In vitro and in vivo self-cleavage of Streptococcus pneumoniae signal peptidase I.

We have previously demonstrated that Streptococcus pneumoniae signal peptidase (SPase) I catalyzes a self-cleavage to result in a truncated product, SPase37-204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. Bacteriol.183, 621-627]. In this study, we investigated the effect of phospholipid on invitro self-cleavage of S. pneumoniae SPase I. In the presence of phospholipid, the self-cleavage predominantly occurred at one cleavage site between Gly36-His37, whereas the self-cleavage occurred at multiple sites in the absence of phospholipid, and two additional self-cleavage sites, Ala65-His66 and Ala143-Phe144, were identified. All three self-cleavage sites strongly resemble the signal peptide cleavage site and follow the (-1, -3) rule for SPase I recognition. Kinetic analysis demonstrated that self-cleavage is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25-fold higher than that in the absence of phospholipid. Biochemical analysis demonstrated that SPase37-204, the major product of the self-cleavage totally lost activity to cleave its substrates, indicating that the self-cleavage resulted in the inactivation of the enzyme. More importantly, the self-cleavage was demonstrated to be happening in vivo in all the growth phases of S. pneumoniae cells. The bacterial cells keep the active SPase I at the highest level in exponential growth phase, suggesting that the self-cleavage may play an important role in regulating the activity of the enzyme under different conditions.     

Analysis of a Streptococcus pneumoniae gene encoding signal peptidase I and overproduction of the enzyme

The spi gene of Streptococcus pneumoniae was cloned and its nucleotide sequence was determined. It encodes a protein of 204 amino acids that is homologous to bacterial signal peptidase I proteins. The S. pneumoniae protein contains all of the conserved amino acid sequence motifs previously identified in this enzyme from both prokaryotic and eukaryotic sources. Sequence comparisons revealed several additional motifs characteristic of the enzyme. The cloned S. pneumoniae gene complemented an Escherichia coli mutant defective in its leader peptidase gene. Expression of the spi gene in S. pneumoniae appeared to be essential for viability. The cloned gene was shown to produce a polypeptide of approximately 20 kDa. Overproduction of the S. pneumoniae spi gene in an E. coli expression system gave a native protein product, soluble in the presence of a non-ionic detergent, which should be amenable to structural determination.     

Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae

The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.     

Development of an internally quenched fluorescent substrate and a continuous fluorimetric assay for Streptococcus pneumoniae signal peptidase I

Signal peptidase (SPase) I is responsible for the cleavage of signal peptides of many secreted proteins in bacteria and serves as a potential target for the development of novel antibacterial agents due to its unique physiological and biochemical properties. In this paper, we describe a novel fluorogenic substrate, KLTFGTVK(Abz)PVQAIAGY(NO2)EWL, in which 2-aminobenzoic acid (Abz) and 3-nitrotyrosine (Y(NO2)) were used as the fluorescent donor and acceptor, respectively. The substrate can be cleaved by both Streptococcus pneumoniae and Escherichia coli SPase I. Upon cleavage of the fluorogenic substrate by SPase I, the fluorescent intensity increases and can be monitored continuously by spectrofluorometer. Kinetic analysis with S. pneumoniae SPase I demonstrated that the K(m) value for the substrate is 118.1 microM, and the k(cat) value is 0.032 s(-1). Mass spectrometric analysis and peptide sequencing of the two cleaved products confirmed that the cleavage occurs specifically at the predicted site. More interestingly, the positively charged lysine in the N-terminus of the substrate was demonstrated to be important for effective cleavage. Phospholipids were found to stimulate the cleavage reaction. This stimulation by phospholipids is dependent upon the N-terminal charge of the substrate, indicating that the interaction of the positively charged substrate with anionic phospholipids is important for maintaining the substrate in certain conformation for cleavage. The substrate and assay described here can be readily automated and utilized for the identification of potential antibacterial agents.     

Cloning and characterization of archaeal type I signal peptidase from Methanococcus voltae

Archaeal protein trafficking is a poorly characterized process. While putative type I signal peptidase genes have been identified in sequenced genomes for many archaea, no biochemical data have been presented to confirm that the gene product possesses signal peptidase activity. In this study, the putative type I signal peptidase gene in Methanococcus voltae was cloned and overexpressed in Escherichia coli, the membranes of which were used as the enzyme source in an in vitro peptidase assay. A truncated, His-tagged form of the M. voltae S-layer protein was generated for use as the substrate to monitor the signal peptidase activity. With M. voltae membranes as the enzyme source, signal peptidase activity in vitro was optimal between 30 and 40°C; it was dependent on a low concentration of KCl or NaCl but was effective over a broad concentration range up to 1 M. Processing of the M. voltae S-layer protein at the predicted cleavage site (confirmed by N-terminal sequencing) was demonstrated with the overexpressed archaeal gene product. Although E. coli signal peptidase was able to correctly process the signal peptide during overexpression of the M. voltae S-layer protein in vivo, the contribution of the E. coli signal peptidase to cleavage of the substrate in the in vitro assay was minimal since E. coli membranes alone did not show significant activity towards the S-layer substrate in in vitro assays. In addition, when the peptidase assays were performed in 1 M NaCl (a previously reported inhibitory condition for E. coli signal peptidase I), efficient processing of the substrate was observed only when the E. coli membranes contained overexpressed M. voltae signal peptidase. This is the first proof of expressed type I signal peptidase activity from a specific archaeal gene product.     

Biochemical characterization of the CDP-D-arabinitol biosynthetic pathway in Streptococcus pneumoniae 17F

Streptococcus pneumoniae is a major human pathogen associated with many diseases worldwide. Capsular polysaccharides (CPSs) are the major virulence factor. The biosynthetic pathway of D-arabinitol, which is present in the CPSs of several S. pneumoniae serotypes, has never been identified. In this study, the genes abpA (previously known as abp1) and abpB (previously known as abp2), which have previously been reported to be responsible for nucleoside diphosphate (NDP)-D-arabinitol (the nucleotide-activated form of D-arabinitol) synthesis, were cloned. The enzyme products were overexpressed, purified, and analyzed for their respective activities. Novel products produced by AbpA- and AbpB-catalyzing reactions were detected by capillary electrophoresis, and the structures of the products were elucidated using electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. As a result, abpA was identified to be a D-xylulose-5-phosphate cytidylyltransferase-encoding gene, responsible for the transfer of CTP to D-xylulose-5-phosphate (D-Xlu-5-P) to form CDP-D-xylulose, and abpB was characterized to be a CDP-D-xylulose reductase-encoding gene, responsible for the conversion of CDP-D-xylulose to CDP-D-arabinitol as the final product. The kinetic parameters of AbpA for the substrates D-Xlu-5-P and CTP and those of AbpB for the substrate CDP-D-xylulose and the cofactors NADH or NADPH were measured, and the effects of temperature, pH, and cations on the two enzymes were analyzed. This study confirmed the involvement of the genes abpA and abpB and their products in the biosynthetic pathway of CDP-D-arabinitol.     

Solubilization and characterization of yeast signal peptidase

An efficient post-translational assay for solubilized yeast signal peptidase has been developed. The enzyme can be solubilized in nonionic detergent (0.5% Nikkol) without added salt, but salt increased the efficiency of solubilization. Radiosequencing of the cleaved substrate revealed that the enzyme removed the signal peptide. The substrate (prepro-alpha-factor) must be pretreated with sodium dodecyl sulfate to be cleaved. The enzyme displays a broad, alkaline pH optimum, retaining activity at pH 12. Moderately high temperatures (35 degrees C), excess detergent (greater than 0.5% Nikkol), or high salt (greater than 300 mM KOAc) will inactivate the enzyme. Phosphatidylcholine is necessary for optimal activity. The optimal ratio of Nikkol:lipid:sodium dodecyl sulfate is 6.4:2.2:1. The membrane association of yeast signal peptidase is resistant to carbonate extraction, indicating that it is an integral membrane protein.     

Mammalian signal peptidase: partial purification and general characterization of the signal peptidase from microsomal membranes of porcine pancreas

Signal peptidase has been enriched extensively from microsomal membranes of porcine pancreas. Microsomal membranes were washed with 1 M KCl and Brij 35, and then solubilized with 1% Nonidet P-40. The solubilized signal peptidase was purified by DEAE-cellulose chromatography and Sepharose CL-6B filtration. Cleavage of pre-human placental lactogen with the partially purified enzyme gave the mature form, whose NH2-terminus was identified as valine. The signal peptidase is heat-labile and approximately 90% of the enzymatic activity was lost at 60 degrees C within 1 min. The pH optimum of the activity was 7 to 8. Chymostatin and o-phenanthroline at concentrations of 2.5 mM inhibited the signal peptidase activity by 62% and 30%, respectively.     

The kinetic mechanism of 5-enolpyruvylshikimate-3-phosphate synthase from a gram-positive pathogen Streptococcus pneumoniae

The Streptococcus pneumoniae 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a potential novel antibacterial target. The enzyme catalyzes a reversible transfer of an enolpyruvyl group from phospho(enol)pyruvate (PEP) to shikimate 3-phosphate (S3P) to give EPSP with the release of inorganic phosphate (Pi). Understanding the kinetic mechanism of this enzyme is crucial to the design of novel inhibitors of this enzyme that may have potential as antibacterial agents. Steady-state kinetic studies of product inhibition and inhibition by glyphosate (GLP) have demonstrated diverse inhibition patterns of the enzyme. In the forward reaction, GLP is a competitive inhibitor with respect to PEP, but an uncompetitive inhibitor relative to S3P. Product inhibition shows that EPSP is a competitive inhibitor versus both PEP and S3P, suggesting that the forward reaction follows a random sequential mechanism. In the reverse reaction, GLP is an uncompetitive inhibitor versus EPSP, but a noncompetitive inhibitor versus Pi. This indicates that a non-productive quaternary complex might be formed between the enzyme, EPSP, GLP and Pi. Product inhibition in the reverse reaction has also been investigated. The inhibition patterns of the S. pneumoniae EPSP synthase are not entirely consistent with those of EPSP synthases from other species, indicating that EPSP synthases from different organisms may adopt unique mechanisms to catalyze the same reactions.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Functional characterization of penicillin-binding protein 1b from Streptococcus pneumoniae

The widespread use of antibiotics has encouraged the development of drug resistance in pathogenic bacteria. In order to overcome this problem, the modification of existing antibiotics and/or the identification of targets for the design of new antibiotics is currently being undertaken. Bifunctional penicillin-binding proteins (PBPs) are membrane-associated molecules whose transpeptidase (TP) activity is irreversibly inhibited by beta-lactam antibiotics and whose glycosyltransferase (GT) activity represents a potential target in the antibacterial fight. In this work, we describe the expression and the biochemical characterization of the soluble extracellular region of Streptococcus pneumoniae PBP1b (PBP1b*). The acylation efficiency for benzylpenicillin and cefotaxime was characterized by stopped-flow fluorometry and a 40-kDa stable TP domain was generated after limited proteolysis. In order to analyze the GT activity of PBP1b*, we developed an electrophoretic assay which monitors the fluorescence signal from PBP1b*-bound dansylated lipid II. This binding was inhibited by the antibiotic moenomycin and was specific for the GT domain, since no signal was observed in the presence of the purified functional TP domain. Binding studies performed with truncated forms of PBP1b* demonstrated that the first conserved motif of the GT domain is not required for the recognition of lipid II, whereas the second motif is necessary for such interaction.     

Purification and characterization of the RecA protein from Streptococcus pneumoniae

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.     

Type I signal peptidase: an overview

The signal hypothesis suggests that proteins contain information within their amino acid sequences for protein targeting to the membrane. These distinct targeting sequences are cleaved by specific enzymes known as signal peptidases. There are various type of signal peptidases known such as type I, type II, and type IV. Type I signal peptidases are indispensable enzymes, which catalyze the cleavage of the amino-terminal signal-peptide sequences from preproteins, which are translocated across biological membranes. These enzymes belong to a novel group of serine proteases, which generally utilize a Ser-Lys or Ser-His catalytic dyad instead of the prototypical Ser-His-Asp triad. Despite having no distinct consensus sequence other than a commonly found 'Ala-X-Ala' motif preceding the cleavage site, signal sequences are recognized by type I signal peptidase with high fidelity. Type I signal peptidases have been found in bacteria, archaea, fungi, plants, and animals. In this review, I present an overview of bacterial type I signal peptidases and describe some of their properties in detail.     

Phenotypic characterization of Streptococcus pneumoniae biofilm development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.     

Maturation of Streptococcus pneumoniae lipoproteins by a type II signal peptidase is required for ABC transporter function and full virulence

Cell surface lipoproteins are important for the full virulence of several bacterial pathogens, including Streptococcus pneumoniae. Processing of prolipoproteins seems to be conserved among different bacterial species, and requires type II signal peptidase (Lsp) mediated cleavage of the N-terminal signal peptide to form the mature lipoprotein. Lsp has been suggested as a target for new antibiotic therapies, but at present there are only limited data on the function of Lsp for Gram-positive bacterial pathogens. We have investigated the function and role during disease pathogenesis of the S. pneumoniae Lsp, which, blast searches suggest, is encoded by the gene Sp0928. Expression of Sp0928 protected Escherichia coli against the Lsp antagonist globomycin, and proteomics and immunoblot analysis demonstrated that deletion of Sp0928 prevented processing of S. pneumoniae prolipoproteins to mature lipoproteins. These data strongly suggest that Sp0928 encodes the S. pneumoniae Lsp. However, immunoblots of membrane-associated proteins, immunoelectron microscopy and flow cytometry assays all confirmed that in the absence of Lsp, immature lipoproteins were still attached to the cell surface. Despite preservation of lipoprotein attachment to the cell membrane, loss of S. pneumoniae Lsp resulted in several phenotypes associated with impaired lipoprotein function and reduced S. pneumoniae replication in animal models of infection.     

Regulation of signal peptidase by phospholipids in membrane: characterization of phospholipid bilayer incorporated Escherichia coli signal peptidase

Prokaryotic signal peptidases are membrane-bound enzymes. They cleave signal peptides from precursors of secretary proteins. To study the enzyme in its natural environment, which is phospholipid bilayers, we developed a method that allows us effectively to incorporate full-length Escherichia coli signal peptidase I into phospholipid vesicles. The membrane-bound signal peptidase showed high activity on a designed substrate. The autolysis site of the enzyme is separated from its catalytic site in vesicles by the lipid bilayer, resulting in a dramatic decrease of the autolysis rate. Phosphotidylethanolamine, which is the most abundant lipid in Escherichia coli inner membrane, is required to maintain activity of the membrane-incorporated signal peptidase. The maximal activity is achieved at about 55% phosphotidylethanolamine. Negatively charged lipids, which are also abundant in Escherichia coli inner membrane, enhances the activity of the enzyme too. Its mechanism, however, cannot be fully explained by its ability to increase the affinity of the substrate to the membrane. A reaction mechanism was developed based on the observation that cleavage only takes place when the enzyme and the substrate are bound to the same vesicle. Accordingly, a kinetic analysis is presented to explain some of the unique features of phospholipid vesicles incorporated signal peptidase, including the effect of lipid concentration and substrate-vesicle interaction.