Crystal structures of CbpF complexed with atropine and ipratropium reveal clues for the design of novel antimicrobials against Streptococcus pneumoniae

Background

Streptococcus pneumoniae is a major pathogen responsible of important diseases worldwide such as pneumonia and meningitis. An increasing resistance level hampers the use of currently available antibiotics to treat pneumococcal diseases. Consequently, it is desirable to find new targets for the development of novel antimicrobial drugs to treat pneumococcal infections. Surface choline-binding proteins (CBPs) are essential in bacterial physiology and infectivity. In this sense, esters of bicyclic amines (EBAs) such as atropine and ipratropium have been previously described to act as choline analogs and effectively compete with teichoic acids on binding to CBPs, consequently preventing in vitro pneumococcal growth, altering cell morphology and reducing cell viability.

Methods

With the aim of gaining a deeper insight into the structural determinants of the strong interaction between CBPs and EBAs, the three-dimensional structures of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, complexed with atropine and ipratropium, have been obtained.

Results

The choline analogs bound both to the carboxy-terminal module, involved in cell wall binding, and, unexpectedly, also to the amino-terminal module, that possesses a regulatory role in pneumococcal autolysis.

Conclusions

Analysis of the complexes confirmed the importance of the tropic acid moiety of the EBAs on the strength of the binding, through π–π interactions with aromatic residues in the binding site.

General significance

These results represent the first example describing the molecular basis of the inhibition of CBPs by EBA molecules and pave the way for the development of new generations of antipneumococcal drugs.     

Drastic reduction in the virulence of Streptococcus pneumoniae expressing type 2 capsular polysaccharide but lacking choline residues in the cell wall

The role of capsular polysaccharides and several virulence-related proteins in the pathogenic potential of Streptococcus pneumoniae has been studied extensively. Much less information is available about the role of the pneumococcal cell wall in virulence. In this communication we describe an experimental system that has allowed us to test - in a global way - the role of choline, a structural component of the pneumococcal cell wall, in virulence. We constructed double mutants of S. pneumoniae which have lost the auxotrophic requirement for choline and which were also blocked from utilizing choline from the growth medium. Such a double mutant expressing type 2 capsule but completely lacking choline residues from its cell wall grew well both in vitro and also in the blood of infected mice, but showed striking reduction of virulence approaching that of a capsule-free strain in several models of pneumococcal disease including the capacity to attach and invade a human nasopharyngeal cell line; nasal colonization and intraperitoneal and intravenous inoculation in the mouse. The findings allow one to separate the choline requirement of S. pneumoniae into two sharply defined classes: the need for choline in growth and replication which can be effectively bypassed and the need for choline in pneumococcal virulence that appears to be irreplaceable. The double mutant should be a useful experimental tool to dissect the mechanism of choline requirement in various stages of pneumococcal virulence.     

Structural and thermodynamic characterization of Pal, a phage natural chimeric lysin active against pneumococci

Pal amidase, encoded by pneumococcal bacteriophage Dp-1, represents one step beyond in the modular evolution of pneumococcal murein hydrolases. It exhibits the choline-binding module attaching pneumococcal lysins to the cell wall, but the catalytic module is different from those present in the amidases coded by the host or other pneumococcal phages. Pal is also an effective antimicrobial agent against Streptococcus pneumoniae that may constitute an alternative to antibiotic prophylaxis. The structural implications of Pal singular structure and their effect on the choline-amidase interactions have been examined by means of several techniques. Pal stability is maximum around pH 8.0 (Tm approximately 50.2 degrees C; DeltaHt = 183 +/- 4 kcal mol(-1)), and its constituting modules fold as two tight interacting cooperative units whose denaturation merges into a single process in the free amidase but may proceed as two well resolved events in the choline-bound state. Choline titration curves reflect low energy ligand-protein interactions and are compatible with two sets of sites. Choline binding strongly stabilizes the cell wall binding module, and the conformational stabilization is transmitted to the catalytic region. Moreover, the high proportion of aggregates formed by the unbound amidase together with choline preferential interaction with Pal dimers suggest the existence of marginally stable regions that would become stabilized through choline-protein interactions without significantly modifying Pal secondary structure. This structural rearrangement may underlie in vitro "conversion" of Pal from the low to the full activity form triggered by choline. The Pal catalytic module secondary structure could denote folding conservation within pneumococcal lytic amidases, but the number of functional choline binding sites is reduced (2-3 sites per monomer) when compared with pneumococcal LytA amidase (4-5 sites per monomer) and displays different intermodular interactions.     

肺炎链球菌磷壁酸胆碱成分介导细菌的侵袭

分析肺炎链球菌细胞壁胆碱成分在其侵袭宿主细胞的过程中的作用。通过肺炎链球菌对人脐静脉内皮细胞的侵袭实验 ,观察血小板活化因子受体 (PAF R)拮抗剂BN 5 2 0 2 1对肺炎链球菌侵袭率的变化 ,以及乙醇胺取代细胞壁胆碱后肺炎链球菌侵袭率的变化。研究发现受体拮抗剂BN 5 2 0 2 1处理活化血管内皮细胞后 ,肺炎链球菌的侵袭率显著降低 (P <0 0 1 ) ,乙醇胺取代肺炎链球菌细胞壁成份中的胆碱同样降低了细菌对活化内皮细胞的侵袭 (P<0 0 1 )。实验表明肺炎链球菌可能是通过脂磷壁酸和磷壁酸的胆碱成分与内皮细胞表面的血小板活化因子受体结合来介导侵袭作用的     

Ofloxacin-like antibiotics inhibit pneumococcal cell wall-degrading virulence factors

The search for new drugs against Streptococcus pneumoniae (pneumococcus) is driven by the 1.5 million deaths it causes annually. Choline-binding proteins attach to the pneumococcal cell wall through domains that recognize choline moieties, and their involvement in pneumococcal virulence makes them potential targets for drug development. We have defined chemical criteria involved in the docking of small molecules from a three-dimensional structural library to the major pneumococcal autolysin (LytA) choline binding domain. These criteria were used to identify compounds that could interfere with the attachment of this protein to the cell wall, and several quinolones that fit this framework were found to inhibit the cell wall-degrading activity of LytA. Furthermore, these compounds produced similar effects on other enzymes with different catalytic activities but that contained a similar choline binding domain; that is, autolysin (LytC) and the phage lytic enzyme (Cpl-1). Finally, we resolved the crystal structure of the complex between the choline binding domain of LytA and ofloxacin at a resolution of 2.6 Angstroms. These data constitute an important launch pad from which effective drugs to combat pneumococcal infections can be developed.     

Versatility of choline metabolism and choline-binding proteins in Streptococcus pneumoniae and commensal streptococci

The pneumococcal choline-containing teichoic acids are targeted by choline-binding proteins (CBPs), major surface components implicated in the interaction with host cells and bacterial cell physiology. CBPs also occur in closely related commensal species, Streptococcus oralis and Streptococcus mitis , and many strains of these species contain choline in their cell wall. Physiologically relevant CBPs including cell wall lytic enzymes are highly conserved between Streptococcus pneumoniae and S. mitis . In contrast, the virulence-associated CBPs, CbpA, PspA and PcpA, are S. pneumoniae specific and are thus relevant for the characteristic properties of this species.     

Characterization of LytA-like N-acetylmuramoyl-L-alanine amidases from two new Streptococcus mitis bacteriophages provides insights into the properties of the major pneumococcal autolysin

Two new temperate bacteriophages exhibiting a Myoviridae (phiB6) and a Siphoviridae (phiHER) morphology have been isolated from Streptococcus mitis strains B6 and HER 1055, respectively, and partially characterized. The lytic phage genes were overexpressed in Escherichia coli, and their encoded proteins were purified. The lytAHER and lytAB6 genes are very similar (87% identity) and appeared to belong to the group of the so-called typical LytA amidases (atypical LytA displays a characteristic two-amino-acid deletion signature). although they exhibited several differential biochemical properties with respect to the pneumococcal LytA, e.g., they were inhibited in vitro by sodium deoxycholate and showed a more acidic pH for optimal activity. However, and in sharp contrast with the pneumococcal LytA, a short dialysis of LytAHER or LytAB6 resulted in reversible deconversion to the low-activity state (E-form) of the fully active phage amidases (C-form). Comparison of the amino acid sequences of LytAHER and LytAB6 with that of the pneumococcal amidase suggested that Val317 might be responsible for at least some of the peculiar properties of S. mitis phage enzymes. Site-directed mutagenesis that changed Val317 in the pneumococcal LytA amidase to a Thr residue (characteristic of LytAB6 and LytAHER) produced a fully active pneumococcal enzyme that differs from the parental one only in that the mutant amidase can reversibly recover the low-activity E-form upon dialysis. This is the first report showing that a single amino acid residue is involved in the conversion process of the major S. pneumoniae autolysin. Our results also showed that some lysogenic S. mitis strains possess a lytA-like gene, something that was previously thought to be exclusive to Streptococcus pneumoniae. Moreover, the newly discovered phage lysins constitute a missing link between the typical and atypical pneumococcal amidases known previously.     

Inhibition of competence development, horizontal gene transfer and virulence in Streptococcus pneumoniae by a modified competence stimulating peptide

Competence stimulating peptide (CSP) is a 17-amino acid peptide pheromone secreted by Streptococcus pneumoniae. Upon binding of CSP to its membrane-associated receptor kinase ComD, a cascade of signaling events is initiated, leading to activation of the competence regulon by the response regulator ComE. Genes encoding proteins that are involved in DNA uptake and transformation, as well as virulence, are upregulated. Previous studies have shown that disruption of key components in the competence regulon inhibits DNA transformation and attenuates virulence. Thus, synthetic analogues that competitively inhibit CSPs may serve as attractive drugs to control pneumococcal infection and to reduce horizontal gene transfer during infection. We performed amino acid substitutions on conserved amino acid residues of CSP1 in an effort to disable DNA transformation and to attenuate the virulence of S. pneumoniae. One of the mutated peptides, CSP1-E1A, inhibited development of competence in DNA transformation by outcompeting CSP1 in time and concentration-dependent manners. CSP1-E1A reduced the expression of pneumococcal virulence factors choline binding protein D (CbpD) and autolysin A (LytA) in vitro, and significantly reduced mouse mortality after lung infection. Furthermore, CSP1-E1A attenuated the acquisition of an antibiotic resistance gene and a capsule gene in vivo. Finally, we demonstrated that the strategy of using a peptide inhibitor is applicable to other CSP subtype, including CSP2. CSP1-E1A and CSP2-E1A were able to cross inhibit the induction of competence and DNA transformation in pneumococcal strains with incompatible ComD subtypes. These results demonstrate the applicability of generating competitive analogues of CSPs as drugs to control horizontal transfer of antibiotic resistance and virulence genes, and to attenuate virulence during infection by S. pneumoniae.     

A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA.

Choline binding proteins are virulence determinants present in several Gram-positive bacteria. Because anchorage of these proteins to the cell wall through their choline binding domain is essential for bacterial virulence, their release from the cell surface is considered a powerful target for a weapon against these pathogens. The first crystal structure of a choline binding domain, from the toxin-releasing enzyme pneumococcal major autolysin (LytA), reveals a novel solenoid fold consisting exclusively of beta-hairpins that stack to form a left-handed superhelix. This unique structure is maintained by choline molecules at the hydrophobic interface of consecutive hairpins and may be present in other choline binding proteins that share high homology to the repeated motif of the domain.     

Interaction of the pneumococcal amidase with lipoteichoic acid and choline

The choline-containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N-acetyl-muramoyl-L-alanine-amidase (amidase) in aqueous solution [H?ltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690-1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA-Sepharose. The amidase could be eluted from the column with choline only. This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA-Sepharose, the amidase converted from the applied E-form (an inactive form of the amidase) to the active C-form, a process which up to now was known to be mediated only by the pneumococcal choline-containing wall teichoic acid. Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from choline-grown cells: the membrane-associated enzyme was present in the C-form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase activity in membrane fractions derived from ethanolamine-grown pneumococci, in which choline containing LTA is absent. The LTA-Sepharose-associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase-digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA. When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase-deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wall-mediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C-amidase was different: the cell-wall-converted enzyme was of high Mr, whereas the choline-converted and the LTA-Sepharose-eluted enzyme showed an apparent low molecular mass known for the E-form, when analyzed on sucrose gradients.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carboxy-terminal deletion analysis of the major pneumococcal autolysin.

Autolysins are endogenous enzymes that specifically degrade the covalent bonds of the cell walls and eventually can induce bacterial lysis. One of the best-characterized autolysins, the major pneumococcal LytA amidase, has evolved by the fusion of two domains, the N-terminal catalytic domain and the C-terminal domain responsible for the binding to cell walls. The precise biochemical role played by the six repeat units that form the C-terminal domain of the LytA amidase has been investigated by producing serial deletions. Biochemical analyses of the truncated mutants revealed that the LytA amidase must contain at least four units to efficiently recognize the choline residues of pneumococcal cell walls. The loss of an additional unit dramatically reduces its hydrolytic activity as well as the binding affinity, suggesting that the catalytic efficiency of this enzyme can be considerably improved by keeping the protein attached to the cell wall substrate. Truncated proteins lacking one or two repeat units were more sensitive to the inhibition by free choline than the wild-type enzyme, whereas the N-terminal catalytic domain was insensitive to this inhibition. In addition, the truncated proteins were inhibited by deoxycholate (DOC), and the expression of a LytA amidase lacking the last 11 amino acids in Streptococcus pneumoniae M31, a strain having a deletion in the lytA gene, conferred to the cells an atypical phenotype (Lyt+ DOC-) (cells autolysed at the end of the stationary phase but were not sensitive to lysis induced by DOC), which has been previously observed in some clinical isolates of pneumococci. Our results are in agreement with the existence of several choline-binding sites and suggest that the stepwise acquisition of the repeat units and the tail could be considered an evolutionary advantage for the enzyme, since the presence of these motifs increases its hydrolytic activity.     

Cloning, purification, and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin.

Cp-1, a small virulent bacteriophage infecting Streptococcus pneumoniae, encodes its own lytic enzyme (CPL). A fragment of Cp-1 DNA containing the gene cpl coding for CPL was cloned and expressed in high amounts in Escherichia coli. CPL was purified to electrophoretic homogeneity by using affinity chromatography on choline-Sepharose (T. Briese and R. Hakenbeck, Eur. J. Biochem. 146:417-427, 1985), and the enzyme showing a Mr of 39,000 was characterized as a muramidase. This muramidase required for in vivo and in vitro activity the presence of choline in the teichoic acids of the pneumococcal cell walls. Free choline or lipoteichoic acid noncompetitively inhibited the activity of CPL.     

Pneumococcal virulence factors: structure and function.

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.     

Insights into the structure-function relationships of pneumococcal cell wall lysozymes, LytC and Cpl-1

The LytC lysozyme belongs to the autolytic system of Streptococcus pneumoniae and carries out a slow autolysis with optimum activity at 30 degrees C. Like all pneumococcal murein hydrolases, LytC is a modular enzyme. Its mature form comprises a catalytic module belonging to the GH25 family of glycosyl-hydrolases and a cell wall binding module (CBM), made of 11 sequence repeats, that is essential for activity and specifically targets choline residues present in pneumococcal lipoteichoic and teichoic acids. Here we show that the catalytic module is natively folded, and its thermal denaturation takes place at 45.4 degrees C. However, the CBM is intrinsically unstable, and the ultimate folding and stabilization of the active, monomeric form of LytC relies on choline binding. The complex formation proceeds in a rather slow way, and all sites (8.0 +/- 0.5 sites/monomer) behave as equivalent (Kd = 2.7 +/- 0.3 mm). The CBM stabilization is, nevertheless, marginal, and irreversible denaturation becomes measurable at 37 degrees C even at high choline concentration, compromising LytC activity. In contrast, the Cpl-1 lysozyme, a homologous endolysin encoded by pneumococcal Cp-1 bacteriophage, is natively folded in the absence of choline and has maximum activity at 37 degrees C. Choline binding is fast and promotes Cpl-1 dimerization. Coupling between choline binding and folding of the CBM of LytC indicates a high conformational plasticity that could correlate with the unusual alternation of short and long choline-binding repeats present in this enzyme. Moreover, it can contribute to regulate LytC activity by means of a tight, complementary binding to the pneumococcal envelope, a limited motility, and a moderate resistance to thermal denaturation that could also account for its activity versus temperature profile.     

Mutations in the tacF gene of clinical strains and laboratory transformants of Streptococcus pneumoniae: impact on choline auxotrophy and growth rate

The nutritional requirement that Streptococcus pneumoniae has for the aminoalcohol choline as a component of teichoic and lipoteichoic acids appears to be exclusive to this prokaryote. A mutation in the tacF gene, which putatively encodes an integral membrane protein (possibly, a teichoic acid repeat unit transporter), has been recently identified as responsible for generating a choline-independent phenotype of S. pneumoniae (M. Damjanovic, A. S. Kharat, A. Eberhardt, A. Tomasz, and W. Vollmer, J. Bacteriol. 189:7105-7111, 2007). We now report that Streptococcus mitis can grow in choline-free medium, as previously illustrated for Streptococcus oralis. While we confirmed the finding by Damjanovic et al. of the involvement of TacF in the choline dependence of the pneumococcus, the genetic transformation of S. pneumoniae R6 by using S. mitis SK598 DNA and several PCR-amplified tacF fragments suggested that a minimum of two mutations were required to confer improved fitness to choline-independent S. pneumoniae mutants. This conclusion is supported by sequencing results also reported here that indicate that a spontaneous mutant of S. pneumoniae (strain JY2190) able to proliferate in the absence of choline (or analogs) is also a double mutant for the tacF gene. Microscopic observations and competition experiments during the cocultivation of choline-independent strains confirmed that a minimum of two amino acid changes were required to confer improved fitness to choline-independent pneumococcal strains when growing in medium lacking any aminoalcohol. Our results suggest complex relationships among the different regions of the TacF teichoic acid repeat unit transporter.     

Pneumococcal Virulence Factors: Structure and Function

The overall goal for this review is to summarize the current body of knowledge about the structure and function of major known antigens of Streptococcus pneumoniae, a major gram-positive bacterial pathogen of humans. This information is then related to the role of these proteins in pneumococcal pathogenesis and in the development of new vaccines and/or other antimicrobial agents. S. pneumoniae is the most common cause of fatal community-acquired pneumonia in the elderly and is also one of the most common causes of middle ear infections and meningitis in children. The present vaccine for the pneumococcus consists of a mixture of 23 different capsular polysaccharides. While this vaccine is very effective in young adults, who are normally at low risk of serious disease, it is only about 60% effective in the elderly. In children younger than 2 years the vaccine is ineffective and is not recommended due to the inability of this age group to mount an antibody response to the pneumococcal polysaccharides. Antimicrobial drugs such as penicillin have diminished the risk from pneumococcal disease. Several pneumococcal proteins including pneumococcal surface proteins A and C, hyaluronate lyase, pneumolysin, autolysin, pneumococcal surface antigen A, choline binding protein A, and two neuraminidase enzymes are being investigated as potential vaccine or drug targets. Essentially all of these antigens have been or are being investigated on a structural level in addition to being characterized biochemically. Recently, three-dimensional structures for hyaluronate lyase and pneumococcal surface antigen A became available from X-ray crystallography determinations. Also, modeling studies based on biophysical measurements provided more information about the structures of pneumolysin and pneumococcal surface protein A. Structural and biochemical studies of these pneumococcal virulence factors have facilitated the development of novel antibiotics or protein antigen-based vaccines as an alternative to polysaccharide-based vaccines for the treatment of pneumococcal disease.     

Incorporation of choline into Streptococcus pneumoniae cell wall antigens: evidence for choline kinase activity

Abstract The choline-containing teichoic and lipoteichoic acids play an important part in cell wall metabolism of Streptococcus pneumoniae . We propose that a choline kinase enzyme has a role in the synthesis of these antigens. The presence of this enzyme was demonstrated in cell free extracts of S. pneumoniae by measuring the fall in ATP concentration due to phosphorylation of choline. Genomic DNA of S. pneumoniae hybridised with a probe consisting of an internal fragment of the choline kinase gene of Saccharomyces cerevisiae and one consisting of the choline binding domain of lytA .     

α-Enolase of Streptococcus pneumoniae induces formation of neutrophil extracellular traps

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia throughout the world, with high morbidity and mortality rates. A major feature of pneumococcal pneumonia is abundant neutrophil infiltration. In this study, we identified S. pneumoniae α-enolase as a neutrophil binding protein in ligand blot assay and mass spectrometry findings. Scanning electron microscopic and fluorescence microscopic analyses also revealed that S. pneumoniae α-enolase induces formation of neutrophil extracellular traps, which have been reported to bind and kill microbes. In addition, cytotoxic assay results showed that α-enolase dose-dependently increased the release of extracellular lactate dehydrogenase from human neutrophils as compared with untreated neutrophils. Furthermore, an in vitro cell migration assay using Chemotaxicell culture chambers demonstrated that α-enolase possesses neutrophil migrating activity. Interestingly, bactericidal assay findings showed that α-enolase increased neutrophil extracellular trap-dependent killing of S. pneumoniae in human blood. Moreover, pulldown assay and mass spectrometry results identified myoblast antigen 24.1D5 as an α-enolase-binding protein on human neutrophils, whereas flow cytometric analysis revealed that 24.1D5 was expressed on human neutrophils, but not on human monocytes or T cells. Together, our results indicate that α-enolase from S. pneumoniae increases neutrophil migrating activity and induces cell death of human neutrophils by releasing neutrophil extracellular traps. Furthermore, we found that myoblast antigen 24.1D5, which expressed on the surface of neutrophils, bound to α-enolase of S. pneumoniae.     

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N -acetyl-muramoyl- L -alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis , whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.