biochemical properties of IspC are not influenced by its recombinant configuration, which is thus suitable for biochemical, structural, and functional characterization. Characterization of rIspC by N-terminal sequencing and mass spectrometry Biochemical and structural analysis of IspC requires accurate determination of protein concentration in solution. One simple and rapid means to measure the concentration is to use UV–visible spectrophotometry in combination with knowledge of the extinction coefficient
Fig. 3. Alignment of the determined N-terminal sequence of purified rIspC with the deduced N-terminal sequence of IspC precursor. The sequence of the first 12 N-terminal residues (bold) of purified rIspC was experimentally determined as described in Materials and methods. The IspC precursor sequence was from previous studies [40,38]. Cleavage between Thr23 and Thr24 by a signal peptidase leading to formation of the mature IspC is indicated by an arrow.
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Fig. 4. Electrospray ionization (ESI) mass spectrum of the purified IspC protein at a concentration of 10 pmol/ll. The ESI mass spectral data (top) was deconvoluted using MaxEnt1 algorithm to derive the zero charge state spectrum which provided a molecular weight of 84.187 kDa for the purified IspC (bottom).
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the protein from the extracytoplasmic side of the membrane, the mature IspC protein is displayed on the surface via binding of its CWBD to the cell wall component lipoteichoic acid, as demonstrated in a number of other surface proteins from Gram-positive bacteria including Listeria [1,5,17]. The IspC protein was expressed as a product with a Cterminal fusion of eight residues including a 6-histidine tag. Mass spectrometry analysis of the purified rIspC revealed its molecular weight of 84.187 kDa (Fig. 4), which is essentially the same as the deduced molecular weight of 84.219 kDa after the removal of the N-terminal signal peptide and the addition of eight C-terminal residues have been taken into consideration. The mass spectral data is in good agreement with the N-terminal sequencing result. Conclusion By using a combination of altered expression conditions (i.e. different E. coli host, earlier IPTG induction, shorter period of induction at a high temperature, longer period of induction at a low temperature), metal-chelate affinity chromatography, and cation-exchange chromatography, we successfully produced in a sufficient quantity a biologically active, highly purified recombinant form of IspC, a novel immunogenic surface autolysin from L. monocytogenes serotype 4b [40,38]. IspC possesses an N-terminal signal peptide of 23 residues that is cleavable by an E. coli signal peptidase. This homogeneous protein material is being used for biochemical, structural, and functional studies. Acknowledgments We are grateful to the technical support and discussion from H. Dan, W.L. Yu, B.S. Luo, J. Bennett, M.-E. Auclair, H. McRae, D. Todoric, M. Mallory, and E. Trottier. References [1] T. Baba, O. Schneewind, Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus, EMBO J. 15 (1996) 4789–4797. [2] G. Bernadsky, T.J. Beveridge, A.J. Clarke, Analysis of the sodium dodecyl sulfate-stable peptidoglycan autolysins of select Gramnegative pathogens by using renaturing polyacrylamide gel electrophoresis, J. Bacteriol. 176 (1994) 5225–5232. [3] A.M. Berry, J.C. Paton, Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins, Infect. Immun. 68 (2000) 133–140. [4] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [5] L. Braun, S. Dramsi, P. Dehoux, H. Bierne, G. Lindahl, P. Cossart, InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association, Mol. Microbiol. 25 (1997) 285–294. [6] D. Cabanes, O. Dussurget, P. Dehoux, P. Cossart, Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence, Mol. Microbiol. 51 (2004) 1601–1614.
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