Rubens López

Biofilm Formation by Streptococcus pneumoniae: Role of Choline, Extracellular DNA, and Capsular Polysaccharide in Microbial Accretion

Streptococcus pneumoniae colonizes the human upper respiratory tract, and this asymptomatic colonization is known to precede pneumococcal disease. In this report, chemically defined and semisynthetic media were used to identify the initial steps of biofilm formation by pneumococcus during growth on abiotic surfaces such as polystyrene or glass. Unencapsulated pneumococci adhered to abiotic surfaces and formed a three-dimensional structure about 25 microm deep, as observed by confocal laser scanning microscopy and low-temperature scanning electron microscopy. Choline residues of cell wall teichoic acids were found to play a fundamental role in pneumococcal biofilm development. The role in biofilm formation of choline-binding proteins, which anchor to the teichoic acids of the cell envelope, was determined using unambiguously characterized mutants. The results showed that LytA amidase, LytC lysozyme, LytB glucosaminidase, CbpA adhesin, PcpA putative adhesin, and PspA (pneumococcal surface protein A) mutants had a decreased capacity to form biofilms, whereas no such reduction was observed in Pce phosphocholinesterase or CbpD putative amidase mutants. Moreover, encapsulated, clinical pneumococcal isolates were impaired in their capacity to form biofilms. In addition, a role for extracellular DNA and proteins in the establishment of S. pneumoniae biofilms was demonstrated. Taken together, these observations provide information on conditions that favor the sessile mode of growth by S. pneumoniae. The experimental approach described here should facilitate the study of bacterial genes that are required for biofilm formation. Those results, in turn, may provide insight into strategies to prevent pneumococcal colonization of its human host.

Sequence and transcriptional analysis of a DNA region involved in the production of capsular polysaccharide in Streptococcus pneumoniae type 3

The nucleotide (nt) sequence of a 9704-bp EcoRI fragment of Streptococcus pneumoniae (Sp) type-3 DNA has been determined and found to contain one partial and five complete open reading frames (ORFs). One of these ORFs corresponds to the cap3 A gene coding for the UDP-glucose (UDPGlc) dehydrogenase which is directly responsible for the transformation of some unencapsulated serotype-3 Sp mutants to the encapsulated phenotype [Arrecubieta et al., J. Bacteriol. 176 (1994) 6375-6383]. The two ORFs downstream from this gene (cap3B and cap3C) encode proteins with molecular masses of 49 and 34 kDa. Analysis of the deduced amino acid (aa) sequences of Cap3B and Cap3C shows homology to polysaccharide synthases and UDPG1c pyrophosphorylases, respectively. Furthermore, genetic complementation analysis showed that cap3C restored the galU defect of an Escherichia coli mutant. Northern blots have shown that cap3A, cap3B and cap3C constitute a single transcriptional unit, and primer extension analysis has revealed that the transcription start point is preceded by a nt sequence identical to the sigma 70 consensus promoter sequence of E. coli. The sequence upstream from this cluster also has a high degree of similarity with genes postulated to be essential for capsular production in several Gram+ bacteria. However, Northern blot analysis and insertion-duplication mutagenesis indicated that genes located in this region are not necessary for type-3 capsule production in the Sp strain 406.

Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages.

The nucleotide sequences of genes cpl7 and cpl9 of the Streptococcus pneumoniae bacteriophages Cp-7 and Cp-9, encoding the muramidases CPL-7 and CPL-9, respectively, have been determined. The N-terminal domains of CPL-7 and CPL-9 were virtually identical to that previously reported for the CPL-1 muramidase. The C-terminal domain of the CPL-7 muramidase, however, was different from those of the host amidase and the phage Cp-1 and Cp-9 lysozymes. Whereas all enzymes studied are characterized by repeated sequences at their C termini, the repeat-unit lengths are 20 amino acids (aa) in CPL-1, CPL-9 and in the host amidase, but 48 aa in CPL-7. Six repeated sequences represent the C-terminal domains of CPL-1, CPL-9 and the host amidase, and 2.8 perfect tandem repetitions that of CPL-7. The peculiar characteristics of the structure of CPL-7 muramidase correlate with its biochemical and biological properties. Whereas CPL-1, CPL-9 and the pneumococcal amidase strictly depend on the presence of choline-containing cell walls for activity, CPL-7 is able to degrade cell walls containing either choline or ethanolamine. These results support the previously postulated role for the C-terminal domain of these lytic enzymes in substrate recognition and provide further experimental evidence supporting the notion that the proteins have evolved by an exchange of modular units.

Nucleotide sequence and expression of the pneumococcal autolysin gene from its own promoter in Escherichia coli

Autolysins are enzymes that have several important biological functions and also seem to be responsible for the irreversible effects induced by the beta-lactam antibiotics. The pneumococcal autolysin gene (lyt) has been subcloned from the plasmid pGL30 [García et al., Mol. Gen. Genet. 201 (1985) 225-230] and we have found that the E form of the autolysin is synthesized in Escherichia coli using its own promoter. The high amount of autolysin obtained in the heterologous system when the lyt gene is present in different orientations in the recombinant plasmids studied supports the idea that the autolysin promoter could be a strong one. The nucleotide sequence of the HindIII fragment of pGL80 (1213 bp) containing the autolysin structural gene has been determined. A unique open reading frame (ORF) has been found, a consensus ribosome-binding site and -10 and -35 promoter-like sequences as well as A + T-rich regions farther upstream were also identified. The lyt ORF encodes a protein of 318 amino acid residues having a calculated Mr of 36,532, which agrees with previous size estimates based on electrophoretic migration [Höltje and Tomasz, J. Biol. Chem. 251 (1976) 4199-4207; Briese and Hakenbeck, Eur. J. Biochem. 146 (1985) 417-427]. Our results also demonstrate that the lyt-4 marker represents the first example of a mutation in a structural gene of a bacterial autolysin. The polarity profile of the pneumococcal autolysin supports previous suggestions about the localization of this enzyme in the normal cell.

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 β-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.

LytB, a novel pneumococcal murein hydrolase essential for cell separation

Streptococcus pneumoniae is an important human pathogen that has an absolute nutritional requirement for choline. Replacement of this amino alcohol in a synthetic medium by structural analogues, such as ethanolamine (EA cells), leads to alterations in several physiological properties including cell separation (Tomasz, 1968, Proc Natl Acad Sci USA 59: 86–93). Identical changes including chain formation and loss of autolytic properties can also be induced by adding up to 2% choline chloride to the growth medium (Briese and Hakenbeck, 1985, Eur J Biochem 146: 417– 427). Choline has been shown to inhibit the LytA pneumococcal autolysin (an N-acetylmuramoyl-L-alanine amidase) by preventing its attachment to wall teichoic acids (Giudicelli and Tomasz, 1984, J Bacteriol 158: 1188–1190). In addition, it has been shown that the pneumococcal surface protein A (PspA) is anchored to the choline residues of the membrane-associated lipoteichoic acids (Yother and White, 1994, J Bacteriol 176: 2976–2985). Pneumococcus synthesizes several proteins that recognize and bind to the choline residues of the teichoic and lipoteichoic acids through a specialized domain, the choline-binding domain (ChBD) (Sánchez-Puelles et al., 1990, Gene 89: 69–75). ChBD is built up of six or more well-conserved motifs, each about 20-amino-acid residues long (Garcı́a et al., 1988, Proc Natl Acad Sci USA 85: 914– 918; Garcı́a et al., 1998, Microb Drug Resist 4: 25–36). Currently, there is an increasing interest in the study of pneumococcal genes coding for proteins that possess ChBDs. These proteins have been demonstrated to participate in a series of important biological functions such as cell adhesion and division. For many years, we have studied the molecular structure and biological role of the lytic enzymes of pneumococcus and its bacteriophages (López et al., 1997, Microb Drug Resist 3: 199–211). These enzymes, which exhibit different chemical substrate specificities (i.e. lysozymes, amidases and glucosaminidases), display a modular organization in which the catalytic domain and the ChBD are located at the Nand C-terminal moieties of the protein respectively. It is likely that a defective autolytic system might explain the physiological alterations leading to chain formation in S. pneumoniae. Recently, two independent experimental approaches have generated pneumococcal mutants that do not require choline or analogues for growth (Severin et al., 1997, Microb Drug Resist 3: 391–400; Yother et al., 1998, J Bacteriol 180: 2093–2101). These mutants form long chains when growing under choline-free conditions, and it has been claimed that the lack of an active LytA amidase, the main pneumococcal lytic enzyme, could be responsible for impaired cell separation at the end of cell division. Nevertheless, previous studies have demonstrated that the primary biological consequences of the lytA gene deletion were the formation of small chains (six to ten cells) and the absence of lysis in the stationary phase of growth (Sánchez-Puelles et al., 1986, Eur J Biochem 158: 289–293; Ronda et al., 1987, Eur J Biochem 164: 621–624) (Fig. 2A). A recent study of ABC-type Mn permease-defective mutants of pneumococcus also reports chain formation in the stationary phase of growth (psaC and psaD mutants) or the appearance of small conglomerates or simply aberrant morphology (psaA and psaB mutants) (Novak et al., 1998, Mol Microbiol 29: 1285–1296). Nevertheless, a gene involved in the formation of long chains is not yet known in pneumococcus. The fact that most bacteria possess several lytic enzymes makes it difficult to determine the precise physiological role of these enzymes. Overcoming this limitation requires experiments with well-defined, single, or perhaps multiple, mutants with altered peptidoglycan hydrolase. Using a previously published procedure to characterize the pneumococcal glucosaminidase (Garcı́a et al., 1989, Biochem Biophys Res Commun 158: 251–256), we identified, by SDS–PAGE, four protein bands with apparently strong choline binding affinity. One of these bands was excised from the acrylamide gel, and the N-terminal amino acid sequence was found to be Ser-Asp-Gly-ThrTrp-Gln-Gly. This sequence was compared with the translated version of the partial nucleotide sequence of the S. pneumoniae genome (ftp://ftp.tigr.org/pub/data/s_pneumoniae), and a perfect match was found with an internal peptide of a gene product. This gene (hereafter designated lytB), located in the 10 373 bp contig no. 4117, was polymerase chain reaction (PCR) amplified, sequenced and preliminarily characterized (accession no. AJ010312). The putative 76.4 kDa LytB protein (658 amino acid residues) displays a modular organization different from all the ChBD proteins described previously in the pneumococcal system (Fig. 1A). Furthermore, this enzyme contains a 23-aminoacid-long, cleavable signal peptide (predicted Mr of the processed protein 73 800), as in the case of the S. pneumoniae LytC lysozyme, a new murein hydrolase recently Molecular Microbiology (1999) 31(4), 1275–1281

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.

The molecular characterization of the first autolytic lysozyme of Streptococcus pneumoniae reveals evolutionary mobile domains

A biochemical approach to identify proteins with high affinity for choline‐containing pneumococcal cell walls has allowed the localization, cloning and sequencing of a gene (lytC ) coding for a protein that degrades the cell walls of Streptococcus pneumoniae. The lytC gene is 1506 bp long and encodes a protein (LytC) of 501 amino acid residues with a predicted Mr of 58 682. LytC has a cleavable signal peptide, as demonstrated when the mature protein (about 55 kDa) was purified from S. pneumoniae. Biochemical analyses of the pure, mature protein proved that LytC is a lysozyme. Combined cell fractionation and Western blot analysis showed that the unprocessed, primary product of the lytC gene is located in the pneumococcal cytoplasm whereas the processed, active form of LytC is tightly bound to the cell envelope. In vivo experiments demonstrated that this lysozyme behaves as a pneumococcal autolytic enzyme at 30°C. The DNA region encoding the 253 C‐terminal amino acid residues of LytC has been cloned and expressed in Escherichia coli. The truncated protein exhibits a low, but significant, choline‐independent lysozyme activity, which suggests that this polypeptide adopts an active conformation. Self‐alignment of the N‐terminal part of the deduced amino acid sequence of LytC revealed the presence of 11 repeated motifs. These results strongly suggest that the lysozyme reported here has changed the general building plan characteristic of the choline‐binding proteins of S. pneumoniae and its bacteriophages, i.e. the choline‐binding domain and the catalytic domain are located, respectively, at the N‐terminal and the C‐terminal moieties of LytC. This work illustrates the natural versatility exhibited by the pneumococcal genes coding for choline‐binding proteins to fuse separated catalytic and substrate‐binding domains and create new and functional mature proteins.

Purification and Polar Localization of Pneumococcal LytB, a Putative Endo-β-N-Acetylglucosaminidase: the Chain-Dispersing Murein Hydrolase

The DNA region encoding the mature form of a pneumococcal murein hydrolase (LytB) was cloned and expressed in Escherichia coli. LytB was purified by affinity chromatography, and its activity was suggested to be the first identified endo-beta-N-acetylglucosaminidase of Streptococcus pneumoniae. LytB can remove a maximum of only 25% of the radioactivity from [(3)H]choline-labeled pneumococcal cell walls in in vitro assays. Inactivation of the lytB gene of wild-type strain R6 (R6B mutant) led to the formation of long chains but did not affect either total cell wall hydrolytic activity at the stationary phase of growth or development of genetic competence. Longer chains were formed when the lytB mutation was introduced into the M31 strain (M31B mutant), which harbors a complete deletion of lytA, which codes for the major autolysin. Furthermore, the use of this mutant revealed that LytB is the first nonautolytic murein hydrolase of pneumococcus. Purified LytB added to pneumococcal cultures of R6B or M31B was capable of dispersing, in a dose-dependent manner, the long chains characteristic of these mutants into diplococci or short chains, the typical morphology of R6 and M31 strains, respectively. In vitro acetylation of purified pneumococcal cell walls did not affect the activity of LytB, whereas that of the LytA amidase was drastically reduced. On the other hand, the use of a translational fusion between the gene (gfp) coding for the green fluorescent protein (GFP) and lytB supports the notion that LytB accumulates in the cell poles of either the wild-type R6, lytB mutants, or ethanolamine-containing cells (EA cells). The GFP-LytB fusion protein was also able to unchain the lytB mutants but not the EA cells. In contrast, translational fusion protein GFP-LytA preferentially bound to the equatorial regions of choline-containing cells but did not affect their average chain length. These observations suggest the existence of specific receptors for LytB that are positioned at the polar region on the pneumococcal surface, allowing localized peptidoglycan hydrolysis and separation of the daughter cells.

Molecular organization of the genes required for the synthesis of type 1 capsular polysaccharide of Streptococcus pneumoniae: formation of binary encapsulated pneumococci and identification of cryptic dTDP‐rhamnose biosynthesis genes

We report here the molecular organization of the capsular locus (cap1 ) of the type 1 pneumococcus. This locus is located between dexB and aliA and flanked by IS1167 insertion elements. Sequence analysis showed that the cluster contains 11 genes (cap1A to cap1K ), which are apparently arranged as a single transcriptional unit. The presence of a functional promoter (cap1p ) located upstream of cap1A has been demonstrated and the transcription start point was mapped by primer‐extension analysis. A 14.3 kb fragment containing the genes cap1ABCDEFGHIJK and including cap1p was sufficient to allow the synthesis of a type 1 capsule in Streptococcus pneumoniae. An internal deletion of cap1E leads to an unencapsulated phenotype demonstrating that this gene is essential for capsular production. The cap1K gene has been expressed in Escherichia coli resulting in UDP‐glucose dehydrogenase (UDP‐GlcDH) activity. Moreover, this gene was able to restore the synthesis of type 3 capsule when cloned into a plasmid and introduced by transformation into S. pneumoniae cap3A mutants deficient in UDP‐GlcDH. In marked contrast with what was previously thought, recombination between cap1K and cap3A does occur. We provide data on the molecular mechanism that leads to the formation of binary encapsulated pneumococcal cells, i.e. strains that simultaneously produce type 1 and type 3 capsules. Downstream of cap1K, one truncated and three complete open reading frames homologous to those involved in the biosynthesis of dTDP‐rhamnose, a monosaccharide that does not participate in the formation of type 1 polysaccharide, have been identified in all the clinical strains of type 1 pneumococcus tested. Our results provide new insights into the generation of capsule diversity in pneumococci.