Báo cáo khoa học: Enzymatic investigation of the Staphylococcus aureus type I signal peptidase SpsB – implications for the search for novel antibiotics ppt

Enzymatic investigation of the Staphylococcus aureus type I signal peptidase SpsB – implications for the search for novel antibiotics ´ Smitha Rao C.V.1, Katrijn Bockstael2, Sangeeta Nath3, Yves Engelborghs3, Jozef Anne1 and Nick Geukens1,* Laboratory of Bacteriology, Katholieke Universiteit Leuven, Belgium Laboratory for Medicinal Chemistry, Katholieke Universiteit Leuven, Belgium Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Belgium Keywords arylomycin; IsaA; signal peptidase; SpsB; Staphylococcus aureus Correspondence ´ J Anne, Laboratory of Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium Fax: +32 16 337 340 Tel: +32 16 337 371 E-mail: jozef.anne@rega.kuleuven.be Website: http://www.kuleuven.be/ bacteriology/ *Present address PharmAbs, Katholieke Universiteit Leuven Antibody Center, Belgium (Received 11 July 2008, revised 10 March 2009, accepted April 2009) doi:10.1111/j.1742-4658.2009.07037.x Staphylococcus aureus has one essential type I signal peptidase (SPase), SpsB, which has emerged as a potential target in the search for antibiotics with a new mode of action In this framework, the biochemical properties of SpsB are described and compared with other previously characterized SPases Two different substrates have been used to assess the in vitro processing activity of SpsB: (a) a native preprotein substrate immunodominant staphylococcal antigen A and (b) an intramolecularly quenched fluorogenic synthetic peptide based on the sequence of the SceD preprotein of Staphylococcus epidermidis for fluorescence resonance energy transfer-based analysis Activity testing at different pH showed that the enzyme has an optimum pH of approximately The pH-rate profile revealed apparent pKa values of 6.6 and 8.7 Similar to the other SPases, SpsB undergoes self-cleavage and, although the catalytic serine is retained in the self-cleavage product, a very low residual enzymatic activity remained In contrast, a truncated derivative of SpsB, which was nine amino acids longer at the N-terminus compared to the self-cleavage product, retained activity The specificity constants (kcat ⁄ Km) of the full-length and the truncated derivative were 1.85 ± 0.13 · 103 m)1Ỉs)1 and 59.4 ± 6.4 m)1Ỉs)1, respectively, as determined using the fluorogenic synthetic peptide substrate These observations highlight the importance of the amino acids in the transmembrane segment and also those preceding the catalytic serine in the sequence of SpsB Interestingly, we also found that the activity of the truncated SpsB increased in the presence of a non-ionic detergent Staphylococcus aureus is a frequent commensal of the human skin and nose, but is also responsible for a wide array of infections, ranging from minor skin infection to life-threatening conditions such as endocarditis and haemolytic pneumonia [1] This Gram-positive bacterium is the most common cause of nosocomial infections S aureus infections are becoming increasingly difficult to treat because the bacterium has evolved into a highly successful pathogen when it comes to antibiotic resistance [2] The emergence and spread of strains such as methicillin-resistant S aureus, vancomycin-intermediate S aureus and vancomycin-resistant S aureus has become a major concern New drugs are being developed and launched in the market, but most currently Abbreviations CBB, Coomassie brilliant blue; FRET, fluoresence resonance energy transfer; pre-IsaA, immunodominant staphylococcal antigen A precursor; sc-SpsB, self-cleavage product of SpsB; SPase, signal peptidase; tr-SpsB, N-terminally truncated SpsB derivative 3222 FEBS Journal 276 (2009) 3222–3234 ª 2009 The Authors Journal compilation ª 2009 FEBS Rao C V S et al developed antimicrobials are derivatives of well-known and extensively used compound classes [3] and, therefore, the chances that the bacterium would develop cross-resistance to these drugs are quite high However, in the search for novel classes of antibiotics for combating this pathogen, new drug targets [4,5] have been in focus in recent years Proteins that are destined for transmembrane transport are produced in the cell as preproteins with a signal peptide that is recognized and cleaved off by signal peptidases (SPases) [6,7] Bacterial type I SPases are membrane-bound endopeptidases that remove the signal peptide from proteins on translocation across the cytoplasmic membrane [8] SPases are unique serine proteases, and differ from the classical serine proteases in that they act using a serine ⁄ lysine catalytic dyad mechanism [9–11] Both Gram-positive and Gram-negative bacterial SPases have regions of high sequence similarity, which are referred to as boxes A to E [6], although they differ in certain aspects, including size, the number of transmembrane segments and substrate specificity [12] SPases have already been proposed as antibiotic targets because of their essentiality, the easier accessibility of the catalytic domain for potential inhibitors as a result of being located on the outer side of the cytoplasmic membrane, and the different catalytic mechanism employed compared to that used by eukaryotic SPases [8] LepB, the SPase of Escherichia coli, is the most extensively studied SPase The crystal structure of the soluble form of this enzyme has been determined [13–15] and NMR data are also available for the full-length enzyme [16] and the soluble derivative [17] Among the Gram-positive bacteria, functional analysis and biochemical characterization of type I SPases have been described for Bacillus subtilis [18], Bacillus amyloliquefaciens [19], Streptomyces lividans [20] and Streptococcus pneumoniae [21] For S aureus, two genes, designated spsA and spsB, were identified encoding homologues of SPase of which only the latter was shown to be essential [22] SpsB also has been functionally expressed in E coli and was demonstrated to process E coli preproteins in vivo [22] It was predicted that SpsA is an inactive SPase homologue Furthermore, SpsB, but not SpsA, was shown to be responsible for the removal of the N-terminal leader of AgrD, in vitro, which also suggested a role for type I SPases in quorum sensing [23] In the present study, we report the biochemical characteristics of SpsB and describe two different in vitro assays for the enzyme: one with its native substrate immunodominant staphylococcal antigen A precursor (pre-IsaA) and the other with a fluorogenic synthetic peptide, SceD In addition, a nonmembrane-bound, S aureus type I signal peptidase SpsB truncated SpsB (tr-SpsB) was designed to determine the effect of removal of the transmembrane segment of SpsB The specific activities of the full-length and the truncated SpsB were compared using the fluoresence resonance energy transfer (FRET)-based assay involving the SceD peptide Results and discussion Expression and purification of the full-length SpsB and preprotein IsaA The gene encoding SpsB was amplified by PCR using primers that were also designed to bring about two modifications: the incorporation of NdeI and EcoRI restriction sites (at the 5¢ and 3¢ ends, respectively) and a hexa-histidine-encoding sequence for obtaining a His-tag at the N-terminus of the produced protein to facilitate purification The fragments were cloned after the T7 promoter in pET-3a plasmid The proteins expressed in E coli BL21(DE3)pLysS were purified (see Experimental procedures) and analyzed by SDS ⁄ PAGE The purification of the full-length SpsB normally yielded samples of sufficient purity (> 95%) and concentration (30–40 lm) (see Supporting information, Fig S1A) The gene encoding pre-IsaA was amplified by PCR using oligonucleotides that were also designed to incorporate NcoI and EcoRI restriction sites and sequences encoding a hexa-histidine tag and a c-Myc tag to appear at N- and C-terminal ends of the expressed protein, respectively The c-Myc tag was included to facilitate immunodetection of the protein The gene was cloned in pET-23d and expressed in E coli BL21(DE3)pLysS Pre-IsaA (predicted MW = 26.2 kDa, including hexa-his and c-Myc tag) was purified, refolded and used in the in vitro assay after analysis by SDS ⁄ PAGE (see Supporting information, Fig S1B) In vitro preprotein processing by SpsB The choice of the preprotein substrate was made after a preliminary analysis of secreted proteins indicated in the genomic sequence data of S aureus The criteria for selection of the substrate were a good prediction of the presence and location of the signal peptide cleavage site (as indicated by signalp 3.0 server [24]), and non-indication as a general protease The latter is not desirable because it could degrade the SPase itself Pre-IsaA was selected as the substrate for this assay IsaA was first identified as one of the four proteins expressed in vivo during sepsis caused by methicillin- FEBS Journal 276 (2009) 3222–3234 ª 2009 The Authors Journal compilation ª 2009 FEBS 3223 S aureus type I signal peptidase SpsB Rao C V S et al resistant S aureus [25] It is a lytic transglycosylase and was proposed to be important for the virulence of S aureus along with another paralogue, SceD [26], which is also a substrate of SpsB The in vitro assay was carried out in the presence of a protease inhibitor cocktail and the reactions were stopped at different time intervals in the range 0–15 h Analysis of the assay products by means of immunodetection of pre-IsaA ⁄ IsaA revealed the presence of two bands in the sample containing the preprotein substrate and SpsB (Fig 1A) The upper band corresponds to the unprocessed preprotein and the lower one to the mature protein (predicted MW = 22.5 kDa) As shown in Fig 1A, the substrate remained unprocessed in the absence of the enzyme The amount of preprotein processed increased over time (Fig 1A) After 15 h, unprocessed protein remained and the addition of fresh SpsB followed by incubation for h did not result in any significant improvement in processing Similar observations of incomplete processing have been made previously with in vitro assays involving the SPases and preproteins [21,27–29] and it has been suggested that the remaining preprotein is probably in an unprocessible state The addition of arylomycin A2 [15], a known SPase inhibitor, to the reaction mixture containing the enzyme and the substrate did not result in pre-IsaA processing (Fig 1B) These observations confirmed the in vitro activity of the purified SpsB The specificity of the preprotein cleavage by SpsB was confirmed by N-terminal sequence analysis of the mature protein obtained The substrate was cleaved at the predicted site (Fig 2), following the ()1, )3) or ‘Ala-X-Ala’ rule [30] This substrate could also be processed by LepB, the SPase of the Gram-negative bacterium E coli under the same in vitro conditions described in the Fig SPase recognition sequence and cleavage sites of the SpsB substrates used in the present study: Showing part of the sequence of the IsaA precursor (upper row) and the sequence of the SceD peptide (lower row) with the SPase cleavage sites indicated The SPase recognition sequence, which consists of small aliphatic residues at positions )1 and )3 relative to the cleavage sites, is shown in bold present study (data not shown), indicating the broad substrate specificity of the SPases A continuous fluorometric assay for SpsB and measurement of its specific enzymatic activity A FRET-based assay was designed for SpsB The substrate used was an internally quenched peptide based on the sequence of the signal peptide region of Staphylococcus epidermidis SceD preprotein and containing 4-(4-dimethylaminophenylazo)benzoic acid ⁄ 5-((2-aminoethyl)amino)-1-naphthalenesulfonic acid as the FRET pair SpsB was found to cleave this peptide efficiently in the presence of protease inhibitor cocktail, to which the bacterial type I SPases are resistant (see Supporting information, Fig S2) The standardized assays were carried out in microtitre plates in a total volume of 100 lL in the assay buffer (50 mm Tris-HCl pH 8; 0.5% Triton X-100) with a certain concentration of SpsB (final concentration of lm in most cases) and SceD peptide (final concentration of or 10 lm, as indicated) dissolved in dimethylformamide The final concentration of dimethylformamide in the reaction mixtures was 1% The hydrolysis of the peptide was measured by the increase in fluorescence on a Fig Preprotein processing by SpsB (full-length): (A) as function of time and (B) blocked by arylomycin A2 SpsB and pre-IsaA (at final concentrations of and 10 lM, respectively) were incubated at 37 °C in the assay buffer for different time periods The proteins were separated on 12.5% (w ⁄ v) SDS ⁄ PAA gels and analyzed by western blotting and chemiluminescent detection (A) Lane 1, SpsB (control); lane 2, pre-IsaA (control); lane 3, SpsB and pre-IsaA at time = 0; lanes 4–10, pre-IsaA processing by SpsB with increase in time; lane 11, pre-IsaA processing by SpsB after 900 followed by addition of fresh SpsB (final concentration of lM) and further incubation for h (B) SpsB and pre-IsaA (final concentrations of and 10 lM, respectively) were incubated without and with arylomycin A2 (final concentration of 200 lM) for 15 h at 37 °C Lane 1, pre-IsaA processing by SpsB; lane 2, pre-IsaA processing blocked by arylomycin A2 3224 FEBS Journal 276 (2009) 3222–3234 ª 2009 The Authors Journal compilation ª 2009 FEBS Rao C V S et al microplate reader using excitation and emission wavelengths of 340 and 510 nm, respectively As part of the validation of the assay, inhibitor arylomycin A2 was used and no increase in fluorescence was observed in the time-based scan (see Supporting information, Fig S3A), confirming that the peptide remains uncleaved when the enzyme activity is inhibited A dose-dependent response to arylomycin A2 was plotted (see Supporting information, Fig S3B) and the IC50 of the inhibitor against SpsB was found to be lm (0.82 lgỈmL)1) The specificity of the proteolytic reaction of the SceD peptide by SpsB was also analysed by RP-HPLC to determine whether the SceD peptide was cleaved at the expected cleavage site The resulting fractions were subjected to ESI-MS and it was found that the fluorogenic synthetic SceD peptide was cleaved by S aureus SpsB at a single cleavage site and that this cleavage occurred specifically at the predicted site located at the A-S bond (data not shown) The sequence and cleavage site of the SceD peptide are shown in Fig It should be noted that, at high substrate concentrations (> 20 lm), the linear correlation between the fluorescence and the substrate concentration is lost as a result of the inner filter effect The inner filter effect is the phenomenon observed when the fluorescent light is absorbed by quenching groups on neighbouring substrates or cleaved product molecules, allowing only a fraction of light to be detected by the instrument Therefore, only kcat ⁄ Km could be measured using the condition [S]